Anti-FZD7 single-domain antibody and use thereof

Abstract

Provided in the present invention is a single-domain antibody that specifically binds to FZD7. The FZD7 single-domain antibody only binds to human FZD7, and does not bind to human FZD1, FZD2, FZD4, FZD5 or FZD8, thus exhibiting strong specificity. The FZD7 single-domain antibody comprises: CDR1 having an amino acid sequence as shown in SEQ ID NO. 1: GX1X2X3SLYT, wherein X1 is Y, W, T, N, F, A, H, I, L, M, R, V or S, X2 is P, Y, T, S, N, A, V, G, L, F, M, R, D, Q or I, and X3 is L, F, Y, M, I or W; CDR2 having an amino acid sequence as shown in SEQ ID NO. 2: MTRGGIT; and CDR3 having an amino acid sequence as shown in SEQ ID NO. 3: TAKMFTAX4X5NDY, wherein X4 is N, Q, A, D, E, I, K, L, M, R, S, T or V, and X5 is G, A or S. Further provided in the present invention are various mutants and fusion proteins prepared on the basis of the single-domain antibody, and the use thereof.

Description

Anti-fzd7 single-domain antibody and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to anti-human FZD7 single-domain antibody and application thereof. BACKGROUND

[0002] WNT signaling pathway is a class of highly conserved signaling pathways in the process of species evolution, which plays a crucial role in the early development of animal embryos, organ formation and tumor occurrence and development. WNT signaling pathway includes several different signaling pathways, such as the classic β-catenin pathway, non-classical planar cell polarity (PCP) pathway and WNT / Ca 2+ pathway, which affect the physiological function of cells through different mechanisms (Niehrs, C. Nat Rev Mol Cell Biol 13, 767-779 (2012)). Over-activation of WNT signaling pathway in tumor stem cells will cause abnormal accumulation of β-catenin in cytoplasm and nucleus, and then activate downstream proteins such as c-Myc, survivin, cyclin D1 and Wisp1, thereby promoting the proliferation of tumor stem cells. At the same time, WNT signaling pathway will act as an immune regulator, inhibit the activity of macrophages, natural killer (NK) cells and dendritic cells, prevent T cells from infiltrating into tumor cells, and then inhibit the recognition and killing of the immune system to tumor, leading to tumor cell immune tolerance and immune escape.

[0003] The WNT protein family binds to the N-terminal extracellular region CRD (cysteine-rich domain) of the receptor Frizzled (FZD), interacts with DVL (disheveled) protein and activates downstream signaling pathways. FZD proteins are a family of WNT receptors with seven transmembrane molecular structures, and there are 10 member proteins in this family, each of which can interact with more than one WNT protein of 19 WNT subtypes. However, the physiological functions of different FZDs still have regulatory bias (Y Wang, et al., Curr Top Dev Biol. 2016; 117: 113-139). FZD7 is one of the FZDs protein members, and studies have shown that WNT receptor FZD7 is abnormally expressed in different types of cancer (including triple-negative breast cancer TNBC, non-small cell lung cancer NSCLC, liver cancer HCC, etc.); FZD7 induces tumor occurrence and metastasis by activating the canonical WNT pathway (WNT / β-catenin). In addition, the expression of FZD7 is mainly limited to embryonic development and is not expressed or expressed in small amounts in most normal adult tissues. On the contrary, FZD7 is observed to be overexpressed in a large number of cancer types, which indicates that FZD7 specific targeting strategies may provide treatment opportunities for multiple cancer types.

[0004] WNT pathway is involved in tumor invasion and metastasis, and also has a regulatory effect on tumor angiogenesis. Knocking out FZD7 can significantly inhibit tumor cell proliferation, transplanted tumor growth and angiogenesis. There is a close relationship between WNT signaling and tumor stem cell behavior, and FZD7 is one of the tumor stem cell markers, and the up-regulation of FZD7 in these tumor stem cells is significantly related to tumor chemotherapy resistance and low survival rate of patients. In addition, the "context-specific functions" of FZD7 suggest that selective targeting of FZD7 can treat cancer without affecting the homeostasis of normal tissues. In summary, FZD7 is a potential therapeutic target for multiple tumors. However, FZD7 is similar in structure to other members of the WNT receptor family, and developing FZD7 targeting drugs with strong specificity faces many difficulties, which is also the main reason why FZD related targets of the WNT pathway are difficult to develop drugs.

[0005] Currently, the main methods targeting FZD7 are RNA interference, small molecule inhibitors, small interfering peptides, soluble recombinant FZD7 protein and anti-FZD7 monoclonal antibodies. Fzd7 shRNA can effectively inhibit the proliferation, invasion and tumor growth of TNBC cells; small interfering peptide RHPDs or small molecule inhibitor FJ9 interfere with the binding of FZD7 and DVL to block WNT signaling and effectively inhibit the growth of mouse transplanted tumors; using the extracellular region recombinant protein of FZD7 to compete with cell surface FZD7 for binding to WNT3 ligand to block WNT / β-catenin signaling also inhibits tumor development; the above methods are still in the preclinical research stage. In contrast, a more clinically applicable method is to use antibodies to specifically target FZD7. OncoMed has developed a monoclonal antibody targeting the CRD domain of FZD, OMP-18R5 (vantictumab), studies have shown that OMP-18R5 binds to multiple FZD proteins in the FZD receptor family, specifically FZD1, FZD2, FZD5, FZD7 and FZD8, and has cross-reactivity. In its phase Ib clinical study of metastatic pancreatic cancer, some patients developed drug-induced brittle bone fractures, which was presumably caused by the cross-reactivity of OMP-18R5. Therefore, obtaining a highly specific FZD7-targeting antibody is an effective way to improve patient clinical benefit. SUMMARY

[0006] The present application utilizes the characteristics of single domain antibodies (sdAb) composed of variable regions of natural heavy chain antibodies lacking light chains in Camelidae (camels, llamas, alpacas and their close species), and screens a plurality of anti-FZD7 single domain antibodies that specifically target FZD7 and have cross-binding with human, monkey and mouse FZD7. Since the WNT signaling pathway is highly involved in bone development and metabolism, and the homology between some proteins in this pathway is very high, drugs targeting the WNT pathway have strong side effects, especially affecting the WNT pathway in normal organs and tissues, thereby causing serious bone toxicity. The highly specific FZD7 single domain antibodies developed in the present application only bind to FZD7 and do not bind to other proteins in the FZD receptor family, which can greatly reduce the toxic side effects caused by non-specific drugs. In addition, the antibodies have comparable cross-reactivity in humans, mice and primates, which can be used for human clinical trials in animal models, avoiding the uncertainty brought by the use of specific model substitutes. For example, chFZD7-1791 (US10766962B2) or F7-Ab (Gumber, Do, et al. eLife 2020, 9: e63060) of the University of California can bind to human FZD7 but cannot bind to mouse FZD7, resulting in the need to use transgenic mice in animal experiments. The present application selects a specific clone (5B6 clone) from the screened FZD7 single domain antibodies, and the CDRs thereof include: CDR1: GSILSLYT, CDR2: MTRGGIT, and CDR3: TAKMFTANGNDY, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO. 32, the amino acid sequence of CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 115. And on the basis of 5B6 and its humanization, a plurality of mutants with improved or unchanged affinity are obtained by site-directed mutagenesis. Specifically, the present applicant finds that any mutation (i.e., S27, I28, L29, N103 and / or G104 can be substituted by any natural amino acid or unnatural amino acid, including but not limited to) at the S27I28L29 site of CDR1 or the N103G104 site of CDR3 does not affect the affinity of the antibody.

[0007] The first aspect of the present application provides an anti-FZD7 single domain antibody. The anti-FZD7 single domain antibody includes CDR1-CDR3 as shown below:

[0008] CDR1: GX1X2X3SLYT, wherein X1 is Y, W, T, N, F, A, H, I, L, M, R, V or S, X2 is P, Y, T, S, N, A, V, G, L, F, M, R, D, Q or I, and X3 is L, F, Y, M, I or W, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO. 1.

[0009] CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2: MTRGGIT;

[0010] CDR3 with an amino acid sequence as set forth in SEQ ID NO. 3: TAKMFTAX4X5NDY, wherein X4 is N, Q, A, D, E, I, K, L, M, R, S, T or V, and X5 is G, A or S.

[0011] In some embodiments of the present application, the combination of X1, X2 and X3 is SIL, YPL, WPL, WTL, WYL, WSL, WNL, WAL, WPF, TVW, NAF, FPF, TPF, AAF, AGF, APL, AVW, AYF, FAW, FAY, FIL, FLL, FLW, FPW, FSW, FVW, FVY, HIF, HLL, HPF, HPL, HVF, IFL, LYL, MPF, NLY, NMF, NSF, RAL, SAW, SIF, SIW, SLW, SPF, SVF, SVL, SYW, TAL, TAW, TFF, TGL, TLL, TPL, TRL, TTL, TVF, TYY, VIL, VYL, WAM, WDL, WGL, WIL, WLL, WLW, WPF, WPI, WPY, WQL, WRL, WSL, WVL, YGL, YIL, YLF, YLL, YML, YSL, YTL, YVL, YVW, YYL or FTF.

[0012] Accordingly, the anti-FZD7 single-domain antibody of the present application has a CDR1 sequence as set forth in any one of SEQ ID NO. 32-114.

[0013] In some embodiments of the present application, the combination of X4 and X5 is NG, QG, AG, DG, EG, IG, KG, LG, MG, RG, SG, TG, VG, NA or NS.

[0014] Accordingly, the anti-FZD7 single-domain antibody of the present application has a CDR3 sequence as set forth in any one of SEQ ID NO. 115-129.

[0015] In some embodiments, when the combination of X4 and X5 is NG, QG, SG, DG or AG,

[0016] X1 is S, X2 is I, X3 is L; X1 is F, X2 is T, X3 is F; X1 is Y, X2 is P, X3 is L; X1 is W, X2 is P, X3 is L; X1 is W, X2 is T, X3 is L; X1 is W, X2 is Y, X3 is L; X1 is W, X2 is S, X3 is L; X1 is W, X2 is N, X3 is L; X1 is W, X2 is A, X3 is L; X1 is W, X2 is P, X3 is F; X1 is T, X2 is V, X3 is W; X1 is N, X2 is A, X3 is F; X1 is F, X2 is P, X3 is F; or X1 is T, X2 is P, X3 is F.

[0017] In other specific implementations, when the combination of X1, X2, and X3 is SIL or FTF...

[0018] X4 is N, X5 is G; X4 is Q, X5 is G; X4 is A, X5 is G; X4 is D, X5 is G; X4 is E, X5 is G; X4 is I, X5 is G; X4 is K, X5 is G; X4 is L, X5 is G; X4 is M, X5 is G; X4 is R, X5 is G; X4 is S, X5 is G; X4 is T, X5 is G; X4 is V, X5 is G; X4 is N, X5 is A; or X4 is N, X5 is S.

[0019] In an embodiment of the present invention, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the following: CDR1 with an amino acid sequence as shown in any of SEQ ID NO. 32 to 114, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in any of SEQ ID NO. 115 to 129.

[0020] Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the following: CDR1 with an amino acid sequence as shown in any of SEQ ID NO.32 to 114, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.115.

[0021] Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the following: CDR1 with an amino acid sequence as shown in any of SEQ ID NO. 32 to 114, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 116.

[0022] Preferably, the anti-FZD7 single-domain antibody comprises CDR1~CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as shown in any one of SEQ ID NO. 32~114, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 118.

[0023] Preferably, the anti-FZD7 single-domain antibody comprises CDR1~CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as shown in any one of SEQ ID NO. 32~114, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 119.

[0024] Preferably, the anti-FZD7 single-domain antibody comprises CDR1~CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as shown in any one of SEQ ID NO. 32~114, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 126.

[0025] Preferably, the anti-FZD7 single-domain antibody comprises CDR1~CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as shown in SEQ ID NO. 32, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in any one of SEQ ID NO. 115~129.

[0026] Preferably, the anti-FZD7 single-domain antibody comprises CDR1~CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as shown in SEQ ID NO. 33, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in any one of SEQ ID NO. 115~129.

[0027] In some embodiments of the present application, the anti-FZD7 single-domain antibody comprises CDR1~CDR3 selected from the group consisting of:

[0028] (1) CDR1 as shown in SEQ ID NO. 32, CDR2 as shown in SEQ ID NO. 2, and CDR3 as shown in SEQ ID NO. 115; or

[0029] (2) CDR1 as shown in SEQ ID NO. 33, CDR2 as shown in SEQ ID NO. 2, and CDR3 as shown in SEQ ID NO. 115; or

[0030] (3) a CDR1 as depicted in SEQ ID NO. 33, a CDR2 as depicted in SEQ ID NO. 2, a CDR3 as depicted in SEQ ID NO. 118; or

[0031] (4) a CDR1 as depicted in SEQ ID NO. 33, a CDR2 as depicted in SEQ ID NO. 2, a CDR3 as depicted in SEQ ID NO. 116; or

[0032] (5) a CDR1 as depicted in SEQ ID NO. 33, a CDR2 as depicted in SEQ ID NO. 2, a CDR3 as depicted in SEQ ID NO. 119; or

[0033] (6) a CDR1 as depicted in SEQ ID NO. 33, a CDR2 as depicted in SEQ ID NO. 2, a CDR3 as depicted in SEQ ID NO. 126.

[0034] The anti-FZD7 single-domain antibody provided in the application is a llama antibody. The anti-FZD7 single-domain antibody described in the application further comprises a framework region FR, which comprises FR1-FR4 as shown in the following amino acid sequence:

[0035] FR1 as shown in SEQ ID NO. 130, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 132, and FR4 as shown in SEQ ID NO. 133.

[0036] The anti-FZD7 single-domain antibody provided by the application can be further humanized to become a humanized antibody. In order to further reduce the potential immunogenicity risk of the single-domain antibody in the process of disease treatment, the specific amino acid sequence in the camel antibody framework can be replaced with the amino acid sequence at the corresponding position of the human antibody as much as possible. In the present application, the process of antibody humanization refers to the chapter of humanization of camel single-domain antibody in the book Single-Domain Antibodies written by Greg Hussack and Kevin A. Henry, and the candidate sites that need to be replaced of the FZD7 single-domain antibody are predicted by using immunogenicity analysis software (http: / / tools.iedb.org / deimmunization / ) and homology modeling software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ), and the amino acids of the candidate sites are replaced by humanization according to the general framework grafting method of VHH humanization established by Ce'cile Vincke et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. J Biol Chem. 2009; 284 (5): 3273-3284). The framework region FR of the anti-FZD7 single-domain antibody described in the application includes FR1-FR4 with the amino acid sequences as shown below:

[0037] FR1 as shown in SEQ ID NO. 134, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135; FR4 as shown in SEQ ID NO. 136; or

[0038] FR1 as shown in SEQ ID NO. 134, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 137; FR4 as shown in SEQ ID NO. 136; or

[0039] FR1 as shown in SEQ ID NO. 139, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135; FR4 as shown in SEQ ID NO. 136; or

[0040] FR1 as shown in SEQ ID NO. 138, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135; FR4 as shown in SEQ ID NO. 136; or

[0041] FR1 as shown in SEQ ID NO. 140, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135; FR4 as shown in SEQ ID NO. 136.

[0042] The anti-FZD7 single domain antibody provided by the present application comprises an amino acid sequence as shown in any one of SEQ ID NO. 141-185. In some embodiments, the anti-FZD7 single domain antibody provided by the present application comprises an amino acid sequence having 80% or more sequence identity to any one of SEQ ID NO. 141-185. More specifically, the anti-FZD7 single domain antibody provided by the present application can have 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NO. 141-185. In some embodiments, the amino acid sequence of the anti-FZD7 single domain antibody provided by the present application is obtained by substituting, deleting, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to any one of SEQ ID NO. 141-185, or by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus. The anti-FZD7 single domain antibody can specifically bind to FZD7.

[0043] The present application provides an anti-FZD7 single domain antibody that binds to an epitope different from prior art. The anti-FZD7 single domain antibody provided by the present application can bind to positions 33-49, 66-70, and / or 110-116 of the amino acid sequence as shown in SEQ ID NO. 10.

[0044] In some embodiments, the anti-FZD7 single domain antibody can bind to one or more discontinuous epitopes of positions 33-49, 66-70, and / or 110-116 of the amino acid sequence as shown in SEQ ID NO. 10.

[0045] In some embodiments, the anti-FZD7 single domain antibody binds to at least one of the following residues of the amino acid sequence as shown in SEQ ID NO. 10: H36, V43, F48, C49, I66, P68, N69, C110, D114, or A116. Preferably, the anti-FZD7 single domain antibody binds to at least one of the following residues of the amino acid sequence as shown in SEQ ID NO. 10: F48, C49, I66, D114, or A116.

[0046] The anti-FZD7 single-domain antibody comprises CDR1-CDR3 as shown below: CDR1: amino acid sequence as shown in SEQ ID NO. 1: GX1X2X3SLYT, wherein X1 is Y, W, T, N, F, A, H, I, L, M, R, V or S, X2 is P, Y, T, S, N, A, V, G, L, F, M, R, D, Q or I, and X3 is L, F, Y, M, I or W; CDR2: MTRGGIT; CDR3: TAKMFTAX4X5NDY, wherein X4 is N, Q, A, D, E, I, K, L, M, R, S, T or V, and X5 is G, A or S.

[0047] In some embodiments, the anti-FZD7 single-domain antibody comprises CDR1-CDR3 selected from the group consisting of: CDR1: amino acid sequence as shown in any one of SEQ ID NO. 32-114, CDR2: amino acid sequence as shown in SEQ ID NO. 2, and CDR3: amino acid sequence as shown in any one of SEQ ID NO. 115-129.

[0048] In some embodiments, the anti-FZD7 single-domain antibody comprises an amino acid sequence as shown in any one of SEQ ID NO. 141-185, or an amino acid sequence having 80% or more sequence identity to any one of SEQ ID NO. 141-185.

[0049] The second aspect of the present application provides a fusion protein comprising the anti-FZD7 single-domain antibody of the first aspect of the present application.

[0050] In some embodiments, the fusion protein further comprises an Fc region of an immunoglobulin. In some preferred embodiments, the Fc region of the immunoglobulin is selected from a human immunoglobulin Fc region. In some preferred embodiments, the Fc region of the immunoglobulin is selected from a complete Fc region, a fragment of the Fc region, or a mutant of the Fc region. For example, the Fc region can or can not comprise a hinge region; the lysine at the C-terminus of the Fc region can be removed to facilitate the uniformity of the product; the Fc region can comprise mutations for altering the Fc-mediated effector functions, including one or more of a combination of Fc receptor binding, CDC activity, ADCC activity, and ADCP activity.

[0051] In some embodiments, the Fc region of the immunoglobulin is selected from one or more of a combination of IgG, IgA1, IgA2, IgD, IgE, and IgM. In some preferred embodiments, the IgG is selected from one or more of a combination of IgG1, IgG2, IgG3, and IgG4 subtypes.

[0052] In some embodiments, the immunoglobulin Fc region is selected from the group consisting of the amino acid sequence of any one of SEQ ID NO. 22-25, SEQ ID NO. 233.

[0053] In some embodiments, the fusion protein has an amino acid sequence as set forth in any one of SEQ ID NO. 186-232 or an amino acid sequence having 80% or more sequence identity to any one of SEQ ID NO. 186-232. More specifically, the anti-FZD7 single-domain antibody provided by the present application can have 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NO. 186-232.

[0054] In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NO. 141-185 or an amino acid sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NO. 141-185.

[0055] The third aspect of the present application provides an isolated polynucleotide encoding the single-domain antibody of the first aspect of the present application or encoding the fusion protein of the second aspect of the present application.

[0056] The fourth aspect of the present application provides an expression vector comprising the isolated polynucleotide of the third aspect of the present application.

[0057] The fifth aspect of the present application provides an expression system comprising the expression vector of the fourth aspect of the present application or the isolated polynucleotide of the third aspect of the present application integrated into the genome of the expression system.

[0058] The sixth aspect of the present application provides a method for preparing the single-domain antibody or the fusion protein, comprising the steps of: culturing the expression system of the single-domain antibody or the fusion protein under conditions suitable for expression of the single-domain antibody or the fusion protein, thereby expressing the single-domain antibody or the fusion protein, and purifying and isolating the single-domain antibody or the fusion protein.

[0059] The seventh aspect of the present application provides a pharmaceutical composition comprising the single-domain antibody of the first aspect of the present application or the fusion protein of the second aspect of the present application.

[0060] In an eighth aspect, the present application provides use of the single-domain antibody of the first aspect, or the fusion protein of the second aspect, or the pharmaceutical composition of the seventh aspect in the manufacture of a medicament for the diagnosis, treatment or prevention of a tumor positive for FZD7 expression. The present application also provides a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one or more of the following: the single-domain antibody of the first aspect, or the fusion protein of the second aspect, or the pharmaceutical composition of the seventh aspect. In some embodiments of the present application, the tumor is selected from the group consisting of one or more of a tumor in which FZD7 expression is upregulated, including but not limited to liver cancer, urothelial cancer, cervical cancer, breast cancer (e.g., triple-negative breast cancer), gastric cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), thyroid papillary carcinoma, ovarian cancer, endometrial cancer, prostate cancer, colorectal cancer, head and neck squamous cell carcinoma, and renal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 is the binding ability of Anti-FZD7-Fc fusion protein to human FZD7 (CRD) recombinant protein;

[0062] Figure 2A is the binding activity of Anti-FZD7-Fc fusion protein to monkey FZD7 overexpression cells;

[0063] Figure 2B is the binding activity of Anti-FZD7-Fc fusion protein to mouse FZD7 overexpression cells;

[0064] Figure 3 is the flow cytometry binding activity of OMP18R5 analog and SHH002-hul analog to CHOK1Q-FZD7 cells;

[0065] Figure 4A is the binding activity of OMP18R5 analog to different FZD family members;

[0066] Figure 4B is the binding activity of Anti-FZD7-5B6-Fc fusion protein to different FZD family members;

[0067] Figure 5A is the binding curve of humanized Anti-FZD7-5B6-Fc to human FZD7 overexpression cells;

[0068] Figure 5B is the binding curve of humanized Anti-FZD7-5B6-Fc to monkey FZD7 overexpression cells;

[0069] Figure 5C is the binding curve of humanized Anti-FZD7-5B6-Fc to mouse FZD7 overexpression cells;

[0070] Figure 6A is the binding curve of humanized Anti-FZD7-5B6-Fc to human FZD1 overexpression cells;

[0071] Figure 6B is a binding curve of humanized Anti-FZD7-5B6-Fc to human FZD2 overexpressing cells;

[0072] Figure 6C is a binding curve of humanized Anti-FZD7-5B6-Fc to human FZD5 overexpressing cells;

[0073] Figure 7A is the ADCC activity of the test substances on FZD7 expression positive cells;

[0074] Figure 7B is the ADCC activity of the test substances on NCI-H1975 cells;

[0075] Figure 7C is the ADCC activity of the test substances on MDAMB231 cells;

[0076] Figure 7D is the ADCC activity of the test substances on HPAF-II cells;

[0077] Figure 8A is a binding curve of hu5B6v17-Fc to Expi293F-FZD7 cells;

[0078] Figure 8B is a binding curve of hu5B6v17-Fc to Expi293F-FZD7 cells;

[0079] Figure 9A is a binding curve of hu5B6v17-Fc to human FZD1 overexpressing cells;

[0080] Figure 9B is a binding curve of hu5B6v17-Fc to human FZD2 overexpressing cells;

[0081] Figure 9C is a binding curve of hu5B6v17-Fc to human FZD5 overexpressing cells;

[0082] Figure 9D is a binding curve of hu5B6v17-Fc to human FZD8 overexpressing cells;

[0083] Figure 10 is a binding curve of CDR3 mutants derived from hu5B6v17 to CHOK1Q-FZD7 cells;

[0084] Figure 11A is the binding activity of hu5B6v10-Fc to different FZD family members;

[0085] Figure 11B is the binding activity of hu5B6v23-Fc to different FZD family members;

[0086] Figure 12 is the binding activity of hu5B6v10-Fc, hu5B6v23-Fc and chFZD7-1791 to human FZD7 (CRD) recombinant protein;

[0087] Figure 13 shows the binding activity of hu5B6v23-Fc, hu5B6v24-Fc and hu5B6v25-Fc to human FZD7;

[0088] Figure 14 shows the binding activity of hu5B6v10-Fc, hu5B6v17-Fc and hu5B6v21-Fc to human FZD7;

[0089] Figure 15 shows the endocytosis ability of FZD7 antibodies on CHOK1Q-FZD7 cells;

[0090] Figures 16A-16D show the binding activity of Anti-FZD7-hu5B6v25-Fc to wild-type FZD7 and mutant FZD7;

[0091] Figure 17 shows the tumor inhibition effect of the test substances on CHOK1-FZD7 tumor-bearing mice;

[0092] Figure 18 shows the effect of the test substances on the body weight of CHOK1-FZD7 tumor-bearing mice. DETAILED DESCRIPTION

[0093] The present inventors, through extensive screening and in-depth research, provide a class of anti-FZD7 single-domain antibodies with extremely high specificity to FZD7; further provided are fusion proteins comprising the single-domain antibodies, and humanized antibodies and fusion proteins prepared based on the single-domain antibodies; the anti-FZD7 single-domain antibodies have extremely high specificity to FZD7, good affinity, and have a significant tumor inhibition effect.

[0094] The term "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen can be a polypeptide, protein, nucleic acid, cell, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments herein, the target antigen is the FZD protein family, including FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, etc.

[0095] The term "antibody" or "immunoglobulin" is used herein as a general term to include full-length antibodies, individual chains thereof, and all portions, domains or fragments thereof, including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively, whether referring to heavy chain antibodies or to conventional antibodies having four chains. Furthermore, the term "sequence" as used herein (e.g., in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is to be understood generally as including both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding said sequence, unless a more limited interpretation is required herein.

[0096] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies display single binding specificity and affinity to a particular epitope.

[0097] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. A discontinuous epitope refers to a conformational epitope on a protein antigen that is composed of at least two separate regions of the primary sequence of the protein.

[0098] The term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of the protein, and in many cases can be added, removed, or transferred to other proteins without loss of function of the remainder of the protein and / or the domain.

[0099] The term "single domain antibody" (often abbreviated as sdAb) refers to a functional antigen binding fragment consisting of only one antibody variable region, which can accomplish antigen recognition without the assistance of light chain; its sources are extensive, including heavy chain antibodies (missing light chain, consisting of only heavy chain) of Camelidae (camels, llamas, etc.), IgNAR (new antigen receptor) of cartilaginous fishes such as sharks, and variable regions (obtained by genetic engineering, such as VH or VL) of traditional antibodies of human or murine origin. While VHH (Variable Domain of Heavy Chain of Heavy-chain Antibody) refers to the heavy chain variable region of heavy chain antibody, which only exists in heavy chain antibodies of Camelidae (Harmsen MM, et al. Appl Microbiol Biotechnol. 2007 Nov;77(1): 13-22), more specifically refers to an immunoglobulin consisting of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, which are interrupted by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of VHH can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. VHH confers the specificity of an antibody for an antigen due to the presence of an antigen binding site. VHH is the smallest antigen binding fragment with full functionality.

[0100] In this article, the terms "single-domain antibody", "heavy chain single-domain antibody", "VHH domain", "VHH", "VHH antibody fragment" and "VHH antibody" are used interchangeably.

[0101] The term "IMGT numbering system" is an integrated information system specifically for immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complexes (MHC) in humans and other vertebrates. (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). The antibody light and heavy chain genes were analyzed using IMGT (http: / / www.imgt.org / IMGT_vquest) to determine the framework regions (FRs) and complementarity determining regions (CDRs) of the variable domain. The “position” of CDRs within the structure of the immunoglobulin variable domain is conserved across species and is located in structures called loops. Therefore, CDRs and framework residues are easily identified using a numbering system that aligns variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into the acceptor framework, typically derived from human antibodies. Unless otherwise stated, in this specification, claims, and drawings, anti-FZD7 single-domain antibodies are numbered according to the IMGT numbering method to determine the CDR and FR regions.

[0102] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding module to bind specific antigenic determinants can be demonstrated by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on BIAcore instruments) (Liljeblad et al., Glyco J17, 323-329 (2000)), and immunofluorescence techniques.

[0103] The term "humanized antibody" refers to a molecule that has an antigen-binding site substantially from a non-human immunoglobulin, wherein the remainder of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site can comprise the entire variable domain fused to a constant domain, or only the complementarity determining regions (CDRs) grafted into the appropriate framework regions of a variable domain. The antigen-binding site can be wild-type, or modified by one or more amino acid substitutions, for example to more closely resemble a human immunoglobulin. Certain forms of humanized antibodies retain all of the CDR sequences (e.g., humanized single domain antibodies containing all three CDRs from a llama). Other forms have one or more CDRs that are altered relative to the original antibody.

[0104] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bound on Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to target cells that display antigen and subsequently kill those target cells with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for the killing. NK cells are the primary cells for mediating ADCC, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcRs are known to be expressed on leukocytes, including lymphocytes, monocytes, neutrophils, and macrophages (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9:457-92). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay (see, e.g., US Patent Nos. 5,500,362 and 5,821,337) can be performed. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

[0105] In the present application, the term "antibody endocytosis" or "antibody endocytosis ability" or "antibody endocytosis activity" refers to the ability of an antibody to enter the interior of a cell through endocytosis after specifically binding to an antigen on the surface of the cell.

[0106] The term“blocking activity of an antibody” generally refers to the ability of an antibody to bind to a specific antigen and prevent or reduce the normal interaction of the antigen with its receptor or ligand. This blocking effect can be used for various biological and medical purposes, for example, (1) neutralization: in viral infection, an antibody can bind to a specific protein on the surface of the virus, preventing the virus from binding to the host cell receptor, thereby neutralizing the infection ability of the virus; (2) signal transduction blocking: in the process of cell signal transduction, an antibody can bind to a specific signal molecule on the surface of a cell or a specific signal molecule, blocking the transmission of signals, affecting cell behavior such as proliferation, differentiation, etc.; (3) immune regulation: antibodies can be used to regulate the immune system, for example, by blocking specific immune cell surface molecules, changing the strength or direction of the immune response.

[0107] The term“WNT protein” or“WNT ligand” refers to a family of cysteine-rich glycoproteins that play a crucial role in the development of organisms and tumorigenesis. Currently, 19 WNT genes have been identified in humans, each encoding a corresponding 19 WNT proteins. WNT3a, as a representative WNT signaling protein, plays a key role in embryonic development, cell growth, differentiation, polarity formation, neural development, and carcinogenesis. WNT proteins can bind to receptors, i.e., Frizzled proteins, which contain seven transmembrane domains, and ten Frizzled receptor proteins have been found in humans (Sun Y, et al., Oncol Res. 2021 Mar 16; 28(6): 661-674.).

[0108] The term“WNT signaling pathway” or“WNT pathway” refers to a series of intracellular signaling pathways mediated by WNT proteins to regulate normal physiological processes of organisms, which are involved in various physiological and pathological processes, including cell proliferation, differentiation, migration, tumorigenesis, etc. When the WNT signaling pathway is abnormally activated, it can lead to the occurrence of pathological conditions such as tumors. Therefore, blocking the activity of the WNT pathway can be used as a treatment strategy, especially in cancer treatment. The WNT signaling pathway can be divided into canonical pathway (i.e., WNT / β-catenin signaling pathway) and non-canonical pathway (including WNT / PCP pathway and WNT / Ca 2+ pathway) according to whether it depends on the transcriptional activation of β-catenin. Wnt / β-catenin signaling pathway, also known as canonical Wnt pathway. In this pathway, WNT ligand protein binds to Frizzled receptor on the cell surface, activates a series of downstream signaling events, and ultimately leads to the accumulation of β-catenin protein in the cytoplasm and translocation into the nucleus, activating specific gene expression. WNT / PCP (Planar Cell Polarity) pathway and WNT / Ca 2+The pathway is independent of β-catenin, and WNT and FZD receptor binding activates Disheveled (DVL) which in turn participates in regulating intracellular calcium levels through a G-protein signaling cascade.

[0109] The term“FZDs family” or“FZD” refers to the Frizzled protein family, which are cell membrane receptors in the WNT signaling pathway, and they belong to the G protein-coupled receptor (GPCR) family. The FZDs family consists of 10 members (FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10), each FZD subtype contains an N-terminal signal peptide, an extracellular cysteine-rich domain (CRD), a 7-transmembrane domain and a discoidin homology region (PDZ). The human 10 FZDs (FZD1-10) proteins contain 500-700 amino acids, and according to sequence homology, FZDs can be divided into four subfamilies: FZD1 / 2 / 7 (amino acid identity AAI: 97%), FZD4 / 9 / 10 (AAI: 65%), FZD5 / 8 (AAI: 70%) and FZD3 / 6 (AAI: 50%) (Zhong L. Hua, et. al., Development. 2014 Oct; 141(20): 3944-3954.). FZD1 and FZD3 are the major expressed FZD proteins in the nervous system, FZD1 is enriched in the central nervous system and has neuroprotective effects, and the deletion of FZD3 in the spinal cord will cause defects in the transmission of sensory information between the limbs and the brain. The main function of FZD2 is to regulate cell cycle, cytochrome, cell growth and cell migration, and it is abnormally highly expressed in gastric cancer, liver cancer, endometrial cancer and oral squamous cell carcinoma. FZD4 plays an important role in blood vessels and the cardiovascular system, and experiments have shown that the deletion of FZD4 will affect the formation of organ blood vessels. FZD5 and FZD7 jointly regulate the homeostasis of the gastrointestinal system, FZD5 mainly regulates the secretion of antibacterial peptides and immunomodulatory proteins by Paneth cells to regulate intestinal flora, and FZD7 is the only member of the FZDs family that regulates the development of the intestinal system, and is very important for cell stem type maintenance, intestinal epithelial homeostasis and regeneration. FZD6 is the only protein in the FZDs family that does not contain a PDZ domain adjacent to the C-terminal, and it is widely reported to play an important role in the non-canonical WNT pathway. FZD1, FZD4, FZD8 and FZD9 jointly regulate the formation and absorption of bone cells and maintain bone homeostasis (Kushwaha P, et. al., J Cell Physiol. 2020 Oct; 235(10): 6673-6683; Albers J, et al. J Cell Biol, 2013, 200(4):537-49; Albers J, et al., J Cell Biol, 2011, 192(6): 1057-72; Yu S, et. al., Biochem J, 2015, 465(3):395-404).

[0110] "Sequence identity" indicates the percentage of identical amino acids between two polypeptide sequences. Methods for evaluating the degree of sequence identity between amino acid sequences are known to those skilled in the art. Amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program in the NCBI database can be used to determine sequence identity.

[0111] The term "treatment / prevention" (and its grammatical variations) refers to an attempt to alter the natural course of a disease in an individual, and can be a clinical intervention implemented for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow the progression of symptoms.

[0112] The abbreviations used in the examples have the following specific meanings:

[0113] L-WNT3a CM: Conditioned culture medium for L-WNT3a cells;

[0114] CM: Conditioned Medium;

[0115] CRD: cysteine-rich domain;

[0116] BSA: Bovine Serum Albumin;

[0117] PBS: Phosphate-Buffered Saline;

[0118] PBST: Phosphate-Buffered Saline with Tween-20;

[0119] TMB: 3,3',5,5'-Tetramethylbenzidine;

[0120] MSX: L-aminosulfoxide methionine;

[0121] EGFP: Enhanced Green Fluorescent Protein;

[0122] HRP: Horseradish Peroxidase;

[0123] Goat Anti-Human IgG, Monkey ads-HRP: Goat anti-human IgG secondary antibody, adsorbed with monkey serum-HRP labeled;

[0124] Anti-M13 Antibody (HRP): HRP labeled antibody against M13 bacteriophage;

[0125] DAR: Dye-to-Antibody Ratio, the ratio of dye molecules to antibody molecules in a fluorescently labeled antibody;

[0126] Goat Anti-Mouse IgG H&L 650)preadsorbed: Goat anti-mouse IgG H&L 650)pre-adsorbed secondary antibody.

[0127] Example 1 Anti-FZD7 single domain antibody library construction

[0128] FZD7 33-185 (CRD sequence, SEQ ID NO: 4) was mixed with 0.5 mg of FZD7(CRD)-llamaFc (SEQ ID NO: 5) fusion protein of alpaca immunoglobulin G-Fc and 0.5 ml of Gerbu water-soluble adjuvant (GERBU biotechnik) (non-emulsification) to immunize a healthy alpaca (Vicugna pacos), and the alpaca was immunized again after 21 days, for a total of 7 times, to stimulate B cells to express antigen-specific single-domain antibodies. Blood samples were collected on days 0, 49, 70, 91, 112, 133 for blood tests, and alpaca serum was detected by ELISA to determine the antibody titer in the alpaca serum. After 7 times of immunization with the antigen protein, the constructed overexpression human FZD7 cell line was used for an additional 4 times of immunization, wherein the construction of the overexpression human FZD7 cell line was performed as follows: an expression plasmid with a gene sequence of human FZD7 (SEQ ID NO: 10) linked to an EGFP fluorescent protein gene sequence was constructed, the expression plasmid was electroporated in CHOK1Q cell strain (Guangdong Zhongshan Kangsheng, item number A13101), and MSX pressure screening and unlimited dilution method were used to obtain the overexpression cell strain CHOK1Q-FZD7. 30 ml of alpaca blood was collected in a vacuum blood collection tube, and lymphocytes were separated by lymphocyte separation medium (Tianjin Haoyang Huake Biological Technology Co., Ltd.). Total RNA was extracted by Trizol method. 5 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Invitrogen) according to the instructions, and VHH was amplified by nested PCR, and the last round of PCR added vector homologous recombination arms to VHH. The target VHH nucleic acid fragment was recovered and recombined into the phage display vector pcomb3xss (Addgene plasmid #63890; RRID: Addgene_63890) using a homologous recombination enzyme (Suzhou Jinan Protein Technology Co., Ltd.). The homologous recombination product was transformed into electrocompetent cells XL1-Blue (Nanjing Heming Yinggu Biological Technology Co., Ltd.) to construct an Anti-FZD7 single-domain antibody library. The library capacity was determined to be greater than 10 7 . More than 24 clones were randomly picked for Sanger sequencing, and the results showed that the insertion rate of the constructed library was 100%, and the sequence diversity of the library was 100%.

[0129] Example 2 Screening and identification, expression and purification, and affinity verification of Anti-FZD7 single-domain antibodies

[0130] 2.1 Screening of Anti-FZD7 single-domain antibodies

[0131] The constructed anti-FZD7 single-domain antibody library was packaged with helper phage M13KO7 (NEB) to obtain a recombinant phage display library. FZD7(CRD)-llamaFc was diluted to 5 μg / ml with 100 mM NaHCO3pH 8.2 coating solution and added to 100 μl / well of coated enzyme-labeled plates (beaverbio) at 4°C overnight. The next day, after adding 200 μl of 3% BSA and blocking at 37°C for 2 h, about 1 x 1011PFU / well of the recombinant phage display library was added and incubated at 37°C for 2 h. Then, non-specifically bound phages were removed by washing 5 times with PBST (0.05% Tween 20 in PBS) and 10 times with PBS. The washed ELISA wells were eluted with 100 μl / well of 0.1 M Gly-HCl 1 mg / ml BSA (pH 2.2) buffer at 37°C for 8 min and neutralized with 1 M pH 8.0 Tris-HCl. The eluted phage titer was determined, and the above phage eluate was amplified. A fusion protein FZD7(CRD)-TS-His (SEQ ID NO. 6) composed of the FZD7 cysteine-rich domain (CRD) sequence and the Twin-Strep-His8 tag was used as the coating protein, the coating plate amount was reduced to 200 ng / well, 3% ovalbumin (OVA) was used for blocking, the incubation time was shortened to 37°C x 1 h, the washing conditions were enhanced to 10 times with PBST and 20 times with PBS, and the second round of affinity enrichment was performed. Based on the above phage panning basic ideas and methods, a total of two or three rounds of enrichment panning were performed, and various panning conditions were introduced, including liquid phase affinity panning, construction of a human FZD7 overexpression cell line for cell affinity panning, construction of a human FZD5 overexpression cell line for cell negative panning to remove FZD5 cross-binding antibodies, construction of a fusion protein FZD5(CRD)-llamaFc (SEQ ID NO. 8) and FZD5(CRD)-TS-His (SEQ ID NO. 9) of FZD5 27-167 (CRD sequence, SEQ ID NO: 7) and llama immunoglobulin G-Fc or Twin-Strep-His8 for negative panning to remove FZD5 cross-binding antibodies, etc. 11 PFU / well of the recombinant phage display library, and incubated at 37°C for 2 h. Then, non-specifically bound phages were removed by washing 5 times with PBST (0.05% Tween 20 in PBS) and 10 times with PBS. The washed ELISA wells were eluted with 100 μl / well of 0.1 M Gly-HCl 1 mg / ml BSA (pH 2.2) buffer at 37°C for 8 min and neutralized with 1 M pH 8.0 Tris-HCl. The eluted phage titer was determined, and the above phage eluate was amplified. A fusion protein FZD7(CRD)-TS-His (SEQ ID NO. 6) composed of the FZD7 cysteine-rich domain (CRD) sequence and the Twin-Strep-His8 tag was used as the coating protein, the coating plate amount was reduced to 200 ng / well, 3% ovalbumin (OVA) was used for blocking, the incubation time was shortened to 37°C x 1 h, the washing conditions were enhanced to 10 times with PBST and 20 times with PBS, and the second round of affinity enrichment was performed. Based on the above phage panning basic ideas and methods, a total of two or three rounds of enrichment panning were performed, and various panning conditions were introduced, including liquid phase affinity panning, construction of a human FZD7 overexpression cell line for cell affinity panning, construction of a human FZD5 overexpression cell line for cell negative panning to remove FZD5 cross-binding antibodies, construction of a fusion protein FZD5(CRD)-llamaFc (SEQ ID NO. 8) and FZD5(CRD)-TS-His (SEQ ID NO. 9) of FZD5 27-167 (CRD sequence, SEQ ID NO: 7) and llama immunoglobulin G-Fc or Twin-Strep-His8 for negative panning to remove FZD5 cross-binding antibodies, etc.

[0132] 2.2 Phage ELISA for identifying Anti-FZD7 single-domain antibodies

[0133] From the different elution conditions of the panning, 2090 single clones were picked from the plate of the determination of the titer of the eluted phage and cultured in 96-well plates, and then infected and packaged with M13KO7 helper phage to obtain the accumulation of the recombinant phage in the supernatant. FZD7(CRD)-llamaFc was coated at 100 ng / well, and blocked with 3% BSA at 37°C for 2 hours, or the liquid phase detection was performed by coating recombinant Strep-Tactin (containing cysteine) (r-STc, Hangzhou Nuolong Biotechnology Co., Ltd.) to capture FZD7(CRD)-TS-His. The supernatant of the single clone recombinant phage was mixed with 2% BSA at an equal ratio in a 96-well plate, and then 100 μl / well was incubated in the 96-well plate coated with FZD7(CRD)-llamaFc or coated with r-STc to capture FZD7(CRD)-TS-His (wherein, the well only added with M13KO7 helper phage was used as a negative control), and incubated at 37°C for 1 hour. After 5 times of PBST washing, 100 μl of 0.1 μg / mL Anti-M13 Antibody (HRP), Mouse Monoclonal (SinoBiological) was added to each well, and incubated at 37°C for 1 hour. After 5 times of PBST washing, TMB color developing working solution (Huzhou Yingchuang Biotechnology Co., Ltd.) was added, and color development was performed after incubation at 37°C for 5 minutes, and then 1M sulfuric acid was added to terminate the reaction, and OD450nm was read. Among them, the M13KO7 negative control well showed weak or basically no color development (OD450nm value was lower than 0.3), and the FZD7(CRD) positive well showed obvious color development or a significant difference from the negative well (OD450nm value was higher than 2.5). The positive clone combined with FZD7(CRD) was selected for Sanger sequencing, and the repeated sequences were removed.

[0134] After multiple rounds of screening, 5 strong positive or relatively strong positive anti-FZD7 single domain antibody clones of FZD7(CRD) binding were finally picked: 2B1, 3D11, 5B6, 11B1 and 12H4. The full-length sequence of 5B6 clone will be taken as the research object for research, and the CDR region is indicated by underlining in Table 1.

[0135] Table 1

[0136] The framework region (FR) and the complementarity determining region (CDR region) of each antibody are shown in Table 2.

[0137] Table 2

[0138] 2.3 Expression and purification of anti-FZD7 single domain antibody in E. coli

[0139] The candidate single-domain antibodies obtained by screening were subjected to gene synthesis, and an ompA signal peptide sequence was introduced at the 5' end of the sequence, and a histidine tag coding sequence was introduced at the 3' end, which was inserted into a pET32a+ vector (Novagen) to construct an E. coli periplasmic expression plasmid. The expression plasmid was transformed into an E. coli expression host Rosetta (DE3) to construct an E. coli expression strain. Recombinant clones were picked on ampicillin-resistant plates, cultured, and induced to express at 30°C overnight using 1 mM IPTG. The bacteria solution induced to express overnight was subjected to ultrasonic disruption, and after centrifugation at 12,000 g at 4°C for 10 minutes, the supernatant containing the soluble single-domain antibody secreted into the periplasm was purified using a nickel column (Bogu Biotech Co., Ltd.), and finally the anti-FZD7 monovalent single-domain antibody with a fusion histidine tag: Anti-FZD7-VHH-his was obtained.

[0140] 2.4 Anti-FZD7-5B6 single-domain antibody verified for affinity by flow cytometry and ELISA

[0141] FZD7(CRD)-llamaFc (SEQ ID NO: 5) was coated at 200 ng / well at 4°C overnight, and blocked with 3% BSA at 37°C for 2 hours. The Anti-FZD7-VHH-his fusion protein was gradient-diluted with 1% BSA, and incubated at 37°C for 1 hour. After 3 PBST washes, 100 μl / well of Anti-his tag 3D5 antibody (mouse IgG1) diluted with 1% BSA was added, and incubated at 37°C for 1 hour. After PBST washing, 100 μl of HRP-Goat anti mouse IgG antibody (Thermo Scientific) diluted 1:10,000 was added per well, and incubated at 37°C for 1 hour. After 5 PBST washes, TMB substrate was added, and incubated at 37°C. After 5 minutes of color development, 1 M sulfuric acid was added to terminate the reaction, and OD450nm was read, and the binding EC 50 value of Anti-FZD7 monovalent single-domain antibody Anti-FZD7-5B6-His to human FZD7(CRD) recombinant protein was calculated as 3.13 nM.

[0142] An expression plasmid with a gene sequence of human FZD7 (SEQ ID NO: 10) linked to an EGFP fluorescent protein gene sequence was constructed, the expression plasmid was electroporated in CHOK1Q cells, and overexpression cell strain CHOK1Q-FZD7 was obtained by MSX pressure screening and infinite dilution method, which was used to infect CHOK1Q cells at a multiplicity of infection of 5 x 10 5Cells were seeded at 1 cell / well in 96-well plates. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and the gradient-diluted Anti-FZD7-5B6-his was added for incubation at 4°C for 1 h. After centrifugation to remove the supernatant, the cells were washed with 200 μl PBS for 3 times, and 100 μl / well of Anti-his tag 3D5 antibody (mouse IgGl) diluted with 1% BSA was added for incubation at 4°C for 1 h. After centrifugation to remove the supernatant, the cells were washed with 200 μl PBS for 3 times, and 100 μl Goat Anti-Mouse IgG H&L (abcam) preadsorbed was added for incubation at 4°C for 1 h. After centrifugation to remove the supernatant, the cells were washed with 200 μl PBS for 3 times, and finally resuspended with 150 μl PBS. The binding rate and mean fluorescence intensity of the antibody were detected by flow cytometry (Agilent 2060R). The binding EC50 of Anti-FZD7-5B6-His to CHOK1Q-FZD7 overexpressing cells was 41.91 nM. 650)preadsorbed(abcam), 4°C for 1 h. After centrifugation to remove the supernatant, the cells were washed with 200 μl PBS for 3 times, and finally resuspended with 150 μl PBS. The binding rate and mean fluorescence intensity of the antibody were detected by flow cytometry (Agilent 2060R). The binding EC50 of Anti-FZD7-5B6-His to CHOK1Q-FZD7 overexpressing cells was 41.91 nM.

[0143] Example 3 Expression and purification of Anti-FZD7-Fc fusion protein

[0144] In order to facilitate the characterization of the affinity, specificity and other aspects of Anti-FZD7 VHH, a detection tag (such as Fc, His, Twin-Strep-tag, Myc, GST, Flag or HA, etc.) can be combined with Anti-FZD7 VHH. In the present application, Fc is selected as the detection tag. FZD7 VHH is fused with Fc to construct a dimer similar to traditional antibodies. Therefore, the selection of which tag protein and which Fc sequence should not be considered as a limitation of the present application. In the present embodiment, Anti-FZD7-5B6 is connected with human IgG4 Fc region (SEQ ID NO. 25) to construct Anti-FZD7-5B6-Fc fusion protein, wherein the Fc region includes hinge. The full-length sequences of Anti-FZD7-5B6-Fc fusion protein, positive control antibody OMP18R5 analog (i.e. Vantictumab analog, derived from WHO Recommended INN list R71), positive control antibody SHH002-hul analog (derived from patent CN111138539B) and positive antibody ch-FZD7 1791 analog (derived from patent US10766962B2) are shown in Table 3, wherein the Fc domain of the heavy chain is indicated by underlining.

[0145] Table 3

[0146] The above sequence was synthesized and inserted into a pcDNA3.1 expression vector, and the expression vector and transfection reagent PEI (Yixing Biotech (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3, and the cells were cultured in a 37°C, 5% CO2 incubator for 7 days. The expressed protein or antibody was recovered by centrifugation, and then purified by a Protein A affinity chromatography column (Bogu Biotech Co., Ltd.). After purity detection and quantitative analysis, the target antibody was obtained.

[0147] Example 4 Binding ability of Anti-FZD7-Fc fusion protein to human FZD7 (CRD) recombinant protein

[0148] FZD7(CRD)-TS-His was plated at 100 ng / well at 4°C overnight, and blocked with 3% BSA at 37°C for 2 hours. Anti-FZD7-5B6-Fc fusion protein was gradient-diluted with 1% BSA, and incubated at 37°C for 1 hour. After 5 washes with PBST, 100 μl of 0.1 μg / mL Goat Anti-Human IgG, Monkey ads-HRP (SouthernBiotech) was added to each well, and incubated at 37°C for 1 hour. After 5 washes with PBST, TMB substrate was added, and incubated at 37°C. After 5 minutes of color development, 1M sulfuric acid was added to terminate the reaction, and OD450nm was read, and the binding EC 50 value of the Anti-FZD7 antibody to human FZD7(CRD) recombinant protein was calculated. The experimental results are shown in FIG. 1 and Table 4.

[0149] Table 4

[0150] Example 5 Binding ability of Anti-FZD7-Fc fusion protein to human FZD7 overexpression cells, rhesus monkey FZD7 overexpression cells, and mouse FZD7 overexpression cells

[0151] An expression plasmid with the gene sequence of human FZD7 (SEQ ID NO: 10) linked to the gene sequence of EGFP fluorescent protein was constructed. The expression vector contains glutamine synthetase (GS), which can be screened with glutamine synthetase inhibitor (MSX) drugs. The gene sequences of rhesus monkey FZD7 (SEQ ID NO: 11) and mouse FZD7 (SEQ ID NO: 12) were synthesized and linked to the gene sequence of EGFP fluorescent protein through an internal ribosome entry site (IRES) to construct the corresponding overexpression vectors.

[0152] The expression plasmids containing the human FZD7 gene sequence were electroporated in CHOK1Q cells, and stable transfected monoclonal cell lines overexpressing FZD7 were obtained by MSX pressure selection and indefinite dilution. Cells transiently overexpressing FZD7 in rhesus monkeys or mice were obtained by transiently transfecting Expi293F cells with the expression plasmids containing the rhesus monkey or mouse FZD7 gene sequence. 5 × 10⁶ cells were seeded per well in 96-well plates. 5 Overexpressing cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and serially diluted test antibody was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μl PBS, and 100 μl of Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody (DyLight™ 650, Invitrogen, diluted 1:300 with 1% BSA) was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μl PBS. The cells were resuspended in 100 μl PBS, and antibody binding rate and mean fluorescence intensity were detected using a flow cytometer (Agilent 2060R). The affinity of the Anti-FZD7-Fc fusion protein for human FZD7 overexpressing cells CHOK1Q-FZD7 is shown in Table 5. The binding of the Anti-FZD7-Fc fusion protein to rhesus monkey FZD7 overexpressing cells Expi293F-FZD7 is shown in Figure 2A (rhesus monkey FZD7). The binding of the Anti-FZD7-Fc fusion protein to mouse FZD7 overexpressing cells Expi293F-FZD7 is shown in Figure 2B (mouse FZD7).

[0153] The Anti-FZD7-5B6-Fc antibody exhibits strong binding ability to FZD7 expressed on the cell surface of humans, rhesus monkeys, or mice, comparable to that of the OMP18R5 analog.

[0154] Table 5. Binding of Anti-FZD7-Fc fusion protein to CHOK1Q-FZD7 cells

[0155] Example 6: Binding ability of OMP18R5 analog and SHH002-hu1 analog to human FZD7 overexpressing cells

[0156] In a 96-well plate, inoculate 5 × 10⁶ cells per well. 5FZD7 overexpressing CHOK1Q cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, 25 μg / ml of the antibody to be tested was added, and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, washed with 200 μl of PBS for 3 times, and 100 μl of 1:300 diluted Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLightTM650 (Invitrogen) was added, and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, washed with 200 μl of PBS for 3 times. The cells were resuspended with 100 μl of PBS, and the binding of the antibody OMP18R5 analog or SHH002-hu1 analog was detected by flow cytometry (Agilent 2060R). The experimental results are shown in Figure 3, in which OMP18R5 analog or SHH002-hu1 analog is abbreviated as OMP18R5 or SHH002.

[0157] The positive control antibody OMP18R5 analog had a high binding level to CHOK1Q-FZD7 cells, while SHH002-hu1 analog did not bind to CHOK1Q-FZD7 cells and had no FZD7 binding activity (which is inconsistent with the results in patent CN111138539B).

[0158] Example 7 Binding activity of Anti-FZD7-5B6-Fc fusion protein to different FZD family members

[0159] The gene sequences of human FZD1 (SEQ ID NO. 13), human FZD2 (SEQ ID NO. 14), human FZD3 (SEQ ID NO. 15), human FZD4 (SEQ ID NO. 16), human FZD5 (SEQ ID NO. 17), human FZD6 (SEQ ID NO. 18), human FZD7 (SEQ ID NO. 10), human FZD8 (SEQ ID NO. 19), human FZD9 (SEQ ID NO. 20), and human FZD10 (SEQ ID NO. 21) were connected to the EGFP fluorescent protein gene sequence through an internal ribosome entry site (IRES) to construct corresponding overexpression vectors.

[0160] Transient overexpression of cells with different FZD family members was obtained by transient transfection of Expi293F cells with the above expression plasmids containing human FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10 gene sequences. 5 x 10 5The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. 10 μg / ml of the antibody Anti-FZD7-5B6-Fc or OMP18R5 analog to be tested was added, and the mixture was incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed with 200 μl of PBS for 3 times. 100 μl of 1:300 diluted Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight™ 650 (Invitrogen) was added, and the mixture was incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed with 200 μl of PBS for 3 times. The cells were resuspended with 100 μl of PBS, and the antibody binding was detected by flow cytometry (Agilent 2060R). The experimental results are shown in FIGS. 4A and 4B.

[0161] As can be seen from the flow cytometry results, the positive antibody OMP18R5 analog can bind to human FZD1, FZD2, FZD5, FZD7, and FZD8, which is consistent with the literature report. The Anti-FZD7-5B6-Fc antibody only binds to human FZD7, and has very strong specificity.

[0162] Example 8 Humanization of Anti-FZD7-5B6

[0163] To further reduce the potential immunogenicity risk of single-domain antibodies in the process of disease treatment, specific amino acid sequences in the camel antibody framework are replaced with amino acid sequences at corresponding positions of human antibodies as much as possible. The antibody humanization method in this application uses the general framework grafting method for VHH humanization established by Ce'cile Vincke et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. J Biol Chem. 2009; 284(5): 3273-3284), and refers to the chapter on humanization of camel single-domain antibodies in the book Single-Domain Antibodies written by Greg Hussack and Kevin A. Henry, uses immunogenicity analysis software (http: / / tools.iedb.org / deimmunization / ) and homology modeling software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ) to predict candidate sites that need to be replaced for Anti-FZD7-5B6-Fc, and humanize the amino acids of the candidate sites of Anti-FZD7-5B6. Only a few humanized sequences are listed in this embodiment as representatives, and there are more humanization possibilities, which can be obtained by a person skilled in the art according to the method of this embodiment or according to the conventional humanization method. The exemplary antibody sequences before and after humanization are shown in Table 6, and "V10" or "V11" represents different humanization versions, which are not case-sensitive. The affinity of the humanized 5B6 single-domain antibody (hu5B6) is verified by flow cytometry and ELISA according to the method of Example 2, and the results show that the affinity of 5B6 before and after humanization does not change.

[0164] The Fc fusion protein of the humanized antibody (here, Fc is IgG1 Fc) is constructed, and the sequence is also shown in Table 6.

[0165] Table 6 Humanized sequence

[0166] The above humanized gene sequences are synthesized, Expi293F cells are transiently transfected for expression, and the expressed antibody supernatant is recovered after centrifugation, and then purified by Protein A affinity chromatography column (Borglunn Biotechnology Co., Ltd.). After purity detection and quantitative analysis, the target humanized antibody is obtained.

[0167] Example 9 Performance of humanized Anti-FZD7-5B6-Fc fusion protein

[0168] 9.1 Binding ability of humanized Anti-FZD7-5B6-Fc fusion protein to human FZD7 overexpression cells, rhesus monkey FZD7 overexpression cells, and mouse FZD7 overexpression cells

[0169] In each well of a 96-well plate, 5 x 10 5 Overexpression cells (overexpressing human FZD7, human FZD5, human FZD1, human FZD2, monkey FZD7, and mouse FZD7, respectively) were centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. Each well was added with 100 μl of 3% BSA for blocking for 0.5 h, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. Gradient-diluted test antibody was added, and incubated at 4°C for 1 h. The supernatant was removed by centrifugation, and the cells were washed with 200 μl of PBS for 3 times. 100 μl of 1:300 diluted Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight™ 650 (Invitrogen) was added, and incubated at 4°C for 1 h. The supernatant was removed by centrifugation, and the cells were washed with 200 μl of PBS for 3 times. The cells were resuspended with 100 μl of PBS, and the antibody binding rate and mean fluorescence intensity were detected by flow cytometry (Agilent 2060R). The experimental results are shown in FIG. 5A (human FZD7), FIG. 5B (rhesus monkey FZD7), FIG. 5C (mouse FZD7), FIG. 6A (human FZD1), FIG. 6B (human FZD2), and FIG. 6C (human FZD5).

[0170] Compared with the humanized Anti-FZD7-5B6-Fc fusion protein before humanization, the humanized Anti-FZD7-5B6-Fc fusion protein hu-5B6-v10 and hu-5B6-v11 also have high binding ability to human FZD7, and are slightly improved, while the affinity to monkey FZD7 and mouse FZD7 is retained. In addition, the cross-binding ability of hu-5B6-v10 and hu-5B6-v11 to human FZD1, human FZD2, and human FZD5 is lower than that before humanization, and the specificity to FZD7 is stronger.

[0171] 9.2 ADCC killing effect of Anti-FZD7-hu5B6-Fc on cells positive for FZD7 expression

[0172] The mechanism of ADCC is that the antibody binds to the target cell membrane antigen through its variable region, and the Fc region binds to the effector cell (mainly NK cell), forming a "target cell-antibody-effector cell" trimer crosslinking, thereby inducing the killing of the effector cell to the target cell. In this process, the affinity of the antibody to the membrane antigen, the binding epitope, and the distance from the cell membrane will all affect the ADCC activity, so the variable region of the antibody plays a key determining role. The ADCC activity of antibodies targeting the same antigen will also be different.

[0173] To detect the ADCC activity of Anti-FZD7-5B6-Fc, a cell line Jurkat-NFAT-CD16a containing CD16a (FcyRIIIA) and NFAT-luciferase reporter gene was constructed as effector cells, and CHOK1Q-FZD7 as target cells. The target cells and effector cells were mixed at a ratio of 3:5 and added to a 96-well plate. After incubation with the antibody to be tested for 18 hours, the detection solution was added, and the luciferase level was detected. The detection results are shown in Figure 7A. Anti-FZD7-5B6-Fc (SEQ ID NO. 231) and Anti-FZD7-hu5B6v10-Fc (SEQ ID NO. 232) both have ADCC activity, and the levels are similar. Humanization does not affect the ADCC activity of Anti-FZD7-5B6-Fc. This indicates that Anti-FZD7-hu5B6v10-Fc can kill FZD7-positive (tumor) cells through ADCC effect in vivo.

[0174] 9.3 Anti-FZD7-hu5B6-Fc ADCC killing effect on NCIH1975 cells, MDAMB231 cells or HPAF-II cells Lentivirus plasmid lenti-EF1a-H_FZD7-PGK-Puro (Genechem) was transfected into NCIH1975 cells, MDAMB231 cells or HPAF-II cells, respectively, and Puromycin (InvivoGen) was used for pressure screening to obtain pool cell strains NCIH1975-FZD7, MDAMB231-FZD7 or HPAF-II-FZD7, respectively. In a 96-well plate, 1.5x10 5 cells / ml, 100ul per well, and incubated in a 96-well plate overnight; pGL4.30 (Ubiobios) and pcDNA3.1-CD16A (158V) (Anhui General Biosystems) were electroporated on Jurkat cells, and Hygro (InvivoGen) and G418 (InvivoGen) were used for pressure screening and monoclonal cell strain Jurkat-NFAT-Luc2p-CD16A was obtained by monoclonal screening. The next day, the old culture medium was discarded, and the effector cells were 5x10 5cells / ml, 50 μl per well, and plated in cell well plates; the sample to be tested was diluted in 5 μg / ml (2 times the concentration prepared), 5-fold gradient dilution, 50 μl was added to the 96-well plate, and incubated for 18 h, then detected by enzyme-labeled instrument (MD SpectraMax i3). The experimental results are shown in Figures 7B-7D. When NCI-H1975, MDAMB231 or HPAF-II was used as the target cell, the ADCC activity of Anti-FZD7-5B6-Fc (SEQ ID NO. 231) or Anti-FZD7-hu5B6v10-Fc (SEQ ID NO. 232) was better than that of ChFZD7-1791 analog, and OMP18R5 analog had no ADCC activity.

[0175] As can be seen, humanization does not affect the affinity, specificity and ADCC activity of 5B6. Since the CDR region sequences of 5B6, hu5B6v10 or hu5B6v11 are the same, next, hu5B6v10 is arbitrarily selected as the parent antibody (WT), and further screening of mutants specifically binding to FZD7 is performed.

[0176] Example 10 CDR1 mutants derived from hu5B6v10

[0177] The CDR sequence of hu5B6v10, i.e., the parent antibody (WT) CDR1: G-S(X1)-I(X2)-L(X3)-S-L-Y-T, is analyzed. The S27I28L29 sites in CDR1 are mutated and screened based on hu5B6v10, and X1, X2, X3 are used to represent.

[0178] The S27I28L29 sites corresponding to the sequence of hu5B6V10 are randomly introduced into the amino acid saturation mutation by designing a "NNNNNNNNN" primer. The PCR product containing the mutant sequence is recombined and connected to the pComb3xss (Addgene) phagemid. The helper phage M13KO7 is used for library rescue. The phage library contains >10 7The insert frequency was about 100%, and the S27I, L28L, and R29L sites were randomly mutated. The mutation frequency of the three sites was similar. The FZD7 (CRD) fusion protein with LlamaFc or his tag and the cells overexpressing FZD7 antigen were used for phage display antibody positive screening, and the specific test steps were the same as those in Example 2. The clones that bound to the FZD7 antigen protein and had high signal values in enzyme-linked immunosorbent assay (ELISA) screening were selected for sequencing. The sequences with high OD450 signal values in the sequencing results and high enrichment (Table 7) were selected as candidate sequences. There were many other mutant sequences in the screening process, and the signal values were also higher than those of hu5B6v10 (SIL), which are not listed one by one.

[0179] Table 7

[0180] Example 11 CDR1 mutant derived from hu5B6v10

[0181] One of the mutants obtained by screening in Example 10 was randomly selected, for example, the mutant with X1X2X3 being FTF, and was named hu5B6V17. The binding affinity of the mutant to the cell surface FZD7 antigen and the binding activity to different FZD family proteins FZD1 / 2 / 5 / 8 were detected by flow cytometry fluorescence technology (FACS), and the detection method was referred to Example 7.

[0182] As can be seen from FIGS. 8A-8B, hu5B6V17 still has a relatively high affinity to the cell surface FZD7, and the affinity is slightly higher than that of hu5B6V10.

[0183] In addition, hu5B6V17 also does not cross-bind FZD1 / 2 / 5 / 8, and still maintains very good FZD7 specificity, as shown in FIGS. 9A-9D.

[0184] Example 12 CDR3 mutant derived from hu5B6V17

[0185] 5B6, hu5B6V10, hu5B6V17 have the same CDR3. To investigate whether mutation of certain sites on CDR3 will affect or further improve the affinity, based on the CDR3 of the parent antibody (WT): T-A-K-M-F-T-A-N (X4)-G (X5)-N-D-Y, the N103G104 site in CDR3 was mutated and screened, represented by X4, X5, the attempted mutants are shown in Table 8. FACS detected the affinity of hu5B6V17-Fc and each mutant to FZD7 on the surface of CHOK1Q-FZD7 stable transfectants, and the specific detection method and steps are the same as in Example 5, and the results are shown in Figure 10. The binding activity of each mutant to CHOK1Q-FZD7 is comparable.

[0186] Table 8

[0187] Example 13 Performance of CDR3 mutants derived from hu5B6v17

[0188] To further investigate whether mutation of the above-mentioned X4, X5 sites in CDR3 will affect the specificity of 5B6 antibody, one of the CDR3 mutants in Example 12, hu5B6V17-MuQG (hereinafter abbreviated as hu5B6V23), was selected to detect the cross-specificity of hu5B6v23 and hu5B6v10 to FZD1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10, and the detection method was the same as in Example 7.

[0189] As can be seen from Figures 11A-11B, hu5B6V10 and hu5B6V23 maintain good specificity to FZD7 and do not cross-bind to other FZD family members.

[0190] Referring to the method of Example 4, the binding EC C values of hu5B6V23-F C , hu5B6V10-F 50 , and the positive control chFZD7-1791 to human FZD7 (CRD) recombinant protein were detected, and the experimental results are shown in Figure 12. hu5B6V23-F C has comparable binding activity to hu5B6V10-F C , and chFZD7-1791 does not bind to human FZD7 (CRD) recombinant protein.

[0191] Example 14 FR-derived antibodies of 5B6

[0192] Without changing the CDR of hu5B6v17 or hu5B6v23, some derived antibodies were obtained by changing certain sites of FR, as shown in Table 9. ELISA detected the binding EC 50The detection method was the same as in Example 4. The experimental results are shown in Figures 13-14. Compared with hu5B6v23-Fc, hu5B6v21-Fc and hu5B6v10-Fc maintained high binding activity, and the binding activity was even improved compared with hu5B6v10-Fc.

[0193] Table 9

[0194] Example 15: Anti-FZD7-hu5B6 endocytosis capability

[0195] Inducing FZD7 endocytosis and degradation is beneficial for tumor growth inhibition. In this example, the endocytosis capacity of hu5B6v10-Fc, hu5B6v23-Fc, hu5B6v24-Fc, hu5B6v25-Fc and chFZD7-1791 analog were compared.

[0196] Take 100ug each of isotype control IgG, Anti-FZD7-hu5B6v10-Fc, Anti-FZD7-hu5B6v23-Fc, Anti-FZD7-hu5B6v24-Fc, Anti-FZD7-hu5B6v25-Fc or chFZD7-1791 from the test sample into a dialysis bag (Thermo Scientific, S9552). Replace the buffer with 10mM NaHCO3 at 4℃, changing the buffer 4 times during the process. After replacement, add 1.2ul of pHAb amine reactive dye (Thermo Scientific, cat:G9845, lot:#600039) and incubate at room temperature for 1 hour. Add 1ul of 1M NH4Cl to terminate the reaction. Dialyze in 1×PBS buffer at 4℃ for 24 hours, changing the 1×PBS buffer 4 times every 2-4 hours. CHOK1Q-FZD7 cells in logarithmic growth phase were collected, centrifuged at 800 rpm for 5 min, resuspended in CD02, counted, and then divided into groups of 1 × 10⁻⁶ cells. 5 Cells were seeded into 24-well plates. pHAb-labeled antibody was added to the seeded plates to a final concentration of 200 nM, and incubated overnight (24 h) at 37°C in a CO2 incubator. The next day, cells were collected, centrifuged at 800 rpm for 5 min, and the drug-containing medium was discarded. Cells were washed twice with 1×PBS, and the red fluorescence was observed and photographed under a microscope. The PE-Texas channel was then analyzed by flow cytometry. The antibody endocytosis level was calculated based on the fluorescence signal, using the formula Mean X / DAR, where Mean X represents the fluorescence intensity, and DAR represents the ratio of dye molecules to antibody molecules in the fluorescently labeled antibody.

[0197] Results are shown in Figure 15. hu5B6v10-Fc, hu5B6v23-Fc, hu5B6v24-Fc, hu5B6v25-Fc have stronger endocytosis level on CHOK1 Q-FZD7 cells, all of which are better than chFZD7-1791 analog.

[0198] Example 16 Epitope mapping of Anti-FZD7-hu5B6 on FZD7

[0199] Discovery Studio analysis software was used to predict the antibody-antigen interaction. The predicted FZD7 CRD binding regions Q33 to C49, I66 to L70, and C110 to A116 were subjected to alanine scanning mutation, in which the original alanine was mutated to glycine. The binding ability of Anti-FZD7-hu5B6v25-Fc to the mutant FZD7 (compared with wild-type FZD7) was used to study the epitope mapping. A significant decrease in binding refers to a decrease in relative antigen protein binding of more than 25% compared to the control; preferably, a decrease of more than 50%; more preferably, a decrease of more than 75%. The above-mentioned alanine scanning mutant FZD7 and wild-type FZD7 gene sequences were synthesized, with an N-terminal FLAG tag to determine the amount of antigen expression, and were connected to the EGFP fluorescent protein gene sequence through an internal ribosome entry site (IRES) to construct the corresponding overexpression vector.

[0200] Expi293F cells were transiently transfected with the above-mentioned expression plasmids of human FZD7 wild-type and alanine scanning mutant full-length gene sequences with an N-terminal FLAG tag to obtain transiently overexpressed FLAG-FZD7 and its mutants. 5 x 105overexpressed cells were inoculated in each well of a 96-well plate. Centrifugation at 1000 rpm for 5 minutes, removal of the supernatant, and addition of 100 μl of 1:300 diluted Alexa 5 ​647Anti-DYKDDDDK Tag Antibody (BioLegend), or 25 nM of Anti-FZD7-hu5B6v25-Fc antibody was added and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed with 200 μΐ of PBS for 3 times. The antibody binding was detected by adding 100 μΐ of 1:300 diluted Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight™ 650 (Invitrogen) to the cells and incubating at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed with 200 μΐ of PBS for 3 times. The cells were resuspended with 100 μΐ of PBS, and the antigen expression and antibody binding were detected by flow cytometry (Agilent 2060R). The results are shown in FIGS. 16A-16D.

[0201] The ratio of the signal of the FLAG tag binding signal of the EGFP positive cell group to the signal of the blank control group was used as the expression amount of the corresponding antigen FZD7 wild type or mutant. Similarly, the ratio of the average fluorescence signal value (MFI) of the Anti-FZD7-hu5B6v25-Fc antibody binding EGFP positive cell group to the signal of the blank control group was used as the antibody binding ability. The ratio of the relative binding ability of the antibody to the relative expression amount of the antigen was used as the relative binding ability of the antibody after removing the influence of the expression amount of the antigen. The results are shown in Table 10.

[0202] The ability of Anti-FZD7-hu5B6v25-Fc to bind FZD7 mutants was evaluated by flow cytometry. According to the analysis results, when the relative expression amount of the antigen (compared with wild type FZD7) did not change significantly (more than 80%), the relative binding ability of the antibody decreased by more than 25% at sites H36A and V43A, and decreased by more than 75% at sites F48A, I66A and D114A. When the relative expression amount of the antigen (compared with wild type FZD7) decreased significantly (less than 40%), the relative binding ability of the antibody decreased by more than 50% at sites P68A, N69A and C110A, and decreased by more than 75% at sites C49A and A116G. It is believed that the epitope of Anti-FZD7-hu5B6v25-Fc on FZD7 includes H36, V43, F48, C49, I66, P68, N69, C110, D114 and A116, and more preferably F48, C49, I66, D114 and A116.

[0203] Table 10

[0204] Example 17 Anti-FZD7-hu5B6-Fc Inhibitory Activity on Tumor Growth in CHOK1-FZD7 Tumor-Bearing Mice Overexpressing FZD7

[0205] CHOK1-FZD7 cells overexpressing FZD7 protein were inoculated subcutaneously in the right anterior flank of CB17-SCID female mice. When the tumors grew to 120 mm3, the mice were randomly divided into 3 groups with 7 mice in each group, and were intraperitoneally injected with normal saline and different doses of Anti-FZD7-hu5B6V10-Fc (SEQ ID NO. 232) according to Table 11. The tumor volume and body weight were measured 3 times per week, and the relationship between the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, and the tumors were stripped, weighed, photographed, and serum and tumors were collected. The tumor growth inhibition rate (TGI) was calculated 3 TV (%) and statistically analyzed.

[0206] Table 11 Grouping and administration scheme design Note: PG is the abbreviation of post grouping, indicating after grouping; tiw indicates 3 times per week; the first three times of Anti-FZD7-hu5B6v10-Fc administration in the G2 group were at a dose of 5 mg / kg, and the dose was adjusted to 30 mg / kg from the fourth time.

[0207] During the treatment period, the mice in each group ate and drank normally, and their body weight was stable, and no animal deaths occurred.

[0208] At the end of the experiment (PG-D18), the tumor growth inhibition rates of the administration 1 group and the administration 2 group were 57% (p<0.01) and 33%, respectively, as shown in Figure 17, and the body weight changes of the mice in each group are shown in Figure 18. The changes in body weight and tumor volume are summarized in Table 12.

[0209] In summary, the test substance Anti-FZD7-hu5B6V10-Fc had a significant anti-tumor effect on the CHOK1-FZD7 tumor-bearing mouse model overexpressing FZD7 (p<0.01), and the tumor inhibition effect was enhanced with increasing dose.

[0210] Table 12 Anti-tumor effect of the test substance on CHOK1-FZD7 tumor-bearing mice (tumor volume and TGI TV ) Note: “**” indicates P<0.01 compared with the Vehicle group.

[0211] ​The above embodiments are only illustrative of the principles of the present application and its efficacy, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. An anti-FZD7 single-domain antibody, characterized in that, The anti-FZD7 single-domain antibody comprises CDR1-CDR3 as shown below: CDR1: amino acid sequence as shown in SEQ ID NO. 1: GX1X2X3SLYT, wherein X1 is Y, W, T, N, F, A, H, I, L, M, R, V or S, X2 is P, Y, T, S, N, A, V, G, L, F, M, R, D, Q or I, and X3 is L, F, Y, M, I or W; CDR2: MTRGGIT; CDR3: TAKMFTAX4X5NDY, wherein X4 is N, Q, A, D, E, I, K, L, M, R, S, T or V, and X5 is G, A or S.

2. The anti-FZD7 single-domain antibody of claim 1, wherein the combination of X1, X2 and X3 is SIL, YPL, WPL, WTL, WYL, WSL, WNL, WAL, WPF, TVW, NAF, FPF, TPF, AAF, AGF, APL, AVW, AYF, FAW, FAY, FIL, FLL, FLW, FPW, FSW, FVW, FVY, HIF, HLL, HPF, HPL, HVF, IFL, LYL, MPF, NLY, NMF, NSF, RAL, SAW, SIF, SIW, SLW, SPF, SVF, SVL, SYW, TAL, TAW, TFF, TGL, TLL, TPL, TRL, TTL, TVF, TYY, VIL, VYL, WAM, WDL, WGL, WIL, WLL, WLW, WPF, WPI, WPY, WQL, WRL, WSL, WVL, YGL, YIL, YLF, YLL, YML, YSL, YTL, YVL, YVW, YYL or FTF.

3. The anti-FZD7 single-domain antibody of claim 1, wherein the combination of X4 and X5 is NG, QG, AG, DG, EG, IG, KG, LG, MG, RG, SG, TG, VG, NA or NS.

4. The anti-FZD7 single-domain antibody of claim 1, wherein, The combination of X4 and X5 is NG, QG, SG, DG or AG, and X1, X2 and X3 are respectively: X1 is S, X2 is I, and X3 is L; X1 is F, X2 is T, and X3 is F; X1 is Y, X2 is P, and X3 is L; X1 is W, X2 is P, and X3 is L; X1 is W, X2 is T, and X3 is L; X1 is W, X2 is Y, and X3 is L; X1 is W, X2 is S, and X3 is L; X1 is W, X2 is N, and X3 is L; X1 is W, X2 is A, and X3 is L; X1 is W, X2 is P, and X3 is F; X1 is T, X2 is V, and X3 is W; X1 is N, X2 is A, and X3 is F; X1 is F, X2 is P, and X3 is F; or X1 is T, X2 is P, and X3 is F. 5.The FZD7 single-domain antibody of claim 1, wherein the combination of X 1, X 2 and X 3 is FTF, X 4 and X 5 are respectively: X 4 is N, X 5 is G; X 4 is Q, X 5 is G; X 4 is A, X 5 is G; X 4 is D, X 5 is G; X 4 is E, X 5 is G; X 4 is I, X 5 is G; X 4 is K, X 5 is G; X 4 is L, X 5 is G; X 4 is M, X 5 is G; X 4 is R, X 5 is G; X 4 is S, X 5 is G; X 4 is T, X 5 is G; X 4 is V, X 5 is G; X 4 is N, X 5 is A; or X 4 is N, X 5 is S. 6.The FZD7 single-domain antibody of claim 1, wherein the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with the amino acid sequence as shown in any one of SEQ ID NO.32-114, CDR2 with the amino acid sequence as shown in SEQ ID NO.2, and CDR3 with the amino acid sequence as shown in any one of SEQ ID NO.115-129. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with the amino acid sequence as shown in any one of SEQ ID NO.32-114, CDR2 with the amino acid sequence as shown in SEQ ID NO.2, and CDR3 with the amino acid sequence as shown in SEQ ID NO.

115. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with the amino acid sequence as shown in any one of SEQ ID NO.32-114, CDR2 with the amino acid sequence as shown in SEQ ID NO.2, and CDR3 with the amino acid sequence as shown in SEQ ID NO.

116. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with the amino acid sequence as shown in any one of SEQ ID NO.32-114, CDR2 with the amino acid sequence as shown in SEQ ID NO.2, and CDR3 with the amino acid sequence as shown in SEQ ID NO.

118. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with the amino acid sequence as shown in any one of SEQ ID NO.32-114, CDR2 with the amino acid sequence as shown in SEQ ID NO.2, and CDR3 with the amino acid sequence as shown in SEQ ID NO.

119. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with the amino acid sequence as shown in any one of SEQ ID NO.32-114, CDR2 with the amino acid sequence as shown in SEQ ID NO.2, and CDR3 with the amino acid sequence as shown in SEQ ID NO.

126. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as set forth in SEQ ID NO. 32, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 115 to 129. Preferably, the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: CDR1 with an amino acid sequence as set forth in SEQ ID NO. 33, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 115 to 129.

7. The FZD7 single-domain antibody of claim 1, wherein the anti-FZD7 single-domain antibody comprises CDR1 to CDR3 selected from the group consisting of: (1) CDR1 with an amino acid sequence as set forth in SEQ ID NO. 32, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 115; (2) CDR1 with an amino acid sequence as set forth in SEQ ID NO. 33, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 115; (3) CDR1 with an amino acid sequence as set forth in SEQ ID NO. 33, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 118; (4) CDR1 with an amino acid sequence as set forth in SEQ ID NO. 33, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 116; (5) CDR1 with an amino acid sequence as set forth in SEQ ID NO. 33, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO. 119; or (6) CDR1 with an amino acid sequence as set forth in SEQ ID NO. 33, CDR2 with an amino acid sequence as set forth in SEQ ID NO. 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO.

126.

8. The anti-FZD7 single-domain antibody of claim 1, wherein the anti-FZD7 single-domain antibody is a llama antibody or a humanized antibody.

9. The FZD7 single-domain antibody of claim 8, wherein the anti-FZD7 single-domain antibody comprises FR1 to FR4 selected from the group consisting of: (1) FR1 with an amino acid sequence as set forth in SEQ ID NO. 130, FR2 with an amino acid sequence as set forth in SEQ ID NO. 131, FR3 with an amino acid sequence as set forth in SEQ ID NO. 132, and FR4 with an amino acid sequence as set forth in SEQ ID NO. 133; (2) FR1 as shown in SEQ ID NO. 134, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135, and FR4 as shown in SEQ ID NO. 136; (3) FR1 as shown in SEQ ID NO. 134, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 137, and FR4 as shown in SEQ ID NO. 136; (4) FR1 as shown in SEQ ID NO. 139, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135, and FR4 as shown in SEQ ID NO. 136; (5) FR1 as shown in SEQ ID NO. 138, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135, and FR4 as shown in SEQ ID NO. 136; (6) FR1 as shown in SEQ ID NO. 140, FR2 as shown in SEQ ID NO. 131, FR3 as shown in SEQ ID NO. 135, and FR4 as shown in SEQ ID NO.

136.

10. The anti-FZD7 single-domain antibody of claim 9, comprising an amino acid sequence as shown in any one of SEQ ID NO. 141-185, or an amino acid sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% or more sequence identity to any one of SEQ ID NO. 141-185.

11. An anti-FZD7 single-domain antibody, which binds to positions 33-49, 66-70, and or 110-116 of the amino acid sequence as shown in SEQ ID NO.

10.

12. The anti-FZD7 single-domain antibody of claim 11, which binds to at least one of the following residues of the amino acid sequence as shown in SEQ ID NO. 10: H36, V43, F48, C49, I66, P68, N69, C110, D114, or A116; preferably, at least one of F48, C49, I66, D114, or A116.

13. The anti-FZD7 single-domain antibody of any one of claims 11-12, comprising CDR1- CDR3 as shown below: CDR1: amino acid sequence as set forth in SEQ ID NO. 1: GX1X2X3SLYT, wherein X1 is Y, W, T, N, F, A, H, I, L, M, R, V or S, X2 is P, Y, T, S, N, A, V, G, L, F, M, R, D, Q or I, X3 is L, F, Y, M, I or W; CDR2: MTRGGIT; CDR3: TAKMFTAX4X5NDY, wherein X4 is N, Q, A, D, E, I, K, L, M, R, S, T or V, X5 is G, A or S.

14. The anti-FZD7 single-domain antibody of claim 13, comprising CDR1-CDR3 selected from the group consisting of: CDR1: amino acid sequence as set forth in any one of SEQ ID NO. 32-114, CDR2: amino acid sequence as set forth in SEQ ID NO. 2, CDR3: amino acid sequence as set forth in any one of SEQ ID NO. 115-129.

15. The anti-FZD7 single-domain antibody of claim 13, comprising an amino acid sequence as set forth in any one of SEQ ID NO. 141-185 or an amino acid sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NO. 141-185.

16. A fusion protein comprising the anti-FZD7 single-domain antibody according to any one of claims 1-15, characterized in that, The fusion protein further comprises an Fc region of an immunoglobulin; preferably selected from a human immunoglobulin Fc region; preferably selected from a complete Fc region, an Fc region fragment or an Fc region mutant.

17. The fusion protein of claim 16, wherein, The immunoglobulin Fc region is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM; preferably, the IgG is selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes.

18. The fusion protein of claim 16 or 17, wherein, The immunoglobulin Fc region is selected from an amino acid sequence as set forth in any one of SEQ ID NO. 22-25, SEQ ID NO.

233.

19. The fusion protein of any one of claims 16 to 18, wherein, The fusion protein has an amino acid sequence as set forth in any one of SEQ ID NO. 186-232 or an amino acid sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NO. 186-232.

20. An isolated polynucleotide encoding the single-domain antibody of any one of claims 1-15 or encoding the fusion protein of any one of claims 16-19.

21. An expression vector comprising the isolated polynucleotide of claim 20.

22. An expression system comprising the expression vector or the polynucleotide of claim 20 integrated into the genome of the expression system of claim 21.

23. A method for preparing the single-domain antibody of any one of claims 1-15 or the fusion protein of any one of claims 16-19, comprising the steps of culturing the expression system of claim 22 under conditions suitable for expression of the single-domain antibody or the fusion protein, thereby expressing the single-domain antibody or the fusion protein, and purifying and isolating the single-domain antibody or the fusion protein.

24. A pharmaceutical composition comprising the single-domain antibody of any one of claims 1-15 or the fusion protein of any one of claims 16-19.

25. The pharmaceutical composition of claim 24, wherein, Also included are pharmaceutically acceptable carriers.

26. Use of the single-domain antibody of any one of claims 1-15, the fusion protein of any one of claims 16-19, or the pharmaceutical composition of any one of claims 24-25 in the manufacture of a medicament for the diagnosis, treatment or prevention of a tumor positive for FZD7 expression.

27. The use of claim 26, wherein, The tumor is selected from the group consisting of liver cancer, urothelial cancer, cervical cancer, breast cancer, gastric cancer, pancreatic cancer, lung cancer, thyroid papillary cancer, ovarian cancer, endometrial cancer, prostate cancer, colorectal cancer, head and neck squamous cell carcinoma, and renal cancer.

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