Wnt signal path adjusting system and method for activating Wnt signal path

Abstract

The invention discloses a Wnt signal path adjusting system and a method for activating a Wnt signal path. The Wnt signal channel regulation system comprises a first regulator and a second regulator, each regulator comprises an I structural domain and an II structural domain, the first regulator comprises an I-1 structural domain and an II-1 structural domain which are respectively targeted to Wnt receptors Frizzled and LRP6, and the second regulator comprises an I-2 structural domain and an II-2 structural domain which are respectively targeted to Wnt receptors Frizzled and LRP6; the first regulator is different from the second regulator. The Wnt signal channel regulation system provided by the invention has the advantages of high activation capability, small toxic and side effects and selectivity in Wnt signal channel activation, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a Wnt signaling pathway regulating system and a method for activating the Wnt signaling pathway. Background Art

[0002] The Wnt signaling pathway regulates the pluripotent differentiation of stem cells, the development and regeneration of organs, and plays an important role in maintaining tissue homeostasis, repairing and regenerating organ damage, and has great potential for application in regenerative medicine. Wnt proteins recruit receptors Frizzled (Fzd) and co-receptors low-density lipoprotein receptor-related protein 5 or 6 (LRP5 / 6), and the three form a complex that activates the β-catenin-dependent canonical Wnt signaling pathway through a series of signal transductions. However, the palmitoleylation modification and substructure stability of Wnt proteins give them poor physical and chemical properties, making them difficult to purify on a large scale; the 19 highly conserved Wnt proteins in the human body have cross-reaction characteristics with 10 Fzd receptors in signal recognition, which limits the application of Wnt proteins in basic research and medicine. The field is in urgent need of alternatives to Wnt proteins to help research in this field.

[0003] In 2017, Garcia's research group first proposed the concept of Wnt functional substitutes, using natural proteins or single-chain antibody fragments with better stability and water solubility as components, targeting Fzd and LRP6, replacing the function of Wnt protein, and proving its ability to activate the Wnt signaling pathway at the cellular level, organoid level and mouse model. Each research group selected various split forms of antibodies as basic elements, such as the Fab fragment at the antigen-binding end, the Fc segment at the effector end of the antibody, the "heavy chain antibody" lacking the light chain, and nano antibodies, etc., to optimize the physical and chemical properties and effects of Wnt functional substitutes in terms of stability, molecular weight, permeability, etc. On the other hand, researchers have modified antibodies from the construction form, and bivalent, trivalent, and tetravalent multispecific antibodies have been developed to improve the ability to bind to antigens and signal activation. To date, Wnt functional substitutes have been continuously optimized and innovated in terms of components and construction forms to achieve more significant signal activation capabilities. Summary of the invention

[0004] In order to solve the defect that the activation ability of Wnt signaling pathway by Wnt functional substitutes in the prior art still needs to be improved, the present invention provides a Wnt signaling pathway regulation system and a method for activating the Wnt signaling pathway, wherein the Wnt signaling pathway regulation system has a significant Wnt signaling pathway activation ability.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] In the present invention, the term "Wnt signaling pathway regulation system" refers to a composition for regulating the Wnt signaling pathway, including a first regulator and a second regulator. The terms "first regulator" and "second regulator" refer to two regulatory elements. Specifically:

[0007] The first aspect of the present invention provides a Wnt signaling pathway regulation system, which includes a first regulator and a second regulator, both of which include an I domain and an II domain, wherein the first regulator includes an I-1 domain and a II-1 domain that target Wnt receptors Frizzled and LRP6, respectively, and the second regulator includes an I-2 domain and a II-2 domain that target Wnt receptors Frizzled and LRP6, respectively; the first regulator and the second regulator are different.

[0008] In some embodiments of the present invention, both the II-1 domain and the II-2 domain target the extracellular region of LRP6.

[0009] In some preferred embodiments of the present invention, the II-1 domain and the II-2 domain target different extracellular domains respectively, and the extracellular domains include E1, E2, E3 and E4.

[0010] In some more preferred embodiments of the present invention, the II-1 domain targets the extracellular region E1 and / or E2 of LRP6, and the II-2 domain targets the extracellular region of LRP6.

[0011] In some more preferred embodiments of the present invention, the II-1 domain targets the extracellular region of LRP6, and the II-2 domain targets the extracellular region E3 and / or E4 of LRP6.

[0012] In some specific embodiments of the present invention, the II-1 domain targets the extracellular region E1 and / or E2 of LRP6, and the II-2 domain targets the extracellular region E3 and / or E4 of LRP6.

[0013] In some embodiments of the present invention, both the I-1 domain and the I-2 domain target the extracellular region of the Wnt receptor Frizzled; the extracellular region includes a cysteine-rich domain.

[0014] In some preferred embodiments of the present invention, the extracellular region further includes a linker and a hinge region.

[0015] In the present invention, the cysteine-rich domain is a region that directly interacts with the I-1 domain and the I-2 domain, and is connected to the transmembrane region of Frizzled via a linker and / or a hinge region.

[0016] In some embodiments of the present invention, the regulator further comprises Fc.

[0017] In the present invention, "Fc" refers to a part of the constant region of the heavy chain, which is located in the crystallizable region at the C-terminus of the antibody, away from the antigen binding site. Different antibody subclasses (such as IgG1, IgG2, IgA, etc.) have different Fc region sequences. The Fc used in the present invention is the Fc portion of the antibody IgG1.

[0018] In some preferred embodiments of the present invention, the regulator comprises domain I, domain II and Fc, or domain II, domain I and Fc, which are sequentially connected from N-terminus to C-terminus.

[0019] In some more preferred embodiments of the present invention, the connection is carried out using a linker connection or direct connection, and the linker is, for example, (GGSGS)2 (as shown in SEQ ID NO:12) and MVRS (as shown in SEQ ID NO:13).

[0020] In some further preferred embodiments of the present invention, domain I and domain II are connected using a linker (GGSGS) 2, and / or, domain I or domain II is connected to Fc using a linker MVRS.

[0021] In some specific embodiments of the present invention, the nucleotide sequence encoding linker (GGSGS) 2 is shown as SEQ ID NO:21.

[0022] In some specific embodiments of the present invention, the nucleotide sequence encoding the linker MVRS is shown in SEQ ID NO:22.

[0023] In some embodiments of the present invention, the regulator further comprises a signal peptide and a tag protein, wherein the signal peptide is preferably a Kappa signal peptide, and the tag protein is preferably a His6 tag.

[0024] In the present invention, the nucleotide sequence encoding the Kappa signal peptide is shown in SEQ ID NO: 23; the nucleotide sequence encoding the His6 tag is shown in SEQ ID NO: 24.

[0025] In some preferred embodiments of the present invention, the regulator includes a Kappa signal peptide, a His6 tag, a I domain, a II domain and an Fc, or a Kappa signal peptide, a His6 tag, a II domain, a I domain and an Fc connected sequentially from the N-terminus to the C-terminus.

[0026] In some embodiments of the present invention, the I domain and the II domain are selected from antibodies and natural proteins.

[0027] In some embodiments of the present invention, the antibody is an IgG antibody, a Fab fragment, a scFv or a heavy chain antibody, and the heavy chain antibody is, for example, a nanobody; the antibody is a monovalent or multivalent antibody, and the multivalent antibody is, for example, a bivalent, trivalent or tetravalent antibody; and / or the natural protein is selected from Dickkopf-1 protein such as DKK1c, Sclerostin and MESD such as MESDpep.

[0028] In some embodiments of the present invention, the regulator includes a nanobody targeting Frizzled, a nanobody targeting LRP6; or a nanobody targeting Frizzled, a DKK1c targeting LRP6; or a nanobody targeting Frizzled, a SOST targeting LRP6; or a nanobody targeting Frizzled, a MESDpep targeting LRP6.

[0029] In some embodiments of the present invention, the regulator further comprises Fc.

[0030] In the present invention, the Fc sequence can be selected by those skilled in the art, for example, the Fc is an Fc with an amino acid sequence as shown in SEQ ID NO: 19.

[0031] In some specific embodiments of the present invention, the nucleotide sequence encoding the Fc is shown in SEQ ID NO:20.

[0032] In some embodiments of the present invention, the Frizzled targeting Nanobody comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and / or, the Nanobody can bind to one or more of Frizzled 1, 2, 4, 5, 7 and 8.

[0033] In some preferred embodiments of the present invention, the Nanobody can bind to Frizzled 1, 2, 4, 5, 7 and 8.

[0034] In some more preferred embodiments of the present invention, the amino acid sequence of the Nanobody is as shown in SEQ ID NO:4.

[0035] In some embodiments of the present invention, the Nanobody targeting LRP6 comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0036] In some preferred embodiments of the present invention, the amino acid sequence of the Nanobody is as shown in SEQ ID NO:8.

[0037] In some embodiments of the present invention, the amino acid sequence of DKK1c is shown as SEQ ID NO:9; the amino acid sequence of SOST is shown as SEQ ID NO:10; and / or the amino acid sequence of MESDpep is shown as SEQ ID NO:11.

[0038] In some specific embodiments of the present invention, the nucleotide sequence encoding the DKK1c is shown as SEQ ID NO:16; the nucleotide sequence encoding the SOST is shown as SEQ ID NO:17; and / or the nucleotide sequence of the MESDpep is shown as SEQ ID NO:18.

[0039] In this application, the term "antibody" generally refers to a protein comprising a portion that binds an antigen, and optionally a scaffold or framework portion that allows the portion that binds the antigen to adopt a conformation that promotes antibody binding to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.

[0040] The "heavy chain antibody" in this application generally refers to a new type of antibody molecule composed only of heavy chains. Although heavy chain antibodies lack light chains compared to ordinary antibodies, they still retain the ability to bind to antigens. Unlike conventional antibody molecules composed of four polypeptide chains, heavy chains and light chains, heavy chain antibodies are composed of two homologous heavy chain peptides. The heavy chain molecule only contains the variable region, CH2 region and CH3 region, with a relative molecular mass of 90kDa, which is much smaller than the conventional IgG antibody molecule (150kDa).

[0041] The term "nanoantibody" in the present invention, also known as "single-domain antibody", contains only one heavy chain variable region VHH, with a molecular weight of about 15 kDa and a diameter of about 10 nanometers. It retains all antigen binding ability and is the smallest fragment that retains complete antigen binding.

[0042] The amino acid sequences of the CDRs listed in the present invention are determined according to the IMGT definition rules. However, it is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass the complementary determining region defined by any of the above-mentioned known schemes described in the present invention. Various numbering systems corresponding to CDRs are well known to those skilled in the art, as shown in Table 1:

[0043] Table 1 Antibody CDR definition method

[0044]

[0045]

[0046] Note: In Table 1, Haa-Hbb refers to the amino acid sequence starting from the N-terminus of the antibody heavy chain and starting from the aa position to the bb position according to the corresponding coding rules. For example, H31-H35 in the second row and second column of Table 1 refers to the amino acid sequence determined from the 31st to the 35th residue starting from the N-terminus of the antibody heavy chain variable region according to the Kabat coding rules; and the same applies to the others.

[0047] A second aspect of the present invention provides a heavy chain antibody targeting Frizzled, wherein the heavy chain antibody comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0048] In some embodiments of the present invention, the amino acid sequence of the heavy chain antibody is shown in SEQ ID NO:4.

[0049] In some specific embodiments of the present invention, the nucleotide sequence encoding the heavy chain antibody is shown in SEQ ID NO:14.

[0050] In some embodiments of the present invention, the heavy chain antibody further comprises Fc.

[0051] In some specific embodiments of the present invention, the Fc is an Fc with an amino acid sequence as shown in SEQ ID NO:19.

[0052] A third aspect of the present invention provides a heavy chain antibody targeting LRP6, wherein the heavy chain antibody comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0053] In some embodiments of the present invention, the amino acid sequence of the heavy chain antibody is shown in SEQ ID NO:8.

[0054] In some specific embodiments of the present invention, the nucleotide sequence encoding the heavy chain antibody is shown in SEQ ID NO:15.

[0055] In some embodiments of the present invention, the heavy chain antibody further comprises Fc.

[0056] In some specific embodiments of the present invention, the Fc is an Fc with an amino acid sequence as shown in SEQ ID NO:19.

[0057] The fourth aspect of the present invention provides a polynucleotide encoding a heavy chain antibody targeting Frizzled or a heavy chain antibody targeting LRP6.

[0058] In some embodiments of the present invention, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO: 14 or SEQ ID NO: 15, respectively.

[0059] In some embodiments of the present invention, the polynucleotide further comprises a nucleotide sequence encoding the Fc as shown in SEQ ID NO:20.

[0060] The fifth aspect of the present invention provides a recombinant expression vector comprising the polynucleotide of the fourth aspect of the present invention.

[0061] The sixth aspect of the present invention provides a transformant, which comprises the recombinant expression vector of the fifth aspect of the present invention. Preferably, the starting host of the transformant is a eukaryotic cell or a prokaryotic cell. The eukaryotic cell is, for example, a CHO cell, and the prokaryotic cell is, for example, Escherichia coli.

[0062] The seventh aspect of the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the Wnt signaling pathway regulation system as described in the first aspect, and / or the heavy chain antibody as described in the second aspect.

[0063] In some preferred embodiments, the reagent or kit further comprises the polynucleotide as described in the fourth aspect of the present invention, the recombinant expression vector as described in the fifth aspect of the present invention and / or the transformant as described in the sixth aspect of the present invention.

[0064] The eighth aspect of the present invention provides a Wnt signaling pathway regulation system as described in the first aspect, and the use of the heavy chain antibody as described in the second aspect and / or the third aspect in activating the Wnt signaling pathway or preparing a reagent or kit for activating the Wnt signaling pathway.

[0065] The ninth aspect of the present invention provides a method for activating the Wnt signaling pathway, using the Wnt signaling pathway regulatory system as described in the first aspect to contact cells.

[0066] In some embodiments of the present invention, the cell is a mammalian cell.

[0067] In some embodiments of the invention, the method is for non-diagnostic therapeutic purposes.

[0068] The tenth aspect of the present invention provides a use of the Wnt signaling pathway regulation system as described in the first aspect of the present invention, the heavy chain antibody as described in the second aspect, and the reagent or kit as described in the seventh aspect in the preparation of a regenerative medicine product.

[0069] In some embodiments of the present invention, the regenerative medicine is bone regeneration, lung regeneration, hair regeneration or tooth regeneration.

[0070] The eleventh aspect of the present invention provides a method for detecting whether Frizzled and / or LRP6 is present in a sample using the heavy chain antibody described in the second aspect and / or the third aspect of the present invention, which comprises contacting the heavy chain antibody with the sample and determining whether Frizzled and / or LRP6 is present in the sample by conventional techniques in the art.

[0071] The twelfth aspect of the present invention provides a method for diagnosing and / or treating Frizzled and / or LRP6 related diseases using the heavy chain antibody described in the second aspect and / or the third aspect of the present invention, wherein the heavy chain antibody is administered to a patient in need. The Frizzled and / or LRP6 related diseases include diseases caused by mutations and abnormal expression of Frizzled and / or LRP6, such as coronary artery disease (autosomal dominant 2, ADCAD2), tooth agenesis, selective, 7 (STHAG7) or pancreatic ductal carcinoma (PDAC).

[0072] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0073] The reagents and raw materials used in the present invention are commercially available.

[0074] The positive and progressive effects of the present invention are:

[0075] The Wnt signaling pathway regulation system provided by the present invention has the advantage of high activation ability in Wnt signaling pathway activation, and the activation ability is significantly stronger than the activation ability when any regulator in the Wnt signaling pathway regulation system is used alone, and has the advantage of being more selective than the small molecule inhibitor CHIR-99021 of the key protein GSK3 in the Wnt signaling pathway, and can regulate the degree and region of signal activation according to the receptor expression spectrum to reduce the generation of toxic side effects. The Wnt signaling pathway regulation system provided by the present invention has good application prospects and can be applied to basic research and application fields related to Wnt pathway activation, such as the molecular basis of synergistic activation, and regenerative medicine fields such as lung regeneration, bone regeneration, hair and tooth regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1-Figure 3 The following is a diagram showing the binding patterns of two types of Wnt signaling pathway regulators with Fzd and LRP6:

[0077] Figure 1 and Figure 2 It is shown that the Wnt signaling pathway regulator consists of two parts; Figure 3 A potential schematic diagram of the synergistic activation mechanism.

[0078] The grid-filled part indicates the E1E2 domain that targets LRP6 to the extracellular space;

[0079] The dot-filled part indicates the E3E4 domain that targets LRP6 to the extracellular space;

[0080] The vertical line filled part indicates the part targeted to Fzd;

[0081] CRD: Cysteine-rich domain, extracellular cysteine-rich domain.

[0082] Figure 4-Figure 10 The components and construction of two types of Wnt signaling pathway regulators:

[0083] Figure 4-Figure 7 Nanobodies W0529 targeting Fzd1, 2, 4, 5, 7 and 8, including:

[0084] Figure 4 This is the SDS-PAGE result of W0529;

[0085] Figure 5 It is the peak type diagram of molecular sieve;

[0086] Figure 6 This is the result of BLI binding experiment;

[0087] Figure 7 This is the TOPFlash experiment result;

[0088] Figure 8-Figure 10 Nanobody W1114 targeting the E3E4 domain of LRP6, including:

[0089] Figure 8 This is the SDS-PAGE result of W1114;

[0090] Fig. 9 It is the peak type diagram of molecular sieve;

[0091] Fig.10 This is the result of BLI binding experiment;

[0092] Fig.11 Schematic diagram of plasmid construction of four Wnt signaling pathway regulators.

[0093] Figure 12-Figure 15 To simultaneously use two types of Wnt signaling pathway regulators to activate the synergistic effect of the Wnt signaling pathway at the cellular level:

[0094] Fig.12 To compare the activation effects on the Wnt signaling pathway when A1, B1 were added alone, and when A1 and B1 were added simultaneously;

[0095] Fig.13 To compare the activation effects of A1, B2 alone and A1 and B2 simultaneously on the Wnt signaling pathway;

[0096] Fig.14 To compare the activation effects of A2, B1 alone and A2 and B1 simultaneously on the Wnt signaling pathway;

[0097] Fig.15 To compare the activation effects on the Wnt signaling pathway when A2, B2 were added alone or when A2 and B2 were added simultaneously.

[0098] A1:W0529-DKK1c-Fc;

[0099] A2:W0529-W1114-Fc;

[0100] B1:W0529-SOST-Fc;

[0101] B2:W0529-MES Dpep-Fc;

[0102] N: pCDNA3.1-Fc;

[0103] Control group: transfected N+N.

[0104] Figure 16-Figure 19 Corresponding to Example 5, the synergistic effect of using two types of Wnt signaling pathway regulators at the cellular level on the activation of the Wnt signaling pathway:

[0105] Fig.16 To compare the activation effects on the Wnt signaling pathway when A1, B1 were added alone, and when A1 and B1 were added simultaneously;

[0106] Fig.17 To compare the activation effects of A1, B2 alone and A1 and B2 simultaneously on the Wnt signaling pathway;

[0107] Fig.18 To compare the activation effects of A2, B1 alone and A2 and B1 simultaneously on the Wnt signaling pathway;

[0108] Fig.19 To compare the activation effects on the Wnt signaling pathway when A2, B2 were added alone or when A2 and B2 were added simultaneously.

[0109] A1:W0529-DKK1c-Fc;

[0110] A2:W0529-W1114-Fc;

[0111] B1:W0529-SOST-Fc;

[0112] B2:W0529-MES Dpep-Fc;

[0113] Control: pCDNA3.1-Fc. DETAILED DESCRIPTION

[0114] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0115] Example 1 Screening of Nanobodies Using Phage Display Technology and Its Expression and Purification

[0116] The present invention carries out 4 rounds of phage display in total. The purified phage is first incubated with magnetic beads to remove the part that has the ability to bind to the magnetic beads, and then the phage is incubated with the biotinylated antigen (Fzd5 extracellular region or E3E4 of LRP6). Subsequently, another portion of magnetic beads is used to bind to the mixture of the above-mentioned phage and antigen at room temperature, and the non-specific binding part is washed to remove the impurities, and the bound phage is released using 0.2M glycine solution (pH 3). Tris-HCl is added in time to adjust the pH to neutral during the process. The screened phage is amplified in vivo and purified in vitro before a new round of display. The enrichment of the phage after each round of screening is observed by Poly-ELISA experiment, and the single clone of the screening result of the last round is selected for ELISA experiment and sequencing to obtain the nano antibody nucleic acid sequence that can bind to the antigen.

[0117] The screened nanobody gene was cloned into the expression vector pSb and transformed into E.coli MC1061 for expression. The cells were resuspended in TES solution (0.5M sucrose, 0.5mM EDTA, 0.2M Tris-HCl pH 8.0), and milliQ H2O was added after rotation for 30min and continued to rotate. The supernatant collected by centrifugation was incubated with a Ni-NTA affinity column and washed with a buffer containing 30mM imidazole (150mM NaCl, 20mM Tris-HCl pH 8.0), and the protein was eluted with a buffer containing 300mM imidazole. Purified nanobodies W0529 and W1114 were obtained.

[0118] Example 2 Identification of Nanobodies and Analysis of Their Affinity with Antigens

[0119] Nanobody W0529 amino acid sequence (SEQ ID NO: 4):

[0120] AVQLQASGGGFVQPGGSLRLSCAAS GPFFLWSY MGWFRQAPGKEREFVSA QUR YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC ATMGFWDVTMSRHEQY WGQGTQVTVSS

[0121] Nanobody W1114 amino acid sequence (SEQ ID NO: 8):

[0122] AVQLQASGGGFVQPGGSLRLSCAAS GTVDIDIW MGWFRQAPGKEREFVSA INAYPPQW YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC ATWRQTDWYDILSDFY WGQGTQVTVSS

[0123] Nucleotide sequence encoding Nanobody W0529 (SEQ ID NO: 13):

[0124] GCCGTGCAGCTGCAGGCCAGCGGCGGCGGCTTCGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCCCGTTCTTCCTGTGGTCTTACATGGGCTGGTTCAGGCAGGCTCCCGGCAAGGAGAGGGAGTTCGTGAGCGCCATCTGGCAGCCGATCCATCATGAATACTACGCCG ACAGCTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCACCTACTACTGCGCCACTATGGGTTTCTGGGACGTTACTATGTCTCGTCATGAACAGTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0125] Nucleotide sequence encoding Nanobody W1114 (SEQ ID NO: 14):

[0126] GCCGTGCAGCTGCAGGCCAGCGGCGGCGGCTTCGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCACTGTTGACATCGACATCTGGATGGGCTGGTTCAGGCAGGCTCCCGGCAAGGAGAGGGAGTTCGTGAGCGCCATCAACGCTTACCCGCCGCAGTGGTACTACGCCG ACAGCTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCACCTACTACTGCGCCACTTGGCGTCAGACTGACTGGTACGACATCCTGTCTGACTTCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0127] Among them, the CDR sequences of Nanobodies W0529 and W1114 (determined according to the IMGT definition rules) are represented in the "bold + underline" format.

[0128] Specifically, the CDR1 of Nanobody W0529 is shown in SEQ ID NO: 4 from the 26th to 33rd positions from the N-terminus, the CDR2 is shown in SEQ ID NO: 4 from the 51st to 58th positions from the N-terminus, and the CDR3 is shown in SEQ ID NO: 4 from the 97th to 112th positions from the N-terminus.

[0129] The CDR1 of Nanobody W1114 is shown in SEQ ID NO: 8 at positions 26 to 33 from the N-terminus, the CDR2 is shown in SEQ ID NO: 8 at positions 51 to 58 from the N-terminus, and the CDR3 is shown in SEQ ID NO: 8 at positions 97 to 112 from the N-terminus.

[0130] The CDR sequences of Nanobodies and their sequence numbers are shown in Table 2:

[0131] Table 2. CDR sequences of nanobodies and their sequence numbers

[0132]

[0133] The purified nanoantibodies W0529 and W1114 were subjected to SDS-PAGE and size exclusion chromatography (SEC) to detect the protein status, and the affinity of nanoantibody W1114 to the antigen was determined by the BLI method.

[0134] The SDS-PAGE results of nanoantibodies W0529 and W1114 are as follows Figure 4and Figure 8 shown.

[0135] 2.1 Size Exclusion Chromatography Analysis of Nanobodies

[0136] The molecular screening analysis of the nanoantibody was performed using Cytiva's AKTA pure instrument to detect the protein status. The column model was Superdex 75increase 10 / 300GL, and the working solution was a PBS solution filtered through a 0.22 μm filter membrane.

[0137] First, clean the pipeline and complete the column balance, and inject the supernatant of the nanoantibody sample (W0529, W1114) after centrifugation (15000rpm, 10min) into the 1mL sample loop with a 1mL syringe. Start the program with a flow rate of 0.7mL / min and a fixed volume of 0.5mL for collection.

[0138] The results are as follows Figure 5 and Fig. 9 shown.

[0139] 2.2 BLI method to determine the affinity of nanoantibodies to antigens

[0140] Octet RED96 instrument was used to detect the interaction between nanoantibody W1114 and LRP6 extracellular E3E4 and W0529 and Fzd5 CRD using biofilm optical interference (BLI) technology. W1114 or W0529 was diluted to 0nM, 27.8nM, 83.3nM, 250nM, 750nM with PBST buffer (containing 0.05% (v / v) Tween-20). The biotinylated LRP6 extracellular E3E4 or Fzd5 CRD was fixed on the streptavidin probe, and then the probe was inserted into the dilution solution containing different concentrations of W1114 or W0529 to determine the affinity. Among them, k on refers to the binding rate constant, k off refers to the dissociation rate constant, K D (K D =k off / k on ) refers to the equilibrium dissociation constant, which is used to characterize the affinity between antibody and antigen.

[0141] Test results such as Figure 6 , Fig.10 As shown, the binding kinetic parameters of W0529 and Fzd5 CRD are: K D =9.6nM, k off =2.6*10 5 M -1 *s -1 , k off =2.5*10-1 s -1 , indicating that it has good binding ability in the in vitro purification system; the binding kinetic parameters of W1114 and E3E4 are: K D =338nM, k off =3.3*10 5 M -1 *s -1 , k off =

[0142] 1.1*10 -1 s -1 , indicating that it has moderate binding capacity under an in vitro purification system, and the affinity can be further increased through multivalent combination or directed evolution.

[0143] 2.3TOPFlash experiment to detect the binding ability of nanoantibody W0529 to different Wnt receptors Frizzled

[0144] The TOPFlash experiment was used to detect the ability of the Wnt signaling pathway regulator W0529-DKK1c to activate the pathway, and then determine whether W0529 binds to Frizzled. Only when it binds can the downstream signaling pathway be activated.

[0145] HEK293T cells were seeded in 24-well plates with 500 μL of culture medium (DMEM / 10% FBS, 1% PS; FBS supplier is Shuanglu Biotechnology, PS supplier is Shenger Biotechnology), with approximately 1×10 cells per well. 5 cells. 24 hours later, Lipofectamine TM The cells were transfected with 3000 Reagent transfection reagent, 0.75 μL per well. The transfection plasmids included 7×TCL / LEF-firefly luciferase reporter gene plasmid (trade name: M50 Super 8x TOPFlash; supplier: Addgene Plasmid; catalog number: #12456), Renilla luciferase reporter gene plasmid (trade name: pRL Renilla Luciferase Control Reporter Vectors; supplier: Promega; catalog number: #E2261), and a mixture of 100 ng of each W0529-DKK1c-Fc and different Frizzled (1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10). As a control, a parallel operation was performed using plasmid pcDNA3.1-Fc instead of W0529-DKK1c-Fc.

[0146] After culturing for 24 h, the well plate was removed, the culture medium was discarded, and the cells were washed with PBS solution. The cells were treated with lysis buffer and color development buffer provided by Dual-Luciferase@Reporter Assay System (supplier: Promega) to detect Firefly and Renilla luciferase signals and perform comparative analysis.

[0147] The results are as follows Figure 7 As shown, W0529 has strong binding ability to Wnt receptors Frizzled 1, 2, 4, 5, 7, and 8.

[0148] Example 3 Composition and Construction of Regulators of Wnt Signaling Pathway

[0149] The regulator consists of two parts, one part is a component targeting the extracellular E1E2 (or E3E4) domain of LRP6, and the other part is a component targeting Fzd. The present invention uses a synthetic nano antibody targeting Fzd screened by phage display technology, as well as a natural protein that binds to LRP6 and a nano antibody that targets the extracellular E1E2 (or E3E4) domain of LRP6 to construct two types of regulators of the Wnt signaling pathway. Class A is a regulator targeting Fzd and the extracellular E3 and / or E4 of LRP6 (such as Figure 2 Class B is a regulator that targets Fzd and LRP6 extracellular E1 and / or E2 (as shown in Figure 1 The simultaneous administration of these two types of regulators can enhance the ability of the regulators to activate the Wnt signaling pathway (as shown in Figure 3 shown).

[0150] The expression plasmid structure of the regulator constructed in the present invention is as follows Fig.11 As shown (the backbone plasmid is conventional pCDNA3.1, which can be purchased on the market). Nanobody W0529 is connected to the N-terminus of the LRP6 targeting part by 10 amino acids (GGSGS)2. The LRP6 targeting part contains four different proteins: DKK1c that binds to the extracellular E3 domain, nanobody W1114 that binds to the extracellular E3E4 domain, SOST that binds to the extracellular E1E2 domain, and MESDpep, a 38-amino acid peptide at the C-terminus of the MESD protein that binds to the extracellular region. The Fc part of the natural antibody IgG1 is fused to the C-terminus of the four constructs.

[0151] The four Wnt signaling pathway regulators include:

[0152] A1:W0529-DKK1c;

[0153] A2:W0529-W1114;

[0154] B1:W0529-SOST;

[0155] B2:W0529-MESDpep.

[0156] Example 4 TOPFlash assay to detect the ability of Wnt signaling pathway regulators to activate the pathway

[0157] HEK293T cells were seeded in 24-well plates with 500 μL of culture medium (DMEM / 10% FBS, 1% PS; FBS supplier is Shuanglu Biotechnology, PS supplier is Shenger Biotechnology), with approximately 1×10 cells per well. 5 After 24 hours, Lipofectamine TM Transfection was performed using 3000 Reagent transfection reagent, 0.75 μL per well, and the transfection plasmids were 7× TCL / LEF-firefly luciferase reporter gene plasmid (trade name: M50 Super 8x TOPFlash; supplier: Addgene Plasmid; catalog number: #12456), Renilla luciferase reporter gene plasmid (trade name: pRL Renilla Luciferase Control Reporter Vectors; supplier: Promega; catalog number: #E2261), and 100 ng of expression plasmids of class A / B Wnt signaling pathway regulators or plasmid pcDNA3.1-Fc as controls to ensure that the total amount of plasmids transfected in each well was the same.

[0158] After 18 hours of culture, 5 μM GSK3 inhibitor CHIR-99021 (positive control for Wnt signal activation) was added to the positive control wells. After another 6 hours, the wells were removed, the culture medium was discarded, and after washing with PBS solution, the cells were treated with lysis buffer and color development solution provided by Dual-Luciferase@ReporterAssay System (supplier: Promega) to detect Firefly and Renilla luciferase signals and perform comparative analysis.

[0159] Test results such as Figure 12-Figure 15 When A1 or B1 is added alone, it has a certain activation effect on the Wnt signaling pathway. When A1 and B1 are added together, the activation effect on the Wnt signaling pathway increases by about 10 times, which is significantly enhanced compared with the sum of adding A1 or B1 alone (see Fig.12 When A1 and B2 are added simultaneously, there is a synergistic effect similar to that of adding A1 and B1 simultaneously (see Fig.13 When A2 is added alone, the activation effect on the Wnt signaling pathway is very low, and almost no signal can be detected. When B1 is added alone, there is a certain activation effect, which is significantly enhanced when A2 and B1 are added at the same time (see Fig.14When A2 and B2 are added simultaneously, there is a synergistic effect similar to that of adding A2 and B1 simultaneously (see Fig.15 ).

[0160] Example 5 TOPFlash assay to detect the ability of Wnt signaling pathway regulators to activate the pathway

[0161] HEK293T cells were seeded in 48-well plates with 300 μL of culture medium (DMEM / 10% FBS, 1% PS; FBS supplier was Shuanglu Biotechnology, PS supplier was Shenger Biotechnology), with approximately 5×10 cells per well. 4 After 24 hours, Lipofectamine TM Transfection was performed using 3000 Reagent transfection reagent, 0.4 μL per well, and the transfection plasmids were 7×TCL / LEF-firefly luciferase reporter gene plasmid (trade name: M50 Super 8x TOPFlash; supplier: Addgene Plasmid; catalog number: #12456) and Renilla luciferase reporter gene plasmid (trade name: pRL Renilla Luciferase Control Reporter Vectors; supplier: Promega; catalog number: #E2261).

[0162] 24 hours after transfection, conditioned medium containing secreted Wnt signaling pathway regulators was added to the experimental group, and conditioned medium containing secreted pcDNA3.1-Fc was added to the control group. The medium was used to transiently express four Wnt signaling pathway regulators using Expi293F cells (Thermo Fisher Scientific Catalog No.: A14527) and PEI (Trade Name: Transfection Grade Linear PolyethylenimineHydrochloride MW 40,000; Supplier: Polysciences; Catalog No.: 24765-1) transfection reagent. Specifically, Expi293F cells were cultured in Union293 cell culture medium (supplier: Yonglian Bio) at 3×10 6The cells were suspended at a density of 10 cells / mL, and the plasmids of four Wnt signaling pathway regulators were mixed with PEI (mass ratio of 1:3) and added to Union293 medium. After gently mixing, the mixture was incubated at room temperature for 15 minutes to form a transfection complex and slowly added to the culture flask. The final concentration of the plasmid was 1.5 μg / mL. After incubation at 37°C and 8% CO2 for 18 hours, 10 mM sodium butyrate (supplier: Coolaber) was added, and the cells were further cultured for 72 hours at 30°C and 8% CO2. The supernatant was removed by centrifugation and concentrated 10 times to prepare a conditioned medium containing secreted Wnt signaling pathway regulators.

[0163] After HEK293T cells were cultured for 18 hours, 5 μM GSK3 inhibitor CHIR-99021 (positive control for Wnt signal activation) was added to the positive control wells. After another 6 hours, the wells were removed, the culture medium was discarded, and the cells were treated with the lysis solution and color development solution provided by Dual-Luciferase@Reporter AssaySystem (supplier: Promega) to detect Firefly and Renilla luciferase signals and conduct comparative analysis.

[0164] Test results such as Figure 16-Figure 19 When A1 and B1 are added alone, they have a certain activation effect on the Wnt signaling pathway. When A1 and B1 are added at the same time, there is a synergistic effect on the activation of the Wnt signaling pathway (see Fig.16 ). When A1 and B2 are added at the same time (see Fig.17 ), add A2 and B1 (see Fig.18 ), when A2 and B2 are added at the same time (see Fig.19 ), similar phenomena exist.

Claims

1. A Wnt signaling pathway regulation system, characterized in that: It includes a first regulator and a second regulator, both of which include an I domain and an II domain, wherein the first regulator includes an I-1 domain and a II-1 domain that target the Wnt receptor Frizzled and LRP6, respectively, and the second regulator includes an I-2 domain and a II-2 domain that target the Wnt receptor Frizzled and LRP6, respectively; the first regulator and the second regulator are different.

2. The Wnt signaling pathway regulation system according to claim 1, characterized in that: The II-1 domain and the II-2 domain both target the extracellular region of LRP6, preferably target different extracellular domains respectively, and the extracellular domains include E1, E2, E3 and E4.

3. The Wnt signaling pathway regulation system according to claim 2, characterized in that: The II-1 domain targets the extracellular region E1 and / or E2 of LRP6, and the II-2 domain targets the extracellular region of LRP6; or, the II-1 domain targets the extracellular region of LRP6, and the II-2 domain targets the extracellular region E3 and / or E4 of LRP6; Preferably, the II-1 domain targets the extracellular region E1 and / or E2 of LRP6, and the II-2 domain targets the extracellular region E3 and / or E4 of LRP6.

4. The Wnt signaling pathway regulation system according to claim 1 or 2, characterized in that: The I-1 domain and the I-2 domain both target the extracellular region of the Wnt receptor Frizzled; the extracellular region includes a cysteine-rich domain, and preferably also includes a linker and a hinge region.

5. The Wnt signaling pathway regulating system according to any one of claims 1 to 4, characterized in that: The regulator also includes Fc; preferably, the regulator includes domain I, domain II and Fc, or domain II, domain I and Fc connected in sequence from N-terminus to C-terminus; more preferably, the connection is connected using a linker or directly connected, the linker is for example (GGSGS)2 and MVRS; further more preferably, domain I is connected to domain II using a linker (GGSGS)2, and / or, domain I or domain II is connected to Fc using a linker MVRS.

6. The Wnt signaling pathway regulating system according to any one of claims 1 to 5, characterized in that: The I domain and the II domain are selected from antibodies and natural proteins; Preferably, the antibody is an IgG antibody, a Fab fragment, a scFv or a heavy chain antibody, the heavy chain antibody is for example a nanobody; the antibody is a monovalent or multivalent antibody; and / or the natural protein is selected from Dickkopf-1 protein such as DKK1c, Sclerostin and MESD such as MESDpep.

7. The Wnt signaling pathway regulation system according to claim 6, characterized in that: The regulators include nanobodies targeting Frizzled, nanobodies targeting LRP6; or nanobodies targeting Frizzled, DKK1c targeting LRP6; or nanobodies targeting Frizzled, SOST targeting LRP6; or nanobodies targeting Frizzled, MESDpep targeting LRP6; Optionally, the regulator further comprises Fc, and the Fc is, for example, an Fc having an amino acid sequence as shown in SEQ ID NO:

19.

8. The Wnt signaling pathway regulation system according to claim 7, characterized in that: The Frizzled-targeting nanobody comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and / or, the nanobody can bind to one or more of Frizzled 1, 2, 4, 5, 7 and 8, preferably can bind to Frizzled 1, 2, 4, 5, 7 and 8; Preferably, the amino acid sequence of the Nanobody is as shown in SEQ ID NO:

4.

9. The Wnt signaling pathway regulation system according to claim 7, characterized in that: The nanobody targeting LRP6 comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; Preferably, the amino acid sequence of the Nanobody is as shown in SEQ ID NO:

8.

10. The Wnt signaling pathway regulation system according to claim 7, characterized in that: The amino acid sequence of DKK1c is shown in SEQ ID NO:9; the amino acid sequence of SOST is shown in SEQ ID NO:10; and / or the amino acid sequence of MESDpep is shown in SEQ ID NO:

11.

11. A heavy chain antibody targeting Frizzled, characterized in that: The heavy chain antibody comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; Preferably, the amino acid sequence of the heavy chain antibody is as shown in SEQ ID NO: 4; the nucleotide sequence encoding the heavy chain antibody is preferably as shown in SEQ ID NO: 14; More preferably, the heavy chain antibody further comprises Fc, and the Fc is, for example, an Fc having an amino acid sequence as shown in SEQ ID NO:

19.

12. A heavy chain antibody targeting LRP6, characterized in that: The heavy chain antibody comprises CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively; Preferably, the amino acid sequence of the heavy chain antibody is as shown in SEQ ID NO: 8; the nucleotide sequence encoding the heavy chain antibody is preferably as shown in SEQ ID NO: 15; More preferably, the heavy chain antibody further comprises Fc, and the Fc is, for example, an Fc having an amino acid sequence as shown in SEQ ID NO:

19.

13. A reagent or a kit, characterized in that: The reagent or kit comprises the Wnt signaling pathway regulation system according to any one of claims 1 to 10, and / or the heavy chain antibody according to claim 11 or 12.

14. Use of the Wnt signaling pathway regulation system according to any one of claims 1 to 10, or the heavy chain antibody according to claim 11 or 12 in activating the Wnt signaling pathway or preparing a reagent or kit for activating the Wnt signaling pathway.

15. A method for activating a Wnt signaling pathway, characterized in that: Contacting cells with the Wnt signaling pathway regulation system according to any one of claims 1 to 10; Preferably, the cell is a mammalian cell; and / or the method is for non-diagnostic therapeutic purposes.

16. Use of the Wnt signaling pathway regulation system according to any one of claims 1 to 10, the heavy chain antibody according to claim 11 or 12, or the reagent or kit according to claim 13 in the preparation of a regenerative medicine product; Preferably, the regenerative medicine is bone regeneration, lung regeneration, hair regeneration or tooth regeneration.

17. A method for detecting whether Frizzled and / or LRP6 exists in a sample, comprising contacting the heavy chain antibody according to claim 11 or 12 with the sample, and determining whether Frizzled and / or LRP6 exists in the sample based on whether the heavy chain antibody binds to the sample.

18. A method for diagnosing and / or treating Frizzled and / or LRP6 related diseases, comprising administering the heavy chain antibody of claim 11 or 12 to a patient in need thereof; preferably, the Frizzled and / or LRP6 related diseases include diseases caused by Frizzled and / or LRP6 mutations or abnormal expression, such as coronary artery disease, tooth loss or pancreatic ductal carcinoma.

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