A wnt signaling pathway modulation system and a method of activating a wnt signaling pathway

Abstract

The application discloses a Wnt signal pathway regulating system and a method for activating the Wnt signal pathway. The Wnt signal pathway regulating system comprises a first regulator and a second regulator, and the regulators each comprise an I domain and an II domain, wherein the first regulator comprises an I-1 domain and an II-1 domain which are respectively targeted to Wnt receptors Frizzled and LRP6, the second regulator comprises an I-2 domain and an II-2 domain which are respectively targeted to Wnt receptors Frizzled and LRP6, and the first regulator and the second regulator are different. The Wnt signal pathway regulating system provided by the application has the advantages of high activation capacity, small toxic and side effects and selectivity in Wnt signal pathway activation, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a Wnt signaling pathway regulation system and a method for activating the Wnt signaling pathway. Background Technology

[0002] The Wnt signaling pathway regulates the pluripotent differentiation of stem cells, organ development and regeneration, playing a crucial role in maintaining tissue homeostasis and repairing and regenerating damaged organs, and possesses enormous potential for regenerative medicine applications. Wnt proteins recruit the receptor Frizzled (Fzd) and the co-receptor low-density lipoprotein receptor-associated protein 5 or 6 (LRP5 / 6), forming a complex that initiates the β-catenin-dependent classical Wnt signaling pathway through a series of signal transductions. However, the palm oil acylation modification and substructural stability of Wnt proteins result in poor physicochemical properties, making large-scale purification difficult. Furthermore, the highly conserved cross-reactivity between 19 Wnt proteins and 10 Fzd receptors in the human body during signal recognition limits the application of Wnt proteins in basic research and medicine. There is an urgent need for alternatives to Wnt proteins to support research in this field.

[0003] In 2017, Garcia's research group first proposed the concept of Wnt functional substitutes, using natural protein or single-chain antibody fragments with better stability and water solubility as building blocks to target and bind to Fzd and LRP6, replacing the function of the Wnt protein. Their ability to activate the Wnt signaling pathway was demonstrated at the cellular, organoid, and mouse model levels. Various research groups have selected different antibody fragmentation forms as basic building blocks, such as the antigen-binding Fab fragment, the effector Fc fragment, "heavy chain antibodies" lacking the light chain, and nanobodies, optimizing the physicochemical properties and effects of Wnt functional substitutes in terms of stability, molecular weight, and permeability. On the other hand, researchers have modified the antibody construction, developing bivalent, trivalent, and tetravalent multispecific antibodies to improve antigen binding and signal activation capabilities. To date, Wnt functional substitutes continue to be optimized and innovated in terms of building blocks and construction methods to achieve even more significant signal activation capabilities. Summary of the Invention

[0004] To address the shortcomings of existing Wnt functional substitutes in activating the Wnt signaling pathway, which still require improvement, this invention provides a Wnt signaling pathway modulation system and a method for activating the Wnt signaling pathway. The Wnt signaling pathway modulation system has a significant Wnt signaling pathway activation capability.

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

[0006] In this invention, the term "Wnt signal path modulation system" refers to a composition for modulating the Wnt signal path, comprising a first modulator and a second modulator. The terms "first modulator" and "second modulator" refer to two different modulating elements. Specifically:

[0007] The first aspect of the present invention provides a Wnt signaling pathway modulation system, comprising a first regulator and a second regulator, each regulator comprising an I domain and a II domain, wherein the first regulator comprises an I-1 domain and a II-1 domain targeting the Wnt receptor Frizzled and LRP6, respectively, and the second regulator comprises an I-2 domain and a II-2 domain targeting the Wnt receptor 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, including E1, E2, E3 and E4.

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

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

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

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

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

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

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

[0019] In some preferred embodiments of the present invention, the connection is made using connectors or direct connection, such as (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, the I structural domain and the II structural domain are connected using connector (GGSGS)2, and / or the I structural domain or the II structural domain is connected to Fc using connector MVRS.

[0021] In some specific embodiments of the present invention, the nucleotide sequence encoding linker (GGSGS)2 is shown in 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 includes 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 this 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 comprises a Kappa signal peptide, a His6 tag, an I domain, a II domain and an Fc, or a Kappa signal peptide, a His6 tag, an II domain, an 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 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, an scFv, or a heavy chain antibody, the heavy chain antibody being, for example, a nanobody; the antibody is a monovalent or multivalent antibody, the multivalent antibody being, for example, a bivalent, trivalent, or quadrivalent 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 modulator includes a Frizzled nanobody, a LRP6-targeting nanobody; or a Frizzled nanobody, a DKK1c-targeting LRP6-targeting nanobody; or a Frizzled nanobody, a SOST-targeting LRP6-targeting nanobody; or a Frizzled nanobody, a MESDpep-targeting LRP6-targeting nanobody.

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

[0030] In this invention, the Fc sequence can be selected by those skilled in the art, for example, the Fc is 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 nanobody targeting Frizzled comprises amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and / or, the nanobody is capable of binding to one or more of Frizzled 1, 2, 4, 5, 7, and 8.

[0033] In some preferred embodiments of the present invention, the nanobodies are capable of binding Frizzled 1, 2, 4, 5, 7 and 8.

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

[0035] In some embodiments of the present invention, the LRP6-targeting nanobody comprises CDR1, CDR2, and CDR3, respectively, as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

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

[0037] In some embodiments of the present invention, 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.

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

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

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

[0041] The term "nanobody" in this invention, also known as "single-domain antibody," contains only one heavy chain variable region VHH, has a molecular weight of approximately 15 kDa, and a diameter of approximately 10 nanometers. It retains all antigen-binding capacity and is the smallest intact antigen-binding fragment.

[0042] The amino acid sequences of the CDRs listed in this invention are determined according to the IMGT definition rules. However, as is known to those skilled in the art, antibody CDRs can be defined in various ways, such as 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)), Kabat et al. (1987) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass the complementarity-determining region defined by any of the known schemes described above. Various numbering systems and their corresponding CDRs are well known to those skilled in the art, as shown in Table 1:

[0043] Table 1. Definition of Antibody CDR

[0044]

[0045]

[0046] Note: In Table 1, Haa-Hbb refers to the amino acid sequence from position aa to position bb, starting from the N-terminus of the antibody heavy chain and following its corresponding coding rule. For example, H31–H35 in the second row and second column of Table 1 refers to the amino acid sequence from position 31 to position 35, starting from the N-terminus of the variable region of the antibody heavy chain and following the Kabat coding rule; the others follow the same pattern.

[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, as 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 includes Fc.

[0051] In some specific embodiments of the present invention, the Fc is the 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, the heavy chain antibody comprising CDR1, CDR2 and CDR3 with amino acid sequences as 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 includes Fc.

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

[0057] A 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 sequences of the polynucleotide are shown in SEQ ID NO:14 or SEQ ID NO:15, respectively.

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

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

[0061] A sixth aspect of the present invention provides a transformant comprising the recombinant expression vector as described in the fifth aspect of the present invention. Preferably, the originating 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] A seventh aspect of the present invention provides a reagent or kit comprising 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 includes the polynucleotide as described in the fourth aspect of the invention, the recombinant expression vector as described in the fifth aspect of the invention, and / or the transformant as described in the sixth aspect of the invention.

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

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

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

[0067] In some embodiments of the present invention, the method is not for diagnostic or therapeutic purposes.

[0068] The tenth aspect of this invention provides the application of the Wnt signaling pathway regulation system as described in the first aspect of this 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 regenerative medicine products.

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

[0070] The eleventh aspect of the present invention provides a method for detecting the presence of Frizzled and / or LRP6 in a sample using the heavy chain antibody described in the second and / or third aspects of the present invention, wherein the heavy chain antibody is contacted with the sample, and the presence of Frizzled and / or LRP6 in the sample is determined by conventional techniques in the art.

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

[0072] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0073] The reagents and raw materials used in this invention are all commercially available.

[0074] The positive and progressive effects of this invention are as follows:

[0075] The Wnt signaling pathway regulation system provided by this invention has the advantage of high activation capacity in Wnt signaling pathway activation, significantly stronger than the activation capacity of any regulator in the Wnt signaling pathway regulation system used alone. Furthermore, it is more selective than the small molecule inhibitor CHIR-99021, which is a key protein in the Wnt signaling pathway, and can regulate the degree and region of signal activation based on receptor expression profiles, reducing toxic side effects. The Wnt signaling pathway regulation system provided by this invention has promising applications in basic research and applied 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. Attached Figure Description

[0076] Figures 1-3 Schematic diagrams showing the binding patterns of regulators in the two Wnt signaling pathways with Fzd and LRP6:

[0077] Figure 1 and Figure 2 The Wnt signaling pathway modulator shown consists of two parts; Figure 3 This is a schematic diagram of a potential collaborative activation mechanism.

[0078] The mesh filling represents the E1E2 domain targeting the extracellular space of LRP6;

[0079] The dotted portion represents the E3E4 domain targeting the extracellular space of LRP6;

[0080] The vertical line filled portion represents the part targeting Fzd;

[0081] CRD: Cysteine-rich domain, an extracellular domain rich in cysteine ​​residues.

[0082] Figures 4-10 Components and construction of two types of Wnt signaling pathway regulators:

[0083] Figures 4-7 The nanobody W0529 targeting Fzd1, 2, 4, 5, 7, and 8 includes:

[0084] Figure 4 The image shows the SDS-PAGE results for W0529.

[0085] Figure 5 This is a peak shape diagram of molecular sieves;

[0086] Figure 6 The results of the BLI-based experiments;

[0087] Figure 7 These are the experimental results for TOPFlash;

[0088] Figures 8-10 The nanobody W1114 targeting the E3E4 domain of LRP6 includes:

[0089] Figure 8 The image shows the SDS-PAGE results for W1114.

[0090] Figure 9 This is a peak shape diagram of molecular sieves;

[0091] Figure 10 The results of the BLI-based experiments;

[0092] Figure 11 This is a schematic diagram of plasmid construction for four Wnt signaling pathway regulators.

[0093] Figures 12-15 To demonstrate the synergistic effect of simultaneously using two classes of Wnt signaling pathway regulators on Wnt signaling pathway activation at the cellular level:

[0094] Figure 12 To compare the activation effects on the Wnt signaling pathway when A1 and B1 are added individually and when A1 and B1 are added simultaneously;

[0095] Figure 13 To compare the activation effects on the Wnt signaling pathway when A1 and B2 are added individually and when A1 and B2 are added simultaneously;

[0096] Figure 14 To compare the activation effects on the Wnt signaling pathway when A2 and B1 are added alone and when A2 and B1 are added simultaneously;

[0097] Figure 15 The activation effects on the Wnt signaling pathway were compared when A2 and B2 were added individually and 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-MESDpep-Fc;

[0102] N: pCDNA3.1-Fc;

[0103] Control group: N+N transfection.

[0104] Figures 16-19 This corresponds to the synergistic effect of using two types of Wnt signaling pathway regulators simultaneously at the cellular level in Example 5, which activates the Wnt signaling pathway.

[0105] Figure 16 To compare the activation effects on the Wnt signaling pathway when A1 and B1 are added individually and when A1 and B1 are added simultaneously;

[0106] Figure 17 To compare the activation effects on the Wnt signaling pathway when A1 and B2 are added individually and when A1 and B2 are added simultaneously;

[0107] Figure 18 To compare the activation effects on the Wnt signaling pathway when A2 and B1 are added alone and when A2 and B1 are added simultaneously;

[0108] Figure 19 The activation effects on the Wnt signaling pathway were compared when A2 and B2 were added individually and 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-MESDpep-Fc;

[0113] Control: pCDNA3.1-Fc. Detailed Implementation

[0114] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0115] Example 1: Screening and expression / purification of nanobodies using phage display technology

[0116] This invention involved four rounds of phage display. The purified phages were first incubated with magnetic beads to remove the portion capable of binding to the beads. Then, the phages were incubated with biotinylated antigens (the extracellular region of Fzd5 or E3E4 of LRP6). Subsequently, another set of magnetic beads was used to bind the phage and antigen mixture at room temperature. After washing to remove non-specifically bound portions, the bound phages were released using 0.2M glycine solution (pH 3), with Tris-HCl added as needed to adjust the pH to neutral. The selected phages were then amplified in vivo and purified in vitro before undergoing a new round of display. The enrichment of phages from each round of screening was observed using a Poly-ELISA assay. Single clones from the final round of screening were selected for ELISA and sequencing to obtain the nucleic acid sequences of the nanobody capable of binding to the antigen.

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

[0118] Example 2: Identification of nanobodies and analysis of their affinity for antigens

[0119] The amino acid sequence of nanobody W0529 (SEQ ID NO:4):

[0120] AVQLQASGGGFVQPGGSLRLSCAAS GPFFLWSY MGWFRQAPGKEREFVSA IWQPIHHE YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC ATMGFWDVTMSRHEQY WGQGTQVTVSS

[0121] The amino acid sequence of nanobody W1114 (SEQ ID NO:8):

[0122] AVQLQASGGGFVQPGGSLRLSCAAS GTVDIDIW MGWFRQAPGKEREFVSA INAYPPQW YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC ATWRQTDWYDILSDFY WGQGTQVTVSS

[0123] The nucleotide sequence encoding nanobody W0529 (SEQ ID NO:13):

[0124] GCCGTGCAGCTGCAGGCCAGCGGCGGCGGCTTCGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCCCGTTCTTCCTGTGGTCTTACATGGGCTGGTTCAGGCAGGCTCCCGGCAAGGAGAGGGAGTTCGTGAGCGCCATCTGGCAGCCGATCCATCATGAATACTACGCCG ACAGCTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCACCTACTACTGCGCCACTATGGGTTTCTGGGACGTTACTATGTCTCGTCATGAACAGTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0125] The nucleotide sequence encoding nanobody W1114 (SEQ ID NO:14):

[0126] GCCGTGCAGCTGCAGGCCAGCGGCGGCGGCTTCGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCACTGTTGACATCGACATCTGGATGGGCTGGTTCAGGCAGGCTCCCGGCAAGGAGAGGGAGTTCGTGAGCGCCATCAACGCTTACCCGCCGCAGTGGTACTACGCCG ACAGCTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCACCTACTACTGCGCCACTTGGCGTCAGACTGACTGGTACGACATCCTGTCTGACTTCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0127] The CDR sequences of nanobodies W0529 and W1114 (determined according to the IMGT definition rules) are represented in bold + underline format.

[0128] Specifically, the CDR1 of nanobody W0529 is shown as position 26-33 from the N-terminus of SEQ ID NO:4, the CDR2 is shown as position 51-58 from the N-terminus of SEQ ID NO:4, and the CDR3 is shown as position 97-112 from the N-terminus of SEQ ID NO:4.

[0129] The CDR1 of nanobody W1114 is shown as position 26-33 from the N-terminus of SEQ ID NO:8, the CDR2 is shown as position 51-58 from the N-terminus of SEQ ID NO:8, and the CDR3 is shown as position 97-112 from the N-terminus of SEQ ID NO:8.

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

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

[0132]

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

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

[0135] 2.1 Size Exclusion Chromatography Analysis of Nanobodies

[0136] The nanobodies were analyzed using a Cytiva AKTA pure instrument to detect protein state. The column model was Superdex 75increase 10 / 300GL, and the working solution was PBS solution filtered through a 0.22 μm filter membrane.

[0137] First, clean the tubing and equilibrate the column. Then, using a 1 mL syringe, inject the supernatant of the nanobody samples (W0529, W1114) after centrifugation (15000 rpm, 10 min) into 1 mL sample loops respectively. Start the program at a flow rate of 0.7 mL / min and collect a fixed volume of 0.5 mL.

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

[0139] 2.2 Determination of the affinity between nanobodies and antigens using the BLI method

[0140] The interactions between nanobodies W1114 and the extracellular E3E4 of LRP6, and between W0529 and the CRD moiety of Fzd5, were detected using biofilm layer optical interference (BLI) technology on an Octet RED96 instrument. W1114 or W0529 was diluted to 0 nM, 27.8 nM, 83.3 nM, 250 nM, and 750 nM with PBST buffer (containing 0.05% (v / v) Tween-20). Biotinylated LRP6 extracellular E3E4 or the CRD moiety of Fzd5 was immobilized on a streptavidin probe, which was then dipped into dilutions containing different concentrations of W1114 or W0529 to determine affinity. (k...) on The binding rate constant, k off The dissociation rate constant, K D (K D =k off / k on The equilibrium dissociation constant is used to characterize the affinity between an antibody and an antigen.

[0141] Test results as follows Figure 6 , Figure 10 As shown, the bonding kinetic parameters of W0529 and Fzd5 CRD are as follows: K D =9.6nM,k off =2.6*10 5 M -1 *s -1 k off =2.5*10-1 s -1 This indicates 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 This indicates that it has moderate binding capacity in the in vitro purification system, and the affinity can be further increased through multivalent combination or directed evolution.

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

[0144] The activation ability of the Wnt signaling pathway regulator WO529-DKK1c on the pathway was detected by the TOPFlash experiment, thereby determining whether WO529 binds to Frizzled. Only when it can bind can it activate the downstream signaling pathway.

[0145] HEK293T cells were seeded in 500 μL of culture medium (DMEM / 10% FBS, 1% PS; FBS supplier: Shuangru Biotechnology, PS supplier: Shenger Biotechnology) in 24-well plates, approximately 1 × 10⁶ cells per well. 5 1 cell. Lipofectamine was administered 24 hours later. TM Transfection was performed using 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 100 ng each of W0529-DKK1c-Fc mixed with different frizzled (1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10) plasmids. As a control, a parallel operation was performed by replacing W0529-DKK1c-Fc with plasmid pcDNA3.1-Fc.

[0146] After culturing for 24 hours, the well plates were removed, the culture medium was discarded, and the cells were washed with PBS solution. The cells were then treated with lysis buffer and chromogenic solution provided by the Dual-Luciferase@Reporter Assay System (supplier: Promega) to detect and compare the Firefly and Renilla luciferase signals.

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

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

[0149] The regulators consist of two parts: one part is a component targeting the extracellular E1E2 (or E3E4) domains of LRP6, and the other part is a component targeting Fzd. This invention uses phage display technology to screen synthetic nanobodies targeting Fzd, along with natural proteins binding to LRP6 and screened nanobodies targeting the extracellular E1E2 (or E3E4) domains of LRP6, to construct two classes of regulators for the Wnt signaling pathway. Class A consists of regulators targeting Fzd and the extracellular E3 and / or E4 domains of LRP6 (e.g., Figure 2 As shown), class B are regulators that target the extracellular E1 and / or E2 of Fzd and LRP6 (e.g. Figure 1 (As shown). Simultaneous administration of these two types of regulators can enhance the regulators' activation ability on the Wnt signaling pathway (e.g., Figure 3 (As shown).

[0150] The expression plasmid structure of the regulator constructed in this invention is as follows: Figure 11 As shown (the backbone plasmid is standard pCDNA3.1, commercially available). The nanobody W0529 is linked to the N-terminus of the LRP6 targeting region by 10 amino acids (GGSGS)2. The LRP6 targeting region contains four different proteins: DKK1c, which binds to the extracellular E3 domain; nanobody W1114, which binds to the extracellular E3E4 domain; SOST, which binds to the extracellular E1E2 domain; and MESDpep, a 38-amino acid peptide at the C-terminus of the MESD protein, which binds to the extracellular region. All four constructs have the Fc portion of the native antibody IgG1 fused to their C-terminus.

[0151] The four regulators of the Wnt signaling pathway include:

[0152] A1:W0529-DKK1c;

[0153] A2:W0529-W1114;

[0154] B1:W0529-SOST;

[0155] B2:W0529-MESDpep.

[0156] Example 4: TOPFlash experiment to detect the activation ability of Wnt signaling pathway modulators on the pathway.

[0157] HEK293T cells were seeded in 500 μL of culture medium (DMEM / 10% FBS, 1% PS; FBS supplier: Shuangru Biotechnology, PS supplier: Shenger Biotechnology) in 24-well plates, approximately 1 × 10⁶ cells per well. 5 1 cell. 24 hours later, Lipofectamine was used. TM Transfection was performed using 3000 Reagent transfection reagent, 0.75 μL per well. 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 each of expression plasmids of class A / B Wnt signaling pathway regulators or plasmid pcDNA3.1-Fc as controls, ensuring that the total amount of plasmid transfected in each well was the same.

[0158] After 18 hours of culture, 5 μM of the GSK3 inhibitor CHIR-99021 (a 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 washed with PBS solution. The cells were then treated with lysis buffer and chromogenic solution provided by the Dual-Luciferase@ReporterAssay System (supplier: Promega) to detect and compare the Firefly and Renilla luciferase signals.

[0159] Test results as follows Figures 12-15 As shown, the addition of A1 and B1 alone has a certain activating effect on the Wnt signaling pathway. The simultaneous addition of A1 and B1 increases the activation effect on the Wnt signaling pathway by approximately 10 times, which is significantly enhanced compared to the sum of the effects of adding A1 or B1 alone (see...). Figure 12 Adding A1 and B2 simultaneously has a similar synergistic effect as adding A1 and B1 simultaneously (see...). Figure 13 When A2 is added alone, its activation effect on the Wnt signaling pathway is very low, and the signal is almost undetectable. When B1 is added alone, it has a certain activation effect, and when A2 and B1 are added simultaneously, the activation is significantly enhanced (see [link to relevant documentation]). Figure 14Adding A2 and B2 simultaneously has a similar synergistic effect as adding A2 and B1 simultaneously (see...). Figure 15 ).

[0160] Example 5: TOPFlash experiment to detect the activation ability of Wnt signaling pathway modulators on the pathway.

[0161] HEK293T cells were seeded in 300 μL of culture medium (DMEM / 10% FBS, 1% PS; FBS supplier: Shuangru Biotechnology, PS supplier: Shenger Biotechnology) in 48-well plates, approximately 5 × 10⁶ cells per well. 4 1 cell. 24 hours later, Lipofectamine was used. TM Transfection was performed using 3000 Reagent transfection reagent, 0.4 μL per well. 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] Twenty-four hours post-transfection, the experimental group received conditioned medium containing regulators of the Wnt signaling pathway, while the control group received conditioned medium containing pcDNA3.1-Fc. This medium was used to transiently express four Wnt signaling pathway regulators using Expi293F cells (Thermo Fisher Scientific, catalog number: A14527) and PEI (trade name: Transfection Grade Linear Polyethylenimine Hydrochloride MW 40,000; supplier: Polysciences; catalog number: 24765-1) transfection reagent. Specifically, Expi293F cells were transfected in Union293 cell culture medium (supplier: Yonglian Bio) at a concentration of 3 × 10⁻⁶ cells / mL. 6Suspension culture was performed at a density of 10 cells / mL. Plasmids of four Wnt signaling pathway regulators were mixed with PEI (mass ratio 1:3) and added to Union293 medium. After gentle mixing, the mixture was incubated at room temperature for 15 minutes to allow the transfection complex to form. The complex was then slowly added to the culture flask to achieve a final plasmid concentration of 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 culture was continued at 30°C and 8% CO2 for 72 hours. After centrifugation to remove cells and collecting the supernatant, the supernatant was concentrated 10-fold to prepare a conditioned medium containing Wnt signaling pathway regulators.

[0163] After culturing HEK293T cells for another 18 hours, 5 μM of the GSK3 inhibitor CHIR-99021 (a positive control for Wnt signal activation) was added to the positive control wells. After another 6 hours, the plates were removed, the culture medium was discarded, and the cells were treated with lysis buffer and chromogenic solution provided by the Dual-Luciferase@Reporter AssaySystem (supplier: Promega). Firefly and Renilla luciferase signals were detected and compared.

[0164] Test results as follows Figures 16-19 As shown. Adding A1 or B1 alone has a certain activating effect on the Wnt signaling pathway; adding A1 and B1 simultaneously has a synergistic effect on the activation of the Wnt signaling pathway (see...). Figure 16 When A1 and B2 are added simultaneously (see...) Figure 17 Add A2 and B1 (see) Figure 18 When A2 and B2 are added simultaneously (see...), Figure 19 Similar phenomena exist in all of them.

Claims

1. A Wnt signal path conditioning system, characterized in that, It consists of a first regulator and a second regulator; the first regulator and the second regulator are different; Each regulator is connected sequentially from the N end to the C end to the I domain, the II domain, and the Fc domain, and the connection is made using connectors; The first regulator is sequentially linked from the N-terminus to the C-terminus to the I-1 domain targeting the Wnt receptor Frizzled, the II-1 domain targeting the Wnt receptor LRP6, and the Fc region, and these connections are made using connectors; and, The second regulator is sequentially linked from the N-terminus to the C-terminus to the I-2 domain targeting the Wnt receptor Frizzled, the II-2 domain targeting the Wnt receptor LRP6, and the Fc domain, and the links are connected using a connector; wherein, Both the I-1 domain and the I-2 domain target the extracellular region of the Wnt receptor Frizzled, thus forming a Frizzled-targeting nanobody; wherein the Frizzled-targeting nanobody comprises CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The II-1 domain targets the extracellular regions E1 and / or E2 of LRP6, and is either SOST or MESDpep targeting LRP6; the II-2 domain targets the extracellular regions E3 and / or E4 of LRP6, and is either a nanobody or DKK1c targeting LRP6; wherein the LRP6-targeting nanobody comprises the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; the amino acid sequence of MESDpep is shown in SEQ ID NO: 11; The amino acid sequence of CDR is determined according to the IMGT definition rules.

2. The Wnt signal path conditioning system as described in claim 1, characterized in that, The connector is (GGSGS)2 or MVRS.

3. The Wnt signal path adjustment system as described in claim 2, characterized in that, The I structural domain is connected to the II structural domain using connector (GGSGS)2, and / or the I structural domain or the II structural domain is connected to Fc using connector MVRS.

4. The Wnt signal path conditioning system as described in any one of claims 1-3, characterized in that, The Fc is the Fc with the amino acid sequence shown in SEQ ID NO:

19.

5. The Wnt signal path adjustment system as described in claim 1, characterized in that, The amino acid sequence of the targeted Frizzled nanobody is shown in SEQ ID NO:

4.

6. The Wnt signal path conditioning system as described in claim 1, characterized in that, The amino acid sequence of the LRP6-targeting nanobody is shown in SEQ ID NO:

8.

7. The Wnt signal path conditioning system as described in claim 1, characterized in that, The amino acid sequence of the DKK1c is shown in SEQ ID NO: 9; and / or, the amino acid sequence of the SOST is shown in SEQ ID NO:

10.

8. A nanobody targeting Frizzled, characterized in that, The nanobody comprises amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The amino acid sequence of CDR is determined according to the IMGT definition rules.

9. The nanobody as described in claim 8, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

4.

10. An isolated nucleic acid, characterized in that, The nucleic acid encodes the nanobody as described in claim 8 or 9, wherein the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

14.

11. A nanobody targeting LRP6, characterized in that, The nanobody comprises the amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. The amino acid sequence of CDR is determined according to the IMGT definition rules.

12. The nanobody as described in claim 11, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

8.

13. An isolated nucleic acid, characterized in that, The nucleic acid encodes the nanobody as described in claim 11 or 12, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

15.

14. A reagent or kit, characterized in that, The reagent or kit includes the Wnt signaling pathway regulation system as described in any one of claims 1-7, and / or the nanobody as described in claims 8, 9, 11 or 12.

15. The use of the Wnt signaling pathway modulation system according to any one of claims 1-7 in activating the Wnt signaling pathway or in preparing reagents or kits for activating the Wnt signaling pathway; in, The application is for non-diagnostic and non-therapeutic purposes.

16. A method for activating the Wnt signaling pathway, characterized in that, Contact the cells using the Wnt signaling pathway regulation system as described in any one of claims 1-7; The method described herein is not for diagnostic or therapeutic purposes.

17. The method as described in claim 16, characterized in that, The cells in question are mammalian cells.

18. The use of the Wnt signaling pathway modulation system as described in any one of claims 1-7 or the reagent or kit as described in claim 14 in the preparation of products for regenerative medicine.

19. The application as described in claim 18, characterized in that, The regenerative medicine mentioned refers to bone regeneration, lung regeneration, hair regeneration, or tooth regeneration.

20. A method for detecting the presence of Frizzled and / or LRP6 in a sample, characterized in that, The method uses the nanobody as described in claim 8 or 9, and / or the nanobody as described in claim 11 or 12 to contact the sample, and determines whether Frizzled and / or LRP6 are present in the sample based on whether the nanobody binds to the sample. The method described herein is not for diagnostic or therapeutic purposes.

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