Nanobody that specifically binds to LRP5 and / or LRP6 and use thereof

Abstract

Disclosed are a nanobody that specifically binds to LRP5 and / or LRP6 and a use thereof. The nanobody has the ability to selectively target and bind to LRP5 and / or LRP6. Some antibodies can inhibit the interaction between LRP5 and / or LRP6 and Wnt proteins, thereby effectively suppressing the Wnt signaling pathway. The provided nanobody specifically recognizes co-receptors LRP5 and / or LRP6 of the Wnt signaling pathway, exhibits high binding affinity, and can be widely applied to the field of detection and related research of LRP5 and / or LRP6.

Description

A nanobody that specifically binds to LRP5 and / or LRP6 and its applications Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a nanobody that specifically binds to LRP5 and / or LRP6 and its application. Background Technology

[0002] The Wnt signaling pathway plays a crucial role in cell proliferation, differentiation, migration, and gene expression regulation, especially in embryonic development and tissue homeostasis. Low-density lipoprotein receptor-associated protein 5 (LRP5) and LRP6 (LRP6) are key co-receptors in the classic Wnt / β-catenin signaling pathway. Under Wnt mediation, LRP5 and / or LRP6 bind to Frizzled receptors, forming a complex that activates downstream signal transduction, thereby influencing various physiological and pathological processes. Given their critical roles in tumors, osteoporosis, and metabolic diseases, the development of specific nanobodies targeting LRP5 and / or LRP6 has significant scientific value and potential clinical applications.

[0003] LRP5 and LRP6 play crucial roles in the development and progression of various diseases. Studies have shown that aberrant activation of the Wnt signaling pathway is closely related to the occurrence, development, and prognosis of various cancers, such as colorectal cancer, liver cancer, and breast cancer. In the tumor microenvironment, upregulated expression of LRP5 and LRP6 receptors promotes sustained activation of Wnt signaling, thereby promoting tumor cell proliferation, invasion, and metastasis. Furthermore, mutations in the LRP5 and LRP6 genes are significantly associated with an increased incidence of osteoporosis and osteoporosis-related fractures. Therefore, therapeutic strategies targeting LRP5 and LRP6 hold promise for providing new insights into the treatment of various diseases.

[0004] Although many therapeutic strategies targeting the Wnt signaling pathway are under development, these therapies face challenges such as poor selectivity and significant side effects. For example, while some small molecule inhibitors can suppress the Wnt signaling pathway, their unclear targets often affect other important signaling pathways, leading to severe side effects. Compared to drugs that broadly inhibit the Wnt pathway, specific treatments targeting LRP5 and LRP6 can inhibit the activation of signaling pathways under specific pathological conditions while reducing the impact on normal cells, thus showing better clinical application prospects.

[0005] Although monoclonal antibodies (mAbs) have achieved some success in cancer immunotherapy, their large molecular weight, weak penetration, high immunogenicity, and high production cost limit their widespread application in targeted therapy. In contrast, nanobodies (VHHs) are the smallest functional antibody fragments found in camels, sharks, and other animals. They have advantages such as small molecular weight (approximately 1 / 10 the molecular weight of conventional IgG, ~12~15 kDa), high stability, strong penetration, low immunogenicity, and ease of engineering and production. Summary of the Invention

[0006] This invention utilizes phage screening technology to identify multiple LRP5 and / or LRP6-targeting nanobodies from a humanized synthetic phage library. This lays the foundation for advancing the understanding of the Wnt signaling pathway and its application in disease diagnosis and treatment, including but not limited to tumors, osteoporosis, and metabolic diseases. Furthermore, the nanobodies provided by this invention can be used in the assembly of Wnt functional substitute proteins (i.e., fusing elements that bind frizzled to LRP5 and / or LRP6, thereby reducing the distance between them to form a complex, thus replacing the Wnt protein and activating downstream Wnt signaling).

[0007] To better study LRP5 and / or LRP6, this invention provides a nanobody that specifically binds to LRP5 and / or LRP6 and its applications.

[0008] This invention uses the E3E4 region of the biotinylated antigen LRP6 as the antigen, and a synthetically produced humanized nanobody library as the basis. A multi-round panning process combined with magnetic bead sorting is employed to obtain nanobodies with high affinity for the antigen. Enzyme-linked immunosorbent assay (ELISA) is used to assess the enrichment level in each round of screening to evaluate the screening effect. After a certain enrichment level is achieved, phage monoclonal ELISA is performed to identify phages expressing nanobodies with affinity for the antigen. The identified nanobody nucleic acid sequences are transferred to a new expression vector, and periplasmic expression is used to maintain the disulfide bonds within the nanobodies and preserve their stability. Subsequently, affinity chromatography is used for purification to obtain humanized nanobodies with good purity. Biochemical identification was performed using bio-layer interferometry (BLI) to detect single-concentration binding and determine binding kinetic parameters. The relative thermal stability of the nanobodies was assessed using purified Nb and size exclusion chromatography (SEC), and TOP-flash was used to detect the activation of the Wnt / β-catenin signaling pathway.

[0009] In order to solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0010] A first aspect of the present invention provides a nanobody that specifically binds LRP5 and / or LRP6, the nanobody comprising a heavy chain variable region, characterized in that the heavy chain variable region comprises one of the following combinations of CDR1, CDR2 and CDR3:

[0011] (1) The amino acid sequence of CDR1 is shown in SEQ ID NO:56, the amino acid sequence of CDR2 is shown in SEQ ID NO:57, and the amino acid sequence of CDR3 is shown in SEQ ID NO:58; or

[0012] (2) The amino acid sequence of CDR1 is shown in SEQ ID NO:59, the amino acid sequence of CDR2 is shown in SEQ ID NO:60, and the amino acid sequence of CDR3 is shown in SEQ ID NO:61; or

[0013] (3) The amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; or

[0014] (4) The amino acid sequence of CDR1 is shown in SEQ ID NO:5, the amino acid sequence of CDR2 is shown in SEQ ID NO:6, and the amino acid sequence of CDR3 is shown in SEQ ID NO:7; or

[0015] (5) The amino acid sequence of CDR1 is shown in SEQ ID NO:8, the amino acid sequence of CDR2 is shown in SEQ ID NO:9, and the amino acid sequence of CDR3 is shown in SEQ ID NO:10; or

[0016] (6) The amino acid sequence of CDR1 is shown in SEQ ID NO:11, the amino acid sequence of CDR2 is shown in SEQ ID NO:12, and the amino acid sequence of CDR3 is shown in SEQ ID NO:13; or

[0017] (7) The amino acid sequence of CDR1 is shown in SEQ ID NO:14, the amino acid sequence of CDR2 is shown in SEQ ID NO:15, and the amino acid sequence of CDR3 is shown in SEQ ID NO:16; or

[0018] (8) The amino acid sequence of CDR1 is shown in SEQ ID NO:17, the amino acid sequence of CDR2 is shown in SEQ ID NO:18, and the amino acid sequence of CDR3 is shown in SEQ ID NO:19; or

[0019] (9) The amino acid sequence of CDR1 is shown in SEQ ID NO:20, the amino acid sequence of CDR2 is shown in SEQ ID NO:21, and the amino acid sequence of CDR3 is shown in SEQ ID NO:22; or

[0020] (10) The amino acid sequence of CDR1 is shown in SEQ ID NO:23, the amino acid sequence of CDR2 is shown in SEQ ID NO:24, and the amino acid sequence of CDR3 is shown in SEQ ID NO:25; or

[0021] (11) The amino acid sequence of CDR1 is shown in SEQ ID NO:26, the amino acid sequence of CDR2 is shown in SEQ ID NO:27, and the amino acid sequence of CDR3 is shown in SEQ ID NO:28; or

[0022] (12) The amino acid sequence of CDR1 is shown in SEQ ID NO:29, the amino acid sequence of CDR2 is shown in SEQ ID NO:30, and the amino acid sequence of CDR3 is shown in SEQ ID NO:31; or

[0023] (13) The amino acid sequence of CDR1 is shown in SEQ ID NO:32, the amino acid sequence of CDR2 is shown in SEQ ID NO:33, and the amino acid sequence of CDR3 is shown in SEQ ID NO:34; or

[0024] (14) The amino acid sequence of CDR1 is shown in SEQ ID NO:35, the amino acid sequence of CDR2 is shown in SEQ ID NO:36, and the amino acid sequence of CDR3 is shown in SEQ ID NO:37; or

[0025] (15) The amino acid sequence of CDR1 is shown in SEQ ID NO:38, the amino acid sequence of CDR2 is shown in SEQ ID NO:39, and the amino acid sequence of CDR3 is shown in SEQ ID NO:40; or

[0026] (16) The amino acid sequence of CDR1 is shown in SEQ ID NO:41, the amino acid sequence of CDR2 is shown in SEQ ID NO:42, and the amino acid sequence of CDR3 is shown in SEQ ID NO:43; or

[0027] (17) The amino acid sequence of CDR1 is shown in SEQ ID NO:44, the amino acid sequence of CDR2 is shown in SEQ ID NO:45, and the amino acid sequence of CDR3 is shown in SEQ ID NO:46; or

[0028] (18) The amino acid sequence of CDR1 is shown in SEQ ID NO:47, the amino acid sequence of CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of CDR3 is shown in SEQ ID NO:49; or

[0029] (19) The amino acid sequence of CDR1 is shown in SEQ ID NO:50, the amino acid sequence of CDR2 is shown in SEQ ID NO:51, and the amino acid sequence of CDR3 is shown in SEQ ID NO:52; or

[0030] (20) The amino acid sequence of CDR1 is shown in SEQ ID NO:53, the amino acid sequence of CDR2 is shown in SEQ ID NO:54, and the amino acid sequence of CDR3 is shown in SEQ ID NO:55; or

[0031] (21) The amino acid sequence of CDR1 is shown in SEQ ID NO:62, the amino acid sequence of CDR2 is shown in SEQ ID NO:63, and the amino acid sequence of CDR3 is shown in SEQ ID NO:64; or

[0032] (22) The amino acid sequence of CDR1 is shown in SEQ ID NO:65, the amino acid sequence of CDR2 is shown in SEQ ID NO:66, and the amino acid sequence of CDR3 is shown in SEQ ID NO:67; or

[0033] (23) The amino acid sequence of CDR1 is shown in SEQ ID NO:68, the amino acid sequence of CDR2 is shown in SEQ ID NO:69, and the amino acid sequence of CDR3 is shown in SEQ ID NO:70; or

[0034] (24) The amino acid sequence of CDR1 is shown in SEQ ID NO:71, the amino acid sequence of CDR2 is shown in SEQ ID NO:72, and the amino acid sequence of CDR3 is shown in SEQ ID NO:73; or

[0035] (25) The amino acid sequence of CDR1 is shown in SEQ ID NO:74, the amino acid sequence of CDR2 is shown in SEQ ID NO:75, and the amino acid sequence of CDR3 is shown in SEQ ID NO:76; or

[0036] (26) The amino acid sequence of CDR1 is shown in SEQ ID NO:77, the amino acid sequence of CDR2 is shown in SEQ ID NO:78, and the amino acid sequence of CDR3 is shown in SEQ ID NO:79; or

[0037] (27) The amino acid sequence of CDR1 is shown in SEQ ID NO:80, the amino acid sequence of CDR2 is shown in SEQ ID NO:81, and the amino acid sequence of CDR3 is shown in SEQ ID NO:82; or

[0038] (28) The amino acid sequence of CDR1 is shown in SEQ ID NO:83, the amino acid sequence of CDR2 is shown in SEQ ID NO:84, and the amino acid sequence of CDR3 is shown in SEQ ID NO:85; or

[0039] (29) The amino acid sequence of CDR1 is shown in SEQ ID NO:86, the amino acid sequence of CDR2 is shown in SEQ ID NO:87, and the amino acid sequence of CDR3 is shown in SEQ ID NO:88; or

[0040] (30) The amino acid sequence of CDR1 is shown in SEQ ID NO:89, the amino acid sequence of CDR2 is shown in SEQ ID NO:90, and the amino acid sequence of CDR3 is shown in SEQ ID NO:91.

[0041] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:110, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:110.

[0042] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:111, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:111.

[0043] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:92, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:92.

[0044] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:93, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:93.

[0045] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:94, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:94.

[0046] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:95, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:95.

[0047] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:96, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:96.

[0048] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:97, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:97.

[0049] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:98, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:98.

[0050] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:99, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:99.

[0051] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:100, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:100.

[0052] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:101, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:101.

[0053] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:102, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:102.

[0054] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:103, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:103.

[0055] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:104, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:104.

[0056] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:105, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:105.

[0057] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:106, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:106.

[0058] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:107, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:107.

[0059] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:108, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:108.

[0060] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:109, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:109.

[0061] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:112, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:112.

[0062] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:113, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:113.

[0063] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:114, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:114.

[0064] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:115, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:115.

[0065] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:116, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:116.

[0066] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:117, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:117.

[0067] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:118, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:118.

[0068] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:119, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:119.

[0069] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:120, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:120.

[0070] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:121, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:121. In some embodiments of the present invention, the nanobody binds to the E3E4 region of LRP6, the amino acid sequence of which is, for example, shown in SEQ ID NO:1.

[0071] A second aspect of the present invention provides a binding molecule that specifically binds to LRP5 and / or LRP6, said binding molecule comprising the nanobody as described in the first aspect of the present invention.

[0072] In some embodiments of the present invention, the binding molecule is a monovalent antibody, multivalent antibody, or fusion protein comprising one or more of the nanobodies; the fusion protein is preferably a multispecific antibody or a heavy chain antibody fused with Fc.

[0073] A third aspect of the present invention provides an isolated nucleic acid that encodes a nanobody as described in the first aspect of the present invention or a binding molecule as described in the second aspect of the present invention.

[0074] A fourth aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the third aspect of the present invention.

[0075] In some specific embodiments of the present invention, the backbone of the recombinant expression vector is pSB.

[0076] A fifth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the third aspect of the present invention, or a recombinant expression vector as described in the fourth aspect of the present invention.

[0077] In some embodiments of the present invention, the host of the transformant is a prokaryotic cell or a eukaryotic cell.

[0078] In some embodiments of the present invention, the prokaryotic cells are E. coli MC1061F. - strains.

[0079] A sixth aspect of the present invention provides a method for preparing nanobodies as described in the first aspect of the present invention or binding molecules as described in the second aspect of the present invention, the method comprising culturing transformants as described in the fifth aspect of the present invention to obtain the nanobodies or the binding molecules.

[0080] A seventh aspect of the present invention provides a pharmaceutical composition comprising a nanobody as described in the first aspect of the present invention or a binding molecule as described in the second aspect of the present invention, and optionally a pharmaceutically acceptable carrier and / or excipients.

[0081] An eighth aspect of the present invention provides a kit comprising one or more of the following: nanobody as described in the first aspect of the present invention, binding molecule as described in the second aspect of the present invention, and pharmaceutical composition as described in the seventh aspect of the present invention.

[0082] The ninth aspect of the present invention provides the use of one or more of the following in the detection of LRP5 and / or LRP6: nanobodies as described in the first aspect of the present invention, binding molecules as described in the second aspect of the present invention, pharmaceutical compositions as described in the seventh aspect of the present invention, or kits as described in the eighth aspect of the present invention, for non-diagnostic or therapeutic purposes.

[0083] The tenth aspect of the present invention provides a method for detecting LRP5 and / or LRP6, the method comprising contacting a sample with one or more of the following: a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, a pharmaceutical composition as described in the seventh aspect of the present invention, and a kit as described in the ninth aspect of the present invention, to detect whether the sample contains LRP5 and / or LRP6.

[0084] In some embodiments of the present invention, the method is for non-diagnostic purposes.

[0085] In some embodiments of the present invention, the method is based on Western Blot, ELISA, immunohistochemistry, immunocytochemistry or flow cytometry.

[0086] The eleventh aspect of the present invention provides the use of one or more of the following in the preparation of a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, a pharmaceutical composition as described in the seventh aspect of the present invention, and a kit as described in the eighth aspect of the present invention in the preparation of a detection or treatment for LRP5 and / or LRP6-related diseases.

[0087] In some embodiments of the present invention, the LRP5 and / or LRP6-related diseases are diseases related to the Wnt pathway.

[0088] In some embodiments of the present invention, the Wnt pathway-related diseases are diseases related to Wnt signaling deficiency.

[0089] In some embodiments of the present invention, one or two of the nanobody and the binding molecule are fused with the Fizzled protein in the application, thereby bringing one or two of LRP5 and LRP6 closer to the Fizzled protein to form a complex and activating the Wnt downstream signal to achieve the detection or treatment.

[0090] In some embodiments of the present invention, the LRP5 and / or LRP6-related diseases are Wnt pathway-related tumors, osteoporosis, or metabolic diseases.

[0091] 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.

[0092] The reagents and raw materials used in this invention are all commercially available.

[0093] The positive and progressive effects of this invention are as follows: This invention provides LRP5 and / or LRP6-targeting nanobodies, which have the ability to target and bind to LRP5 and / or LRP6. Some of these antibodies can inhibit the binding of LRP5 and / or LRP6 to Wnt proteins (thereby inhibiting the Wnt signaling pathway). For specific inhibitory nanobodies, they can be directly applied to inhibit the binding of LRP5 and / or LRP6 to Wnt proteins, reducing diseases caused by their mediated homeostasis dysregulation, including but not limited to tumors, osteoporosis, and metabolic diseases. For non-inhibitory nanobodies that specifically bind to LRP5 and / or LRP6, they can be combined with various existing therapeutic approaches for LRP5 and / or LRP6-targeted therapy, such as CAR-T and radiotherapy, which can both mediate targeting and potentially provide functional value. Regardless of inhibition, they can be combined with frizzled-targeting elements to form recombinant proteins and other molecules that substitute for Wnt function, thereby activating Wnt signaling. These antibodies can be applied in various forms in the fields of stem cell therapy and regeneration. Attached Figure Description

[0094] Figure 1 is a flowchart of phage screening and subsequent identification.

[0095] Figures 2A and 2B show the results of biochemical identification and kinetic analysis of representative nanobodies, including SDS-PAGE analysis, thermal stability analysis, and affinity identification results.

[0096] Figure 3 shows the affinity distribution after phage screening.

[0097] Figures 4A-4C show the results of the inhibition of Wnt3a-mediated Wnt signaling by representative LRP5 and / or LRP6 inhibitory nanobodies W1134 and W1135.

[0098] Figure 5 shows the SEC and SDS-PAGE analysis results for W1134 and W1135. Detailed Implementation

[0099] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0100] In this invention, the letters in the amino acid sequence represent abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): Alanine: Ala-A (where the letters before the hyphen are three-letter abbreviations of the amino acid, and the letters after the hyphen are single-letter abbreviations, the same below), Arginine: Arg-R, Aspartic acid: Asp-D, Cysteine: Cys-C, Glutamine: Gln-Q, Glutamic acid: Glu-E, Histidine: His-H, Glycine: Gly-G, Asparagine: Asn-N, Tyrosine: Tyr-Y, Proline: Pro-P, Serine: Ser-S, Methionine: Met-M, Lysine: Lys-K, Valine: Val-V, Isoleucine: Ile-I, Phenylalanine: Phe-F, Leucine: Leu-L, Tryptophan: Trp-W, Threonine: Thr-T. As will be known to those skilled in the art, in this invention, amino acids and amino acid residues can be used interchangeably.

[0101] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all shown according to the definition of the IMGT numbering rules. However, it is well known to those skilled in the art that 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 “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0102] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed by this invention is based on the sequence defined according to the IMGT numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0103] In this invention, "LRP5 and / or LRP6 binding molecules" are proteins that have the function of recognizing and binding LRP5 and / or LRP6, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, microbodies, affinity molecules, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0104] In this invention, the terms "single-domain antibody," "anti-LRP5 and / or LRP6 single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," "VHH," and "nanobody" are used interchangeably and all refer to nanobodies that specifically recognize and bind to LRP5 and / or LRP6. A nanobody is the variable region of a heavy chain antibody. Typically, a nanobody contains three CDRs and four FRs. A nanobody is the smallest functional antigen-binding fragment. Typically, an antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting of only one heavy chain variable region.

[0105] In this invention, a binding molecule containing two or more identical nanobodies is a multivalent nanobody; a binding molecule containing two or more nanobodies with different specificities is a multispecific nanobody. Multiple nanobodies are linked together by linkers to form multivalent or multispecific nanobodies. The linkers are typically composed of amino acids selected from G and S, such as (G4S). n n is a natural number less than 10, such as 2, 3, or 4.

[0106] In this invention, "multispecific antibody" refers to a class of antibodies that can simultaneously recognize and bind to at least two different antigenic epitopes, such as bispecific antibodies.

[0107] In this invention, a "fusion protein" refers to a protein formed by linking two or more different protein or polypeptide sequences together through genetic engineering. Specifically, the Fc fusion protein consists of two parts: the Fc segment of an immunoglobulin and the nanobody described in this invention. These two parts have relatively independent structural domains and functions, influencing their physicochemical properties and biological activities from different perspectives; and the two parts can be directly connected or linked through the linkers described herein.

[0108] Fc fusion protein dimerization can form heavy chain antibodies. Compared to full-length antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions (CH2 and CH3). The variable region is linked to the constant region via a hinge-like structure. The antigen-binding fragment of heavy chain antibodies includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

[0109] In this invention, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies.

[0110] In the art, conservative substitution with amino acids of similar or identical properties generally does not alter the function of a protein. This includes substituting amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Therefore, those skilled in the art can modify one or more amino acids in the sequence of the present invention to obtain variants of the antibody or its functional fragment sequence without substantially affecting antibody activity. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, or additions of one or more (typically less than 20, preferably less than 10, more preferably less than 5) amino acids at the C-terminus and / or N-terminus. All of these are considered to be included within the scope of protection of the present invention.

[0111] The variants of the various antibodies described in this invention include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the various antibodies of this invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the various antibodies of this invention.

[0112] In some embodiments, the sequence of the variants described in this invention may have at least 95%, 96%, 97%, 98%, or 99% identity with its source sequence. This sequence identity can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. This invention also includes molecules having a variable region of the antibody heavy chain with a CDR, provided that its CDR has at least 90% (preferably at least 95%, most preferably at least 98%) identity with the CDR identified herein.

[0113] The nanobodies and heavy chain antibodies of the present invention can be prepared using methods conventional in the art, such as phage display technology well known in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the various antibodies of the present invention. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulating polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. In particular, preferred cell lines are selected by identifying which cell lines have high expression levels and produce antibodies with basic LRP5 and / or LRP6 binding properties.

[0114] In this invention, "nucleic acid" refers to a nucleotide chain of any length. A nucleotide can be a deoxyribonucleotide (DNA), a ribonucleotide (RNA), a modified nucleotide or base, and / or its analogues, or any substrate that can be incorporated into the chain by a DNA or RNA polymerase.

[0115] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring nucleotide linkages, or both. Modified or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0116] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0117] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant cell, animal cell, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan. The bacteria may be *Escherichia coli*, the fungus may be a yeast such as *Pichia pastoris*, and the animal cell may be a mammalian cell such as CHO / NSO.

[0118] In this invention, the "non-diagnostic purpose" application scenarios include, but are not limited to: for example, detecting the presence of antigens (containing LRP5 and / or LRP6 proteins) in vitro in the laboratory; or using them as positive antibodies to screen other antibodies targeting LRP5 and / or LRP6; or competing with other antibodies targeting LRP5 and / or LRP6 to detect whether there is competition between the antibodies, i.e., whether the antigen epitopes are the same or similar, etc.

[0119] 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.

[0120] Example 1: Screening, Expression, and Purification of Nanobodies

[0121] Four rounds of phage display experiments were conducted. First, the purified phage (Homemade) was mixed with magnetic beads (Invitrogen Dynabeads). TMM-280 streptavidin (Cat#11206D) was incubated at room temperature for 0.5 h to remove the portion capable of binding to the magnetic beads. Next, the remaining phages were incubated with the E3E4 region of the biotinylated antigen LRP6 (Uniprot ID: O75581) (the preparation of the target protein and the experimental procedures for biotinylation are described in Examples 1 and 3 of WO2023184280A1). Subsequently, another set of identical magnetic beads was used to bind the phage and antigen mixture at room temperature, followed by a washing step to remove non-specifically bound components. The bound phages were eluted with 0.2 M glycine solution (pH 3), with Tris-HCl solution being rapidly added to adjust the pH to neutral. The selected phages were then amplified in vivo and purified in vitro before entering a new round of display. After each round of screening, the enrichment level of the phages was detected by Poly-ELISA. The monoclonal phages selected in the last round were subjected to ELISA and sequencing to obtain the gene sequence of nanobody that can specifically bind to the antigen.

[0122] The amino acid sequence of LRP6 in the E3E4 region is as follows:

[0123] The selected nanobody genes were cloned into the expression vector pSb(addgen Cat#:153525) and transformed into E. coli MC1061F. - Expression was performed in (Cat#:DL1095). Bacterial cells were resuspended in TES solution (0.5 mM EDTA, 0.5 M sucrose, 0.2 M Tris-HCl pH 8.0) and incubated at 150 rpm for 30 minutes, followed by the addition of deionized water and further rotation. The supernatant collected by centrifugation was bound to a Ni-NTA affinity column and washed with a buffer containing 30 mM imidazole (150 mM NaCl, 20 mM Tris-HCl pH 8.0) to remove non-specifically bound proteins. Finally, the target protein was eluted with a buffer containing 300 mM imidazole to obtain purified nanobodies. The specific screening process is shown in Figure 1. The CDR sequences of the screened nanobodies are shown in Table 1, and the full-length sequences are shown in Table 2.

[0124] Table 1. CDR sequences of nanobodies

[0125] The above-mentioned nanobody is a heavy chain variable region constructed by combining the following FR1-FR4 framework regions on the basis of CDR.

[0126] FR1:AVQLQASGGGFVQPGGSLRLSCAASG(SEQ ID NO:122)

[0127] FR2:MGWFRQAPGKEREFVSAI(SEQ ID NO:123)

[0128] FR3:YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA(SEQ ID NO:124)

[0129] FR4:YWGQGTQVTVSS(SEQ ID NO:125)

[0130] Table 2 Full-length sequence of nanobodies

[0131] Example 2: Binding kinetics determination using the BLI method

[0132] The affinity of nanobodies for the E3E4 region of the LRP6 extracellular domain was detected using biofilm layer optical interferometry (BLI) (instrument: Octet RED96). First, the nanobodies were diluted to 500 nM with PBST buffer (containing 0.05% (v / v) Tween-20, pH 7.4). Then, the biotin-labeled LRP6 extracellular domain E3E4 was immobilized on streptavidin sensors (Sartorius, catalog number 18-5019), and these sensors were immersed in a solution containing 500 nM of nanobodies to measure their binding kinetics with the antigen. The binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD = koff / kon) were used to describe the strength of the interaction between the antibody and the antigen. As shown in Figure 3, most of the nanobodies obtained after phage screening had affinity Kd values ​​less than 100 nM. The affinity of some of these antibodies is shown in Table 3 below.

[0133] Table 3 Affinity data for nanobodies

[0134] "*" > 100nM, "**" < 100nM

[0135] Example 3: Evaluation of the relative thermal stability and purity of nanobodies

[0136] The purified Nb was evaluated for relative thermal stability of the nanobodies using size exclusion chromatography (SEC). The specific method was as follows: First, the nanobodies in PBS were divided in two. One portion was heated at 50°C for 20 min, and the other portion was placed at 4°C. Both portions were then centrifuged (21000g, 30 min), and the supernatants were collected. The protein supernatants before and after heating were analyzed by SEC using a fluorescence detector (excitation wavelength 280 nm, emission wavelength 350 nm), and the relative fluorescence intensity (RFU) of the target peak was recorded. The peak height of the target protein was observed, and the relative thermal stability of the nanobodies was assessed by calculating the ratio of the peak intensity after heating to the peak intensity before heating. The closer the ratio was to 1, the better the thermal stability of the nanobodies. Specific results are shown in Figures 2A and 2B.

[0137] The purified Nb (W1134, W1135) was subjected to SEC analysis to assess the homogeneity and purity of the protein. The specific method is as follows: PBS was used as the mobile phase, and 1 mL of sample was injected into a molecular sieve column (Superdex 75 Increase 10 / 300GL) at a flow rate of 0.7 mL / min. The elution peak of the protein was detected at 280 nm using a UV detector, and the absorbance value (mAU) was recorded. The specific results are shown in Figure 5.

[0138] Example 4 TOP-flash

[0139] In a 24-well plate, 500 μL of culture medium (DMEM containing 10% FBS and 1% PS; FBS purchased from Shuangru Biotechnology, PS purchased from Shenger Biotechnology) was added to each well, and approximately 1 × 10^5 HEK293T cells were seeded per well. After 24 hours, Lipofectamine was used to... TMTransfection was performed using 3000 transfection reagents (0.75 μL per well). The plasmids used for transfection included 7×TCL / LEF-firefly luciferase reporter gene plasmid (product name: M50 Super 8×TOPFlash; supplier: Addgene Plasmid; catalog number: #12456) and Renilla luciferase reporter gene plasmid (product name: pRL Renilla Luciferase Control Reporter Vectors; supplier: Promega; catalog number: #E2261). After 24 hours, the corresponding nanobody concentrations (5.5 μM, 3-fold serial dilution or 8.5 μM, 3-fold serial dilution) were added, and the control group received an irrelevant antibody (home-made, no binding Nb). After 2 hours, Wnt3a conditioned medium (L cell secreted, ATCC: L Wnt-3A-CRL-2647) was added. After culturing for another 22 hours, the wells were removed, the medium was discarded, and the cells were washed with PBS. Subsequently, the cells were... Cells were treated with lysis buffer and luminescent reagents provided by the Reporter Assay System (supplier: Promega), and the signal intensities of Firefly and Renilla luciferases were detected and compared.

[0140] In this study, the cells were also replaced with the LRP5 and / or LRP6 double knockout cell line HEK293T, which was then called LRPs-KO cells. If this cell line was used, it was also transfected with LRP5 or LRP6 separately to identify the inhibitory effect of the nanobodies on LRP5 or LRP6 individually.

[0141] Figures 4A-4C show that with increasing W1134 concentration, the RLU value of the experimental group (red curve) was significantly lower than that of the control group (green curve), indicating that W1134 has a concentration-dependent inhibitory effect on the Wnt signaling pathway. Furthermore, the W1134 nanobody showed a strong inhibitory effect on LRP6, but a weaker effect on LRP5. With increasing W1135 concentration, the RLU value of the experimental group (red curve) was significantly lower than that of the control group (green curve), indicating that W1135 has a concentration-dependent inhibitory effect on the Wnt signaling pathway. However, the W1135 nanobody did not show a concentration-dependent inhibitory effect on LRP5, but it did have a certain degree of specific inhibitory effect on LRP6.

[0142] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A nanobody that specifically binds to LRP5 and / or LRP6, said nanobody comprising a heavy chain variable region, characterized in that, The heavy chain variable region includes one of the following combinations of CDR1, CDR2 and CDR3: (1) The amino acid sequence of CDR1 is shown in SEQ ID NO:56, the amino acid sequence of CDR2 is shown in SEQ ID NO:57, and the amino acid sequence of CDR3 is shown in SEQ ID NO:58; or (2) The amino acid sequence of CDR1 is shown in SEQ ID NO:59, the amino acid sequence of CDR2 is shown in SEQ ID NO:60, and the amino acid sequence of CDR3 is shown in SEQ ID NO:61; or (3) The amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; or (4) The amino acid sequence of CDR1 is shown in SEQ ID NO:5, the amino acid sequence of CDR2 is shown in SEQ ID NO:6, and the amino acid sequence of CDR3 is shown in SEQ ID NO:7; or (5) The amino acid sequence of CDR1 is shown in SEQ ID NO:8, the amino acid sequence of CDR2 is shown in SEQ ID NO:9, and the amino acid sequence of CDR3 is shown in SEQ ID NO:10; or (6) The amino acid sequence of CDR1 is shown in SEQ ID NO:11, the amino acid sequence of CDR2 is shown in SEQ ID NO:12, and the amino acid sequence of CDR3 is shown in SEQ ID NO:13; or (7) The amino acid sequence of CDR1 is shown in SEQ ID NO:14, the amino acid sequence of CDR2 is shown in SEQ ID NO:15, and the amino acid sequence of CDR3 is shown in SEQ ID NO:16; or (8) The amino acid sequence of CDR1 is shown in SEQ ID NO:17, the amino acid sequence of CDR2 is shown in SEQ ID NO:18, and the amino acid sequence of CDR3 is shown in SEQ ID NO:19; or (9) The amino acid sequence of CDR1 is shown in SEQ ID NO:20, the amino acid sequence of CDR2 is shown in SEQ ID NO:21, and the amino acid sequence of CDR3 is shown in SEQ ID NO:22; or (10) The amino acid sequence of CDR1 is shown in SEQ ID NO:23, the amino acid sequence of CDR2 is shown in SEQ ID NO:24, and the amino acid sequence of CDR3 is shown in SEQ ID NO:25; or (11) The amino acid sequence of CDR1 is shown in SEQ ID NO:26, the amino acid sequence of CDR2 is shown in SEQ ID NO:27, and the amino acid sequence of CDR3 is shown in SEQ ID NO:28; or (12) The amino acid sequence of CDR1 is shown in SEQ ID NO:29, the amino acid sequence of CDR2 is shown in SEQ ID NO:30, and the amino acid sequence of CDR3 is shown in SEQ ID NO:31; or (13) The amino acid sequence of CDR1 is shown in SEQ ID NO:32, the amino acid sequence of CDR2 is shown in SEQ ID NO:33, and the amino acid sequence of CDR3 is shown in SEQ ID NO:34; or (14) The amino acid sequence of CDR1 is shown in SEQ ID NO:35, the amino acid sequence of CDR2 is shown in SEQ ID NO:36, and the amino acid sequence of CDR3 is shown in SEQ ID NO:37; or (15) The amino acid sequence of CDR1 is shown in SEQ ID NO:38, the amino acid sequence of CDR2 is shown in SEQ ID NO:39, and the amino acid sequence of CDR3 is shown in SEQ ID NO:40; or (16) The amino acid sequence of CDR1 is shown in SEQ ID NO:41, the amino acid sequence of CDR2 is shown in SEQ ID NO:42, and the amino acid sequence of CDR3 is shown in SEQ ID NO:43; or (17) The amino acid sequence of CDR1 is shown in SEQ ID NO:44, the amino acid sequence of CDR2 is shown in SEQ ID NO:45, and the amino acid sequence of CDR3 is shown in SEQ ID NO:46; or (18) The amino acid sequence of CDR1 is shown in SEQ ID NO:47, the amino acid sequence of CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of CDR3 is shown in SEQ ID NO:49; or (19) The amino acid sequence of CDR1 is shown in SEQ ID NO:50, the amino acid sequence of CDR2 is shown in SEQ ID NO:51, and the amino acid sequence of CDR3 is shown in SEQ ID NO:52; or (20) The amino acid sequence of CDR1 is shown in SEQ ID NO:53, the amino acid sequence of CDR2 is shown in SEQ ID NO:54, and the amino acid sequence of CDR3 is shown in SEQ ID NO:55; or (21) The amino acid sequence of CDR1 is shown in SEQ ID NO:62, the amino acid sequence of CDR2 is shown in SEQ ID NO:63, and the amino acid sequence of CDR3 is shown in SEQ ID NO:64; or (22) The amino acid sequence of CDR1 is shown in SEQ ID NO:65, the amino acid sequence of CDR2 is shown in SEQ ID NO:66, and the amino acid sequence of CDR3 is shown in SEQ ID NO:67; or (23) The amino acid sequence of CDR1 is shown in SEQ ID NO:68, the amino acid sequence of CDR2 is shown in SEQ ID NO:69, and the amino acid sequence of CDR3 is shown in SEQ ID NO:70; or (24) The amino acid sequence of CDR1 is shown in SEQ ID NO:71, the amino acid sequence of CDR2 is shown in SEQ ID NO:72, and the amino acid sequence of CDR3 is shown in SEQ ID NO:73; or (25) The amino acid sequence of CDR1 is shown in SEQ ID NO:74, the amino acid sequence of CDR2 is shown in SEQ ID NO:75, and the amino acid sequence of CDR3 is shown in SEQ ID NO:76; or (26) The amino acid sequence of CDR1 is shown in SEQ ID NO:77, the amino acid sequence of CDR2 is shown in SEQ ID NO:78, and the amino acid sequence of CDR3 is shown in SEQ ID NO:79; or (27) The amino acid sequence of CDR1 is shown in SEQ ID NO:80, the amino acid sequence of CDR2 is shown in SEQ ID NO:81, and the amino acid sequence of CDR3 is shown in SEQ ID NO:82; or (28) The amino acid sequence of CDR1 is shown in SEQ ID NO:83, the amino acid sequence of CDR2 is shown in SEQ ID NO:84, and the amino acid sequence of CDR3 is shown in SEQ ID NO:85; or (29) The amino acid sequence of CDR1 is shown in SEQ ID NO:86, the amino acid sequence of CDR2 is shown in SEQ ID NO:87, and the amino acid sequence of CDR3 is shown in SEQ ID NO:88; or (30) The amino acid sequence of CDR1 is shown in SEQ ID NO:89, the amino acid sequence of CDR2 is shown in SEQ ID NO:90, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

91.

2. The nanobody as described in claim 1, characterized in that, The heavy chain variable region includes an amino acid sequence as shown in any of SEQ ID NO:110, 111, 92-109 and 112-121, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any of SEQ ID NO:110, 111, 92-109 and 112-121.

3. A binding molecule that specifically binds to LRP5 and / or LRP6, characterized in that, The binding molecule comprises the nanobody as described in claim 1 or 2; preferably, the binding molecule is a monovalent antibody, multivalent antibody, or fusion protein comprising one or more of the nanobodies; the fusion protein is preferably a multispecific antibody or a heavy chain antibody fused with an Fc.

4. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the nanobody as described in claim 1 or 2, or the binding molecule as described in claim 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 4; preferably, the backbone of the recombinant expression vector is pSB.

6. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 4, or the recombinant expression vector as described in claim 5; preferably, the host of the transformant is a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell is E. coli MC1061F. - strains.

7. A method for preparing the nanobody as described in claim 1 or 2 or the binding molecule as described in claim 3, characterized in that, The method comprises culturing the transformant as described in claim 6 to obtain the nanobody or the binding molecule.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nanobody as described in claim 1 or 2 or a binding molecule as described in claim 3, and optionally a pharmaceutically acceptable carrier and / or excipients.

9. A reagent kit, characterized in that, The kit comprises one or more of the nanobody as described in claim 1 or 2, the binding molecule as described in claim 3, and the pharmaceutical composition as described in claim 8.

10. The use of one or more of the nanobody of claim 1 or 2, the binding molecule of claim 3, the pharmaceutical composition of claim 8, and the kit of claim 9 in the detection of LRP5 and / or LRP6, wherein the use is for non-diagnostic or therapeutic purposes.

11. A method for detecting LRP5 and / or LRP6, characterized in that, The detection method includes contacting a sample with one or more of the following: the nanobody as described in claim 1 or 2, the binding molecule as described in claim 3, the drug combination as described in claim 8, and the kit as described in claim 9, to detect whether the sample contains LRP5 and / or LRP6. Preferably, the detection method is for non-diagnostic purposes; and / or, the detection method is based on Western Blot, ELISA, immunohistochemistry, immunocytochemistry, or flow cytometry.

12. The use of one or more of the nanobody as described in claim 1 or 2, the binding molecule as described in claim 3, the pharmaceutical composition as described in claim 8, and the kit as described in claim 9 in the preparation of a treatment for LRP5 and / or LRP6-related diseases; Preferably, the LRP5 and / or LRP6-related diseases are Wnt pathway-related diseases; more preferably, the Wnt pathway-related diseases are Wnt signaling deficiency-related diseases. More preferably, in the application, one or two of the nanobody and the binding molecule are fused with the Fizzled protein, thereby bringing one or two of LRP5 and LRP6 closer to the Fizzled protein to form a complex and activating the Wnt downstream signal to achieve the detection or treatment; More preferably, the LRP5 and / or LRP6-related diseases are Wnt pathway-related tumors, osteoporosis, or metabolic diseases.

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