Nanometer antibody NB21 targeting LGR4 and application of nanometer antibody NB21 in anti-obesity treatment

Abstract

The invention provides a nano antibody NB21 targeting LGR4 and an application of the nano antibody NB21 in anti-obesity treatment. Specifically, the invention discloses a nano antibody NB21 targeting LGR4 and a derivative protein thereof, a gene sequence for coding the nano antibody NB21 and the derivative protein thereof, and an expression vector and an expression system for producing the nano antibody NB21 and the derivative protein thereof. Besides, in-vitro and in-vivo experiments verify that the nano antibody NB21 for targeted blocking of the LGR4 has high affinity and high specificity, plays the biological functions of promoting heat production and reducing fat, and provides a new strategy for obesity treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. More specifically, the present invention relates to a nanobody NB21 targeting LGR4 and its application in anti-obesity treatment. Background Art

[0002] Compared with traditional antibodies, nanobodies have multiple advantages:

[0003] (1) Smaller in size, only one-tenth of ordinary antibodies, with stronger penetration in animal tissues. It can pass through the human brain tissue and reach the interior of tumors with high density, thus treating certain tumors or brain diseases.

[0004] (2) Stable in efficacy. The time for nanobodies not to be naturally decomposed in vivo is longer than that of ordinary antibodies, which means a longer drug efficacy time. Moreover, it has a wider temperature adaptation range and can still function at a temperature as high as 90°C, while traditional antibodies will be inactivated under such conditions. It is also very stable even under extreme pH values and remains effective even when passing through the human stomach.

[0005] (3) Good antigen specificity and easy to genetically modify; Nanobodies can recognize unique antigenic structural epitopes, have a wider antigen-binding ability than ordinary antibodies, and are convenient for artificial modification to obtain antibodies against different pathogens; Nanobodies can be easily synthesized in microorganisms and can be highly expressed in microorganisms such as phages, Escherichia coli, and yeasts, which is easy for large-scale production.

[0006] Due to the increasing incidence of obesity, various obesity complications such as diabetes, cardiovascular diseases, and cancers have gradually increased. From lifestyle interventions to weight loss surgeries, various methods have been used to prevent and manage obesity, but with low effectiveness or high risks. Discovering new targets is of great significance for weight loss treatment. Currently, G protein-coupled receptors (GPCRs) are the direction of weight loss drug development. For example, currently marketed GLP-1R agonists, GIPR agonists, and GCGR agonists all target GPCRs.

[0007] In 2013, in the Genetics of Obesity in Chinese Youth (GOCY) cohort, a functional activating LGR4 mutation (A750T), also known as GPR48, was first discovered in the leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4), significantly increasing the risk of obesity. Almost simultaneously, studies in Iceland showed that more than 300 people carried a rare loss-of-function LGR4 mutation (p.R126X), showing weight loss. Animal studies have shown that knocking out the LGR4 gene increases energy consumption by promoting the transformation of preadipocytes from white adipocytes to beige adipocytes, and can alleviate obesity caused by high-fat diet and genetic defects. LGR4 is a new member of the GPCR superfamily and plays an important role in maintaining endocrine and metabolic homeostasis. These findings and views have been widely recognized. Genetic and biological evidence suggests that LGR4 may be a potential target for obesity treatment.

[0008] However, it is generally believed that developing small molecule antagonists targeting the extracellular domain (ECD) of LGR4 is challenging because there are two overly smooth curved β-sheets on its concave surface, lacking a drug-binding pocket.

[0009] Therefore, there is an urgent need to develop a safer, more efficient, and easier-to-prepare nanobody against LGR4. Summary of the Invention

[0010] The object of the present invention is to provide a safer, more efficient, and easier-to-prepare nanobody against LGR4.

[0011] Another object of the present invention is to provide the application of the nanobody targeting LGR4, especially its anti-obesity effect.

[0012] In the first aspect of the present invention, a nanobody targeting LGR4 is provided, and the complementarity-determining regions (CDRs) of the VHH chain of the nanobody are selected from the following group:

[0013] The variable region contains any one or more of the CDR1, CDR2, or CDR3 sequences:

[0014] The CDR1 has the amino acid sequence shown in SEQ ID No.1;

[0015] The CDR2 has the amino acid sequence shown in SEQ ID No.2;

[0016] The CDR3 has the amino acid sequence shown in SEQ ID No.3;

[0017] The variable region is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in any one or more of the CDR1, CDR2, and CDR3 sequences, and is an amino acid sequence having the same function as any one or more of the amino acid sequences in CDR1, CDR2, or CDR3.

[0018] In another preferred embodiment, the CDR region of the nanobody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% (such as 96%, 97%, 98% or even more preferably at least 99%) sequence identity with any one of SEQ ID NO: 1-3.

[0019] In another preferred embodiment, the amino acid sequence of the CDR region of the nanobody VHH chain comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NO: 1-3.

[0020] In another preferred embodiment, the nanobody is capable of specifically binding to LGR4.

[0021] In another preferred embodiment, the nanobody is capable of specifically binding to LGR4 and blocking its interaction with RSPO1 / 2.

[0022] In another preferred embodiment, the nanobody is capable of inhibiting the activation of the Wnt signaling pathway.

[0023] In another preferred embodiment, the nanobody is capable of enhancing mitochondrial respiration and thermogenesis in brown adipocytes.

[0024] In another preferred embodiment, the LGR4 is human or non-human mammalian LGR4.

[0025] In another preferred embodiment, the LGR4 is human, mouse or rat LGR4.

[0026] In another preferred embodiment, the variable region has the amino acid sequence shown in SEQ ID No. 7;

[0027] Or an amino acid sequence obtained by substituting, deleting or adding one or more (e.g., 2, 3, 4 or 5) amino acids to the amino acid sequence as described in SEQ ID No. 7.

[0028] In a second aspect of the present invention, there is provided an antibody targeting LGR4, the antibody targeting LGR4 comprising one or more nanobodies targeting LGR4 as described in the first aspect of the present invention.

[0029] In another preferred embodiment, the antibody targeting LGR4 can be a monomer, a bivalent antibody or a multivalent antibody.

[0030] In another preferred embodiment, the antibody targeting LGR4 includes a monospecific antibody, a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0031] In a third aspect of the present invention, there is provided a polynucleotide encoding the nanobody targeting LGR4 as described in the first aspect of the present invention or the antibody targeting LGR4 as described in the second aspect of the present invention.

[0032] In another preferred embodiment, the polynucleotide includes RNA, DNA, or cDNA.

[0033] The nucleic acid molecule has:

[0034] (i) a nucleotide sequence as shown in SEQ ID No. 8; or

[0035] (ii) a complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID No. 8; or

[0036] (iii) a nucleotide sequence that encodes the same protein as the nucleotide sequence of (i) or (ii), but is different from the nucleotide sequence of (i) or (ii) due to the degeneracy of the genetic code; or

[0037] (iv) a nucleotide sequence obtained by substituting, deleting, or adding one or two nucleotide sequences to the nucleotide sequence shown in (i), (ii), or (iii), and having the same or similar function as the nucleotide sequence shown in (i), (ii), or (iii).

[0038] In a fourth aspect of the present invention, there is provided an expression vector containing the polynucleotide as described in the third aspect of the present invention.

[0039] In another preferred embodiment, the vector is selected from the group consisting of: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0040] In a fifth aspect of the present invention, there is provided a host cell containing the expression vector as described in the fourth aspect of the present invention, or having the polynucleotide as described in the third aspect of the present invention integrated into its genome.

[0041] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0042] In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, yeast cells, and mammalian cells.

[0043] In another preferred example, the mammalian cells include (but are not limited to) HEK293F cells and CHO cells.

[0044] In a sixth aspect of the present invention, there is provided an immunoconjugate, which contains:

[0045] (a) a nanobody targeting LGR4 as described in the first aspect of the present invention or an antibody targeting LGR4 as described in the second aspect of the present invention; and

[0046] (b) a conjugate moiety selected from the group consisting of: fluorescein, small molecule compound, PEG, radioisotope, contrast agent, fatty acid chain, protein fragment, or a combination thereof.

[0047] In another preferred example, the components (a) and (b) are operably linked.

[0048] In another preferred example, the conjugate moiety is a chemical label and a biological label.

[0049] In another preferred example, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0050] In another preferred example, the biological label is biotin, avidin, or an enzyme label.

[0051] In another preferred example, the small molecule compound includes (but is not limited to) drugs or toxins with definite or potential therapeutic or adjuvant therapeutic effects on tumors, autoimmune diseases, etc.

[0052] In another preferred example, the radioisotope includes:

[0053] (i) diagnostic isotopes, the diagnostic isotopes are selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0054] (ii) Therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0055] In another preferred embodiment, the radioactive isotopes include, but are not limited to, iodine-131, indium-111, and lutetium-177.

[0056] In another preferred embodiment, the contrast agent is for MRI or CT.

[0057] In another preferred embodiment, the protein fragment includes, but is not limited to, antibody Fc, biotin, avidin, HRP, antibody, enzyme, cytokine, and other bioactive proteins or polypeptides.

[0058] In another preferred embodiment, the coupling moiety is a detectable label.

[0059] In another preferred embodiment, the coupling moiety is selected from the group consisting of fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles that can produce detectable products.

[0060] In a seventh aspect of the present invention, there is provided a fusion protein comprising:

[0061] (i) A nanobody targeting LGR4 as described in the first aspect of the present invention, or an antibody targeting LGR4 as described in the second aspect of the present invention;

[0062] (ii) Optionally, a polypeptide molecule or fragment having a therapeutic function.

[0063] In another preferred embodiment, the polypeptide molecule or fragment with therapeutic function includes but is not limited to: polypeptide molecules or fragments targeting PD-1, LGR4, IL-4R, IL-4Rα, TNF-α, VEGF, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, Trop2, or TIGIT.

[0064] In another preferred embodiment, the polypeptide molecule or fragment with therapeutic function includes but is not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, enteropeptide analog, albumin, antibody fragment, cytokine.

[0065] In another preferred embodiment, the polypeptide molecule or fragment with therapeutic function includes single-chain antibody (scFv), diabody, monoclonal antibody, or chimeric antibody.

[0066] In another preferred embodiment, the fusion protein further contains a tag sequence for assisting expression and / or purification.

[0067] In another preferred embodiment, the tag sequence is selected from the group consisting of: 6His tag, GGGS sequence, FLAG tag.

[0068] In another preferred embodiment, the fusion protein further contains a part selected from the group consisting of: histidine His tag, human influenza hemagglutinin HA tag, FLAG tag, Myc tag, green fluorescent protein, alkaline phosphatase, horseradish peroxidase, luciferase, maltose-binding protein, glutathione transferase, toxin protein, and antibody Fc fragment.

[0069] In another preferred embodiment, the fusion protein includes bispecific antibody, chimeric antibody.

[0070] In the eighth aspect of the present invention, there is provided a pharmaceutical composition, which comprises:

[0071] (ii) a nanobody targeting LGR4 as described in the first aspect of the present invention, an antibody targeting LGR4 as described in the second aspect of the present invention, an immunoconjugate as described in the sixth aspect of the present invention, or a fusion protein as described in the seventh aspect of the present invention;

[0072] (ii) a pharmaceutically acceptable carrier.

[0073] In the ninth aspect of the present invention, there is provided a nanobody complex, which comprises:

[0074] (a) a nanobody targeting LGR4 as described in the first aspect of the present invention, or an antibody targeting LGR4 as described in the second aspect of the present invention; and

[0075] (b) A modifier non-covalently bound to the nanobody, and the modifier is selected from the group consisting of colloidal gold, colloidal silver, or colloidal carbon.

[0076] In the tenth aspect of the present invention, there is provided a use of an active ingredient, and the active ingredient is selected from the group consisting of: the nanobody targeting LGR4 as described in the first aspect of the present invention, the antibody targeting LGR4 as described in the second aspect of the present invention, the immunoconjugate as described in the sixth aspect of the present invention, or the fusion protein as described in the seventh aspect of the present invention, or a combination thereof, (a) for preparing a drug for treating obesity and its related diseases; and / or (b) for preparing a reagent, an affinity medium product, a detection plate, or a kit for detecting LGR4.

[0077] In another preferred example, the obesity is selected from the group consisting of: simple obesity, obesity syndrome, secondary obesity

[0078] In another preferred example, the simple obesity is selected from the group consisting of: hyperphagic obesity, absorptive obesity, and hypometabolic obesity.

[0079] In another preferred example, the obesity syndrome is selected from the group consisting of: Prader Willi syndrome, Bardet-Biedl syndrome, Alstrom syndrome, etc.

[0080] In another preferred example, the reagent is used to detect LGR4 or its fragment in a sample.

[0081] In another preferred example, the detection type includes, but is not limited to, flow cytometry detection, cellular immunofluorescence detection, enzyme-linked immunosorbent assay, and immunoblotting detection.

[0082] In another preferred example, the affinity medium product includes modified polymer microspheres, magnetic microspheres, agarose, dextran, cellulose, and filter membranes.

[0083] In another preferred example, the kit includes an immunochromatography kit, an enzyme-linked immunosorbent assay kit, an immunoturbidimetry kit, and chemical, electrochemical, and bioluminescence kits.

[0084] In the eleventh aspect of the present invention, there is provided a method for in vitro detecting LGR4 or its fragment in a sample, and the method includes the steps of:

[0085] (1) In vitro, contacting the sample with the nanobody targeting LGR4 as described in the first aspect of the present invention, the antibody targeting LGR4 as described in the second aspect of the present invention, or the immunoconjugate as described in the sixth aspect of the present invention;

[0086] (2) Detect whether an antigen-antibody complex is formed, and the formation of the complex indicates the presence of LGR4 or its fragment in the sample.

[0087] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0088] In a thirteenth aspect of the present invention, there is provided a kit, which comprises:

[0089] (1) A first container containing the nanobody targeting LGR4 according to the first aspect of the present invention or the antibody targeting LGR4 according to the second aspect of the present invention; and / or

[0090] (2) A second container containing a secondary antibody against the nanobody targeting LGR4 according to the first aspect of the present invention or the antibody targeting LGR4 according to the second aspect of the present invention.

[0091] In a fourteenth aspect of the present invention, there is provided a method for preparing a recombinant polypeptide, which is characterized in that the method comprises:

[0092] (a) Culturing the host cell according to the fifth aspect of the present invention under suitable expression conditions;

[0093] (b) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the nanobody targeting LGR4 according to the first aspect of the present invention, the antibody targeting LGR4 according to the second aspect of the present invention, or the fusion protein according to the seventh aspect of the present invention.

[0094] In a fifteenth aspect of the present invention, there is provided a method for anti-obesity treatment, which is characterized in that the method comprises: administering to a subject in need the nanobody targeting LGR4 according to the first aspect of the present invention, the antibody targeting LGR4 according to the second aspect of the present invention, the immunoconjugate according to the sixth aspect of the present invention, the fusion protein according to the seventh aspect of the present invention, the pharmaceutical composition according to the eighth aspect of the present invention, or a combination thereof.

[0095] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to the subject in need for combination treatment.

[0096] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1Shows the generation and characterization of the nanobody NB21: a. NB21 showed significant inhibitory effect on LGR4 in the TOPFlash assay; b. NB21 showed high affinity for human LGR4-ECD; c-d. The epitope of NB21 overlapped with the LGR4-ECD binding sites of RSPO1 (c) and RSPO2 (FU) (d).

[0098] Figure 2 Shows the cryo-EM structure of LGR4 and its complex with RSPO2 (FU): a. Structure of the LGR4-MB52 complex, showing the cryo-EM map (left) and atomic model (right); b. Structure of the RSPO2 (FU)-LGR4-MB52 complex, showing the cryo-EM map (left) and atomic model (right); c. Interface between LGR4 and RSPO2 (FU); d. Cryo-EM density map of the interaction interface between LGR4 and RSPO2 (FU). The interacting residues are shown as sticks. LGR4 is in cyan; RSPO2 (FU) is in purple; MB52 is in gray.

[0099] Figure 3 Shows the cryo-EM structure and analysis of the LGR4-NB21 complex: a. Structure of the NB21-LGR4 complex, showing the cryo-EM map (left) and atomic model (right), with NB21 shown in pink; b. Interface between LGR4 and NB21; c. Cryo-EM density map of the interaction interface between LGR4 and NB21; d. Superposition of LGR4-ECD in the LGR4-RSPO2 (FU) complex and the LGR4-NB21 complex.

[0100] Figure 4 Shows that NB21-mFc inhibits the Wnt / β-catenin signaling pathway and promotes the transformation of white adipocytes to brown adipocytes in vitro:

[0101] (a-c) sWAT-SVFs isolated from WT mice were co-cultured with hRSPO1 (FU) (0.1 μg / ml) and / or NB21-mFc (0.1 μM) for 48 h; a. Immunoblot analysis (left) and quantification (right) of non-phosphorylated (active) and total β-catenin (n = 3), with HSP90 used to normalize total protein loading; b. Representative images of immunofluorescence staining of β-catenin (green) and DAPI (blue) (upper) and quantitative analysis of the mean fluorescence intensity of β-catenin in the nucleus (lower), scale bar, 50 μm; c. qPCR analysis of Wnt target genes (such as Axin2, Wisp2, Tcf7l2, Nkd1, CyclinD1, c-Myc and Id2) (n = 3);

[0102] qPCR analysis of induced beige adipocytes after pretreatment with hRSPO1(FU) (0.1 μg / ml) and / or different concentrations of NB21-mFc (0.01, 0.1, and 1.0 μM), including Wnt target genes and thermogenic genes (such as Tcf7l2 (d), Ucp1 (e), Cidea (f), and Cox8b (g)) (n = 3). qPCR, quantitative real-time PCR; sWAT, subcutaneous white adipose tissue; SVFs, stromal vascular fraction; ANOVA, analysis of variance.

[0103] Figure 5 NB21-mFc was shown to increase energy expenditure and promote thermogenesis under cold stimulation:

[0104] (a-d) Eight-week-old female C57BL / 6J mice were fed a high-fat diet (HFD) and treated once daily by intraperitoneal injection of PBS or NB21-mFc (0.2 mg / kg) (n = 8); a, schematic diagram of the treatment protocol; b, representative curves of 24-hour energy expenditure (EE) at room temperature (22 °C) on day 7 (left) and EE normalized to body weight as a covariate (right); c, after the above experiment, the changes in EE of the mice in (a) before and after acute cold exposure (4 °C) were recorded for 7 hours, and the values under cold exposure were taken from the data between 3 - 4 hours; d, after 21 days of treatment, the changes in core body temperature of the mice after 6-hour cold exposure (5 °C).

[0105] (e-k) Eight-week-old female C57BL / 6J mice were fed HFD and intraperitoneally injected with PBS or NB21-mFc (0.2 mg / kg) daily for 7 days under chronic cold stimulation (5 °C) (n = 6); e, schematic diagram of the treatment protocol; f, i, representative images of H&E staining and immunofluorescence staining of UCP1 (green) and Perilipin (red) in vWAT (f) and sWAT (i), scale bar, 100 μm; g, j, qPCR analysis of genes related to heat production and mitochondrial respiratory chain complex genes in vWAT (g) and sWAT (j); h, k, immunoblot analysis of mitochondrial respiratory chain complexes (UQCRC2, MTCO1, and NDUFB8) and heat production gene (UCP1) in vWAT (h) and sWAT (k).

[0106] HFD, high-fat diet; EE, energy expenditure; vWAT, visceral white adipose tissue; sWAT, subcutaneous white adipose tissue; H&E, hematoxylin-eosin staining; ANCOVA, analysis of covariance; ANOVA, analysis of variance.

[0107] Figure 6 NB21-mFc was shown to promote adipose browning in an LGR4-dependent manner:

[0108] (a-d) From WT and littermate Lgr4 m / m (LGR4-deficient) mice-derived beige adipocytes were treated with hRSPO1(FU) (0.1 μg / ml) and / or NB21-mFc (0.1 μM) from day -2 to day 2; a-c, qPCR analysis of thermogenesis-related genes (including Ucp1 (a), Cidea (b), and Cox8b (c)) (n = 4); d, oxygen consumption rate (OCR) (n = 8);

[0109] (e-j) Eight-week-old female WT and Lgr4 m / m mice were fed an HFD and treated by intraperitoneal injection of PBS or NB21-mFc (0.2 mg / kg) every two days for 6 weeks (WT group n = 6, Lgr4 m / m group n = 3); e, schematic diagram of the treatment protocol; f-g, weight gain (f) and tissue weights of vWAT and sWAT (g) of the two genotypes after 6 weeks of treatment; h, representative images of H&E staining and immunofluorescence staining of UCP1 (green) and Perilipin (red) in vWAT, scale bar, 100 μm; i, qPCR analysis of thermogenesis-related genes and mitochondrial respiratory chain complex genes in vWAT; j, immunoblot analysis (left) and quantification (right) of mitochondrial respiratory chain complexes (UQCRC2, MTCO1, and NDUFB8) and thermogenesis gene (UCP1) in vWAT, HSP90 was used to normalize the loading of total protein.

[0110] WT, wild type; OCR, oxygen consumption rate; HFD, high-fat diet; H&E, hematoxylin-eosin staining; vWAT, visceral white adipose tissue; sWAT, subcutaneous white adipose tissue; qPCR, quantitative real-time polymerase chain reaction; ANOVA, analysis of variance. Detailed implementation manners

[0111] Through extensive and in-depth research and a large number of screenings, the present inventors developed a nanobody NB21 targeting LGR4. This nanobody can specifically bind to LGR4 and block its interaction with RSPO1 / 2, thereby inhibiting the activation of the Wnt signaling pathway and enhancing the mitochondrial respiration and thermogenesis of brown (and beige) adipocytes. In addition, in vitro and in vivo experiments verified that the NB21 nanobody has high affinity, high specificity, and anti-obesity biological functions, providing a new strategy for anti-obesity treatment.

[0112] Terms

[0113] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary.

[0114] As used herein, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0115] As used herein, the term "optionally" or "optionally" means that the subsequent described event or situation may occur but is not necessarily required. For example, "optionally comprising 1-3 variable regions of the antibody heavy chain" means that the variable regions of the antibody heavy chain of a specific sequence may or may not be present, and may be 1, 2, or 3.

[0116] antibody

[0117] As used herein, the terms "single-domain antibody", "VHH", "nanobody", and "single-domain antibody" (singledomain antibody, sdAb, or nanobody) have the same meaning and are used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a single-domain antibody (VHH) consisting only of one variable region of the heavy chain, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region (CH1) of the heavy chain, the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting only of one variable region of the heavy chain.

[0118] Nanobody / single-domain antibody (Nanobody), as a novel small-molecule antibody fragment, is obtained by cloning the variable region of the heavy chain of the natural heavy-chain antibody of camelids. Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15 kDa, which is one-tenth of that of a complete antibody, has good tissue penetration, high specificity, and good water solubility. Due to its special structural properties, it combines the advantages of traditional antibodies and small-molecule drugs, almost perfectly overcoming the defects of traditional antibodies such as long development cycle, low stability, and harsh storage conditions, and has gradually become an emerging force in the new generation of antibody therapy, showing broad application prospects in immuno-diagnosis and treatment.

[0119] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody differ in sequence, and it forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions in the variable regions are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs that form connecting loops and can form a partial β-sheet structure in some cases. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0120] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding a drug, toxin, cytokine, radionuclide, enzyme, and other diagnostic or therapeutic molecules to the antibody or its fragment of the present invention. The present invention also includes cell surface markers or antigens that bind to the nanobody or its fragment against the novel coronavirus.

[0121] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0122] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0123] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0124] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0125] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and all refer to a polypeptide that specifically binds to LGR4, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.

[0126] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0127] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called complementarity-determining regions (CDRs), which divide this segment into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures that are brought close to each other in spatial structure by the β-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

[0128] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least some of them are involved in binding to the antigen. Accordingly, the present invention includes those molecules having a heavy chain variable region of an antibody with CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0129] The present invention includes not only intact antibodies but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0130] As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that extends the polypeptide half-life, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6xHis tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0131] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum of the antibody of the present invention.

[0132] The present invention also provides other polypeptides, such as fusion proteins containing the antibody or its fragments. In addition to almost full-length polypeptides, the present invention also includes fragments of the antibody of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0133] In the present invention, "conservative variants of the antibody of the present invention" refer to polypeptides in which, compared with the amino acid sequence of the antibody of the present invention, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0134] Table A

[0135]

[0136]

[0137] Nanobody targeting LGR4

[0138] As used herein, the terms "nanobody of the present invention", "nanobody targeting LGR4 of the present invention", and "LGR4 nanobody of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to LGR4 (including human LGR4).

[0139] In an embodiment of the present invention, a nanobody targeting LGR4 is provided, and the complementarity determining regions (CDRs) of the VHH chain of the nanobody are selected from the group consisting of:

[0140] (a) CDRs determined based on IMGT rules:

[0141] CDR1 with amino acids as shown in SEQ ID No:1,

[0142] CDR2 with amino acids as shown in SEQ ID No:2, and

[0143] CDR3 with amino acids as shown in SEQ ID No:3; or

[0144] (b) CDRs determined based on Kabat rules:

[0145] The CDR1 with amino acids as shown in SEQ ID No: 4,

[0146] the CDR2 with amino acids as shown in SEQ ID No: 5, and

[0147] the CDR3 with amino acids as shown in SEQ ID No: 6;

[0148] (c) A sequence having LGR4 binding affinity, in which any one of the above amino acid sequences is added, deleted, modified and / or substituted by at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

[0149] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0150] The antibody of the present invention can be a double-stranded or single-stranded antibody, and can be selected from animal-derived antibodies (such as camel-derived antibodies), chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0151] The antibody derivative of the present invention can be a single-stranded antibody and / or antibody fragment, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the art, etc., and any one or several of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0152] In a preferred embodiment of the present invention, any one or several of the sequences of SEQ ID NO: 1, 2 and 3 above, or a sequence having the binding affinity for the N-terminus of LGR4 after adding, deleting, modifying and / or substituting at least one amino acid, is located in the CDR region of the heavy chain variable region (VH).

[0153] In a preferred embodiment of the present invention, any one or several of the sequences of SEQ ID NO: 4, 5 and 6 above, or a sequence having the binding affinity for the C-terminus of LGR4 after adding, deleting, modifying and / or substituting at least one amino acid, is located in the CDR region of the heavy chain variable region (VH).

[0154] In the present invention, the number of amino acids added, deleted, modified and / or substituted is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0155] In the present invention, the number of added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, and most preferably 1.

[0156] It should be understood that the antibodies of the present invention also include antibodies that contain one or more mutations in the FR regions and no mutations or only 1 or 2 conservative mutations in the CDR regions, and still retain the N-terminal or C-terminal specific binding and affinity for LGR4.

[0157] Preparation of Antibodies

[0158] Any method suitable for generating antibodies can be used to generate the LGR4-targeting nanobodies of the present invention. For example, animals can be immunized with linked or naturally occurring LGR4 or its fragments. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.

[0159] Any suitable form of LGR4 can be used as an immunogen (antigen) for generating non-human antibodies specific for LGR4 and screening the biological activities of the antibodies. The immunogenic activator can be recombinant LGR4 or its fragment. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic in origin (e.g., cDNA). Suitable genetic vectors can be used to express the DNA encoding the immunogen, and the vectors include but are not limited to: adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, plasmids, and non-viral vectors.

[0160] An exemplary method for producing the LGR4 nanobodies of the present invention is described in Example 1.

[0161] The antibodies of the present invention can be selected from any class of immunoglobulins of any species, including IgG and IgE. Preferred antibodies are IgG antibodies, such as the IgG1 subtype. Optimization of the required constant domain sequences to produce the desired biological activities is readily achieved by screening antibodies using the biological assays described in the examples below.

[0162] Similarly, any class of light chains can be used in the compounds and methods herein. Specifically, κ, λ chains or their variants can be used in the compounds and methods of the present invention.

[0163] The sequences of the DNA molecules of the antibodies or fragments thereof of the present invention can be obtained by conventional techniques, such as methods using PCR amplification or genomic library screening. In addition, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0164] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

[0165] In addition, the relevant sequence can also be synthesized by artificial synthesis, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Then the DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0166] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0167] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.

[0168] The step of transforming a host cell with recombinant DNA described in the present invention can be carried out by techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

[0169] Usually, the transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention. Then, the antibody of the present invention is purified by conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, and other conventional separation and purification means well known to those skilled in the art.

[0170] The obtained monoclonal antibody can be identified by conventional means. For example, the binding specificity of the monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0171] Pharmaceutical composition

[0172] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0173] The pharmaceutical composition of the present invention can be directly used to bind to the LGR4 protein molecule, and thus can be used to treat tumors or cancers. In addition, other therapeutic agents can also be used simultaneously.

[0174] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned single-domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight per day to about 50 milligrams per kilogram of body weight. In addition, the antibody or its active fragment or its fusion protein of the present invention can also be used together with other therapeutic agents.

[0175] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health status, which are within the scope of the skills of a skilled physician.

[0176] Compared with the prior art, the advantages of the present invention are as follows:

[0177] 1. The present invention has developed a specific nanobody NB21 targeting LGR4 and elucidated its role in blocking the binding of LGR4 to RSPO1 / 2 and thus inhibiting the Wnt signaling pathway.

[0178] 2. The present invention unexpectedly discovers that NB21 can enhance the biological effects of brown adipocyte thermogenesis and anti-obesity.

[0179] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0180] Example 1. Construction and Screening of a Nanobody Phage Display Library

[0181] (1) Animal Immunization: The LGR4 antigen was mixed with Freund's adjuvant at a ratio of 1:1, and adult alpacas were immunized by multi-point subcutaneous injection on the back at a dose of 1 mg / time. A total of 4 immunizations were carried out, and the immunization interval was 2 weeks.

[0182] (2) Total RNA Extraction: 10 ml of the peripheral blood of the immunized animal in step (1) was taken, and the peripheral blood of the immunized animal was separated by density gradient centrifugation. 1 ml of the peripheral blood of the immunized animal was taken to extract total RNA, and the RNA concentration was adjusted to 1 μg / μl.

[0183] (3) Obtaining Antibody Variable Region Genes: Reverse transcription cDNA was performed using the RNA obtained in step (2) as a template. Amplification of antibody variable region genes: The cDNA obtained by reverse transcription was used as a template for polymerase chain reaction. The amplification was carried out in two rounds. The primer sequences for the first round of polymerase chain were as follows:

[0184] SEQ ID No.9: GTCCTGGCTGCTCTTCTACAAGG

[0185] SEQ ID No.10: GGTACGTGCTGTTGAACTGTTCC

[0186] The conditions and procedures of the polymerase chain reaction were: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 7 minutes; The band of about 700 bp was recovered using an agarose gel recovery kit, and finally the nucleic acid concentration was adjusted to 5 ng / μl with water.

[0187] The primer sequences for the second round of polymerase chain were as follows:

[0188] SEQ ID No.11: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

[0189] SEQ ID No.12: CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

[0190] The polymerase chain reaction conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, for 15 cycles; 72°C for 7 minutes. The nanobody fragment was recovered by cutting the gel with agarose gel.

[0191] (4) Vector construction and clone selection: The pMECS vector was digested with the restriction endonucleases PstI and BstEII; the antibody fragment in step (3) was digested with PstI and BstEII; the antibody and vector fragments were ligated using T4 ligase. The purified ligation product was electrotransformed into competent Escherichia coli TG1 cells, and clones were randomly selected for colony PCR identification. The library capacity was estimated based on the positive rate of the polymerase chain reaction (library capacity = number of clones × dilution factor × [positive rate]PCR identification × 10). The PCR primer sequences were as follows:

[0192] SEQ ID No.13: TTATGCTTCCGGCTCGTATG

[0193] SEQ ID No.14: CCACAGACAGCCCTCATAG

[0194] (5) Phage screening of nanobodies: An appropriate amount of the bacterial library in step (4) was inoculated into a medium containing tetracycline and ampicillin, and the helper phage M13KO7 was added for infection for 30 minutes. Kanamycin, isopropyl-β-D-thiogalactoside, thiogalactoside, isopropylthio-β-D-galactoside, isopropylthiogalactoside, and galactose were added, and precipitation was carried out with polyethylene glycol 8000 / sodium chloride, and the phage display library was obtained by dissolving with phosphate buffer; LGR4 antigen was coated, blocked with 3% bovine serum albumin, and washed with phosphate buffer; 100 μL of phage was added, incubated at room temperature, washed with phosphate buffer, and the eluted phage was transfected into TG1 for the next round of screening.

[0195] After 3 rounds of screening, the positive clones were verified by enzyme-linked immunosorbent assay. Finally, one clone was selected from 22 clones and sequenced to obtain the nanobody NB21.

[0196] The amino acid sequence of the nanobody NB21 is as follows:

[0197] QVQLQESGGGSVQAGGSLRLSCTASGYTYSKYCMGWFRQVPGKEREGVAGITTGGLSPYYADSVKGRFTISRDNIKNTLYLQMNSLKPEDTAMYYCAASRLSCSALDFKDFRNFVYWGQGTQVTVSS(SEQ ID No.7)

[0198] The coding nucleic acid sequence of the nanobody NB21 is as follows:

[0199] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAAGCTGGGG

[0200] GGTCTCTGAGACTCTCCTGTACAGCCTCTGGATACACCTACAGTAAATAC

[0201] TGCATGGGCTGGTTCCGCCAGGTTCCAGGGAAGGAGCGCGAGGGGGTCG

[0202] CAGGTATTACTACTGGTGGTCTTAGTCCATACTATGCCGACTCCGTGAAG

[0203] GGCCGATTCACCATCTCCCGAGACAATATCAAGAACACGTTGTATCTGCA

[0204] AATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAT

[0205] CCCGCCTATCTTGTAGTGCTCTAGACTTCAAAGACTTTCGGAACTTTGTTT

[0206] ACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID No.8)

[0207] The CDR sequences of the nanobody are shown in Table 1 below.

[0208] Table 1

[0209]

[0210] Expression, purification and affinity determination of the LGR4 antibody in Example 2

[0211] (1) Antibody expression: NB21 was cloned into the pMECS vector with a PelB signal peptide, a C-terminal HA tag, and a His6 tag. It was expressed in the periplasm of Escherichia coli TOP10F' cells.

[0212] (2) Antibody purification: Purification was carried out using Ni-NTA. The treated sample was passed through the column. The specific operation was as follows: After induction was completed, the bacterial cells were lysed and the supernatant was collected. The bacterial cell lysate was diluted equally and loaded onto the column at a flow rate of 10 column volumes per hour, and the flow-through was collected. 15 column volumes of HEPES (pH 7.4), sodium chloride, 5% glycerol, and imidazole were used to wash away the impurity proteins, and 5 column volumes of HEPES (pH 7.4), sodium chloride, 5% glycerol, and imidazole were used for elution, and the eluate was collected.

[0213] (3) Antibody affinity determination: The StrepAvidin sensor was immersed in PBS buffer for 2 minutes to ensure a stable baseline signal. Biotinylated LGR4 was loaded onto the sensor and reacted at room temperature for 2 minutes until a stable binding response signal was reached. After loading was completed, the sensor was rinsed with the wash buffer to remove unbound LGR4 molecules. Different concentrations of NB21 antibody (3.125 nM to 100 nM, diluted in the same reaction buffer) were added sequentially, and the binding response at each concentration was recorded for 1 minute, followed by dissociation. The bimolecular model (1:1) in Octet software was used for curve fitting to analyze the binding and dissociation curves at each concentration, and the association rate constant, dissociation rate constant, and affinity constant were calculated.

[0214] The results showed that: Based on the established LGR4-targeted nanobody phage display library, NB21 was screened out. This antibody showed significant inhibitory effects on LGR4 in the TOPFlash experiment ( Figure 1 a). Its binding kinetics was evaluated by biolayer interferometry, and it was found to have a binding affinity of 1.12 nM and could competitively block the binding of RSPO1 / 2 ( Figure 1 b). Moreover, NB21 had a relatively high affinity for mouse LGR4, with a KD of 0.82 ± 0.01 nM ( Figure 1 c-d).

[0215] Example 3 Structure of the LGR4 Antibody-Stabilized LGR4 Complex

[0216] (1) LGR4 expression: Human LGR4 was expressed in HEK293 GnTI- cells cultured in FreeStyleTM 293 medium.

[0217] (2) Purification of LGR4: Add 1 μM NB21 to form a complex. After lysis, add 1% lauryl maltose neopentyl glycol and 0.1% cholesteryl hemisuccinate in the same buffer and dissolve at 4 °C for 2 hours. Collect the supernatant and apply it to an anti-dykdddddk affinity chromatography column. Wash the column with the same buffer and gradually reduce the detergent concentration to 0.02% LMNG and 0.002% CHS, and finally elute with 20 mM HEPES (pH 7.4), 10 mM sodium chloride, 5% glycerol, 0.02% LMNG, 0.002% CHS, and 0.2 mg / ml FLAG peptide. Use size exclusion chromatography Superose TM 6 for final purification.

[0218] (3) Preparation of LGR4 frozen samples: To prepare cryo-electron microscopy grids, apply 3 μl of the purified complex at a concentration of 2 - 3 mg / mL to freshly discharged 300-mesh R1.2 / R1.3 1.3 ultra-thin holey gold grids or ANTcryoTM Au300-1.2 / 1.3, freeze them into liquid ethane using Vitrobot IV (Thermo Fisher Scientific, FEI), and cool with liquid nitrogen.

[0219] (4) Collection of LGR4 cryo-EM data: 3885 images of LGR4-NB21 were collected on a Titan Krios equipped with a K3 detector.

[0220] (5) Processing of LGR4 cryo-EM data: This dataset was imported into cryoSPARC v3.3.2 (Structural Bioinformatics, Canada). The image stacks were aligned using the patch motion correction module. The contrast transfer function (CTF) parameters of each micrograph without dose weighting were determined by patch CTF estimation. Based on the CTF estimation results and relative ice thickness, high-quality micrographs were selected to ensure that the estimated CTF fitting resolution exceeded 4 Å for subsequent processing. Manual selectors were used, followed by several rounds of 2D classification to generate a specific template for subsequent automatic selection rounds. The automatically picked particles were extracted through two rounds of 2D classification. Subsequently, good particles were selected for 3D ab initio reconstruction and several rounds of 3D non-uniform refinement to eliminate low-density classes. The selected good particles underwent additional steps, including 3D ab initio reconstruction, uniform refinement, and non-uniform refinement, to generate a model and high-quality particles. Then, these particles were subjected to global CTF refinement and local CTF refinement to generate the final map.

[0221] For the NB21-LGR4 complex, approximately 1,836,475 particles were selected. After 2D classification, particles with good features were selected for 3D classification into 5 categories. Four categories of particles were combined and retained, and non-uniform refinement and CTF refinement were performed to obtain the NB21-LGR4 complex map with a final resolution of

[0222] Structural analysis showed that NB21 can block the binding of RSPO1 to LGR4 ( Figure 2 ad). The extracellular domain (ECD) of LGR4 presents a unique horseshoe shape and contains 17 LRR (leucine-rich repeat) domains. The binding mode of NB21 to LGR4 d is as follows ( Figure 3 a, b), the K31 side chain of NB21 forms two salt bridges with D137 and D161 of LGR4 (also the binding residues of RSPO1 / 2) ( Figure 3 c). In addition, K31 also forms a σ-π interaction with the aromatic ring of W159 of LGR4. At the same time, the phenolic hydroxyl group of Y32 of NB21 forms a hydrogen bond with D162 of LGR4 ( Figure 3 c). The aromatic ring of Y32 participates in π-π interaction with H157 of LGR4 and forms a hydrophobic interaction with W159 of LGR4 ( Figure 3 c). In addition, R100 of NB21 forms a salt bridge with E228 of LGR4, while S102 of NB21 forms two hydrogen bonds with D231 and N233 of LGR4 ( Figure 3 c).

[0223] Superposition of the LGR4-RSPO2(FU) complex with the LGR4-NB21 complex revealed that the binding epitope of NB21 overlaps with the binding site of RSPO1 / 2 on LGR4. This overlap effectively blocks the interaction between LGR4 and RSPO1 / 2, which is essential for the function of RSPO1 / 2 to bridge between LGR4 and ZNRF3 / RNF43 to enhance Wnt signaling. Therefore, by disrupting this association, NB21 would theoretically lead to impaired Wnt signaling. In addition, these binding residues are exactly the same in LGR4 of humans and mice, suggesting that NB21 may be a nanobody with cross-species reactivity.

[0224] Example 4 Effect of NB21-Fc fusion protein (NB21-mFc) on adipocyte SVF

[0225] To prolong the half-life of NB21 and improve its bioavailability, NB21-mFc (NB21 fused to mouse IgG2) was designed and expressed and applied in in vitro and in vivo functional experiments.

[0226] (1) Single-cell sequencing and analysis: The single-cell RNA-seq matrix of mouse white adipose tissue was obtained from the Gene Expression Omnibus (GEO) database (GSE176171), and the data was processed using the Seurat v5.0.1 package in the R v4.3.2 programming environment.

[0227] (2) Isolation of SVF and differentiation of beige adipocytes: Mouse stromal vascular fractions (SVFs) were isolated from sWAT of 6-8-week-old mice and subsequently induced to differentiate into beige adipocytes. From day 0 to 2, and then according to the experimental requirements, a growth medium containing insulin, T3, and rosiglitazone was added on days 3-4. To evaluate the effect of NB21 on the browning process, NB21 was administered twice at -2 days and 0 days, before and during the induction program, respectively. In the antagonistic experiment, recombinant human RSPO1 (4645-RS, R&D, USA) or RSPO2 (3266-RS, R&D, USA) protein was co-administered with NB21-mFc.

[0228] (3) Cellular immunofluorescence staining: SVFs were cultured in 8-well glass chamber slides and treated with hRSPO1 (0.1 μg / ml) and / or NB21-mFc (0.1 μM) for 48 hours and fixed with 4% paraformaldehyde. SVFs were incubated with a blocking solution to reduce non-specific binding of antibodies and then incubated overnight at 4°C with a primary antibody against non-phosphorylated (active) β-catenin (Ser33 / 37 / Thr41). After 48 hours, SVFs were incubated with an Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody for 120 minutes at room temperature. Then, the slides were mounted with a coverslip containing 4',6-diamidino-2-phenylindole (DAPI) and imaged using a confocal microscope.

[0229] (4) RNA isolation and qPCR analysis: RNA was isolated from cells or adipose tissue using a total RNA extraction kit. Next, the isolated RNA was reverse transcribed into cDNA templates. cDNA was measured by quantitative real-time PCR (qPCR).

[0230] (5) Protein preparation and WB assay: Total proteins were extracted from SVFs and adipose tissues using cold radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor mixture, and the concentration of the extracted proteins was determined. Protein samples were separated by SDS-PAGE and then transferred onto a 0.45-μm PVDF membrane. The PVDF membrane was blocked with 5% bovine serum albumin (BSA) solution for 90 min and then incubated with primary antibodies overnight at 4 °C. The membrane was incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. HSP90 was used as an internal control. The following primary antibodies were used: non-phosphorylated (active) β-catenin (Ser45), non-phosphorylated (active) β-catenin (Ser33 / 37 / Thr41), total β-catenin, UCP1 antibody, and UQCRC2&MTCO1&NDUFB8. The following secondary antibodies were used: HRP-conjugated rabbit IgG and HRP-conjugated mouse IgG.

[0231] (6) OCR measurement: SVFs were seeded on a Seahorse XF96 V3 PS cell culture microplate coated with polylysine. Under exogenous RSPO1, RSPO2, or NB21 treatment from -2 to 2 days, the cells were induced to differentiate into brown-like adipocytes at 37 °C for 3 days, and then the oxygen consumption rate (OCR) was evaluated at 37 °C. During the measurement, 1 μM oligomycin (to detect coupled respiration), 2 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; to evaluate uncoupled respiration), and 0.5 μM rotenone / antimycin (to measure non-mitochondrial respiration) were injected sequentially.

[0232] Germline gain-of-function germline activating mutations in human RSPO1 and LGR4 partially inhibit the browning ability of white adipocytes through the classical Wnt pathway, thus promoting the occurrence of obesity. To evaluate the possible role of NB21 in this process, the stromal vascular fraction (SVFs) of subcutaneous white adipose tissue (sWAT) was first treated with human RSPO1 (hRSPO1) and / or NB21-mFc. Experiments showed that hRSPO1 treatment significantly increased the levels of activated (non-phosphorylated) and total β-catenin, while this effect was significantly attenuated after the addition of NB21-mFc ( Figure 4 a). Meanwhile, NB21-mFc inhibited the accumulation of nuclear β-catenin induced by hRSPO1 ( Figure 4 b), and weakened its promoting effect on the transcription of Axin2 (a classical Wnt pathway activation marker) and its downstream target genes (such as Wisp2, Tcf7l2, Nkd1, CyclinD1, c-Myc, and Id2) ( Figure 4 c).

[0233] In addition, RSPO2(FU) also activated the Wnt signaling pathway and inhibited the browning process of beige adipocytes. NB21 inhibited the activation of the Wnt pathway induced by RSPO1 or RSPO2(FU) and the expression of thermogenic genes in beige adipocytes in a dose-dependent manner by blocking the shared binding site of RSPO1 / 2 on LGR4( Figure 4 d-g).

[0234] The above results indicate that NB21-mFc can weaken the effects of RSPO1 / 2 on the Wnt signaling pathway and the expression of thermogenic genes in beige adipocytes.

[0235] Example 5 Effects of NB21-mFc on obese mice

[0236] (1) Mouse cultivation: Male and female mice were used in the study and were treated as described in the legend. For the metabolic cage and acute cold exposure experiments, 8-week-old female C57BL / 6J mice were fed a high-fat diet (HFD) and received intraperitoneal injections of PBS or NB21-mFc (0.1 mg / kg) once a day. For chronic cold stimulation, 8-week-old female C57BL / 6J mice on HFD received intraperitoneal injections of PBS or NB21-mFc (0.1 mg / kg) once a day for 7 days in a cage at 4 °C. 8-week-old male and female wild-type (WT) and Lgr4m / m mice were fed a high-fat diet (HFD) and received intraperitoneal injections of PBS or NB21 (0.2 mg / kg) every two days for 6 weeks. For the HFD experiment, mice were fed a 60 kcal% HFD. Male ob / ob mice, 8 weeks old, obtained from the Jackson Laboratory, were fed a normal chow diet (NCD) and received intraperitoneal injections of PBS or NB21 (0.03 mg / kg or 0.1 mg / kg) every two days for 7 weeks.

[0237] (2) Body composition analysis and indirect calorimetry: A Echo MRI-100H composition analyzer was used to measure body composition, including fat and lean mass. To measure energy expenditure (EE), mice were individually housed in a comprehensive laboratory animal monitoring system (Promethion GAFR, Sable systems international, USA) and their food intake, O 2 consumption, CO 2 production, and physical activity were evaluated either at 22 °C or at 4 °C. Respiratory exchange ratio (RER) and EE were calculated based on the O 2 and CO 2 data. EE, O 2 consumption, and CO2 Production. After acclimating to room temperature (22 °C) for 24 hours, basal EE data was collected, and then the cage temperature was reduced to 4 °C, and data was collected over the subsequent 4 hours.

[0238] (3) Rectal temperature measurement: Before the test, the mice were fasted for 6 hours and then transferred to separate cages at 4 °C without food. Rectal temperature was measured using a thermal probe coated with petroleum jelly and a BAT-12 thermometer.

[0239] (4) H&E staining: Fat and liver tissues were separated and fixed in 4% paraformaldehyde solution. After fixation, the tissues were embedded in paraffin and cut into 5-μm thick sections for hematoxylin and eosin (H&E) staining. Tissue sections were scanned using a Tissue FAXS system.

[0240] (5) Tissue immunofluorescence staining: Serial immunostaining of anti-UCP1 antibody and anti-Perilipin antibody was performed on paraffin sections of mouse fat samples. CF-488-Tyramide and CF-594-Tyramide were used as tyramide conversion reagents. Antigen retrieval was performed using 1 mmol pH 9.0 Tris-EDTA solution. Samples were blocked with DAPI. Whole-slide scanning was performed using a TissueFAXS Plus automated acquisition system.

[0241] 5.1 NB21-mFc enhances thermogenesis

[0242] Next, it was evaluated whether NB21-mFc could promote thermogenesis in vivo. Seven days after intraperitoneal injection of NB21-mFc ( Figure 5 a), ANCOVA analysis was performed with body weight as a covariate, and it was found that the total energy expenditure (EE) of mice treated with NB21-mFc increased significantly ( Figure 5 b), while there were no significant differences in cumulative food intake, physical activity, and respiratory exchange ratio (RER) between the two groups.

[0243] Under acute cold exposure conditions, mice treated with NB21-mFc showed higher EE and core temperature ( Figure 5 c, d). After 7 days of chronic cold exposure ( Figure 5 e), a decrease in adipocyte volume was observed in visceral white adipose tissue (vWAT), sWAT, and brown adipose tissue (BAT) in the NB21-mFc treatment group, accompanied by an increase in UCP1 protein expression in vWAT and sWAT ( Figure 5 f, i). Consistent with this, the mRNA levels of thermogenic genes (such as Ucp1, Cidea, Dio2) and mitochondrial respiration genes (such as Pgc1α, Ndufa8) in adipose tissue of the NB21-mFc treatment group were significantly increased ( Figure 5g, j). In addition, the protein levels of UCP1 and mitochondrial respiratory chain complex proteins (including UQCRC2, MTCO1, NDUFB8) in the NB21-mFc treatment group also increased significantly ( Figure 5 h, k).

[0244] The above results indicate that NB21-mFc can enhance the heat production and energy consumption of mouse adipose tissue.

[0245] 5.2 The promotion of browning by NB21-mFc depends on LGR4

[0246] RSPO1 strongly inhibits adipose browning by binding to LGR4, and the effective concentration of RSPO2 is approximately 20 times that of RSPO1, which is consistent with previous studies.

[0247] Through changes in the expression of thermogenic genes and mitochondrial respiratory genes ( Figure 6 a-c) and changes in oxygen consumption capacity ( Figure 6 d), it can be shown that the neutralizing effect of NB21-mFc on the inhibition of adipose browning by the RSPO1-LGR4 complex disappears in brown-like adipocytes lacking LGR4 (Lgr4 m / m ). These results indicate that NB21-mFc enhances adipose thermogenic capacity through an LGR4-dependent mechanism.

[0248] To further verify whether NB21-mFc can combat obesity in vivo, its potential weight loss effect was evaluated in a diet-induced obesity model. Under a high-fat diet (HFD) condition, 8-week-old female and male mice were intraperitoneally injected with NB21-mFc (0.2 mg / kg) or PBS every other day for 4-6 weeks ( Figure 6 e). The results showed that NB21-mFc significantly reduced the body weight of wild-type (WT) female mice ( Figure 6 f), which was mainly attributed to the reduction of vWAT and sWAT ( Figure 6 g), and was accompanied by a decrease in adipocyte volume and stronger staining of the browning marker (UCP1) ( Figure 6 h). Consistent with the in vitro experimental results, NB21-mFc enhanced the expression of thermogenic and mitochondrial respiratory genes in vWAT and sWAT ( Figure 6 i, j). Similar but weaker changes were also observed in BAT.

[0249] In addition to reducing diet-induced obesity, NB21-mFc also reduced hepatic lipid accumulation. Importantly, NB21-mFc lost the above-mentioned browning promotion and anti-obesity effects in Lgr4m / m littermate mice ( Figure 6 e-j).

[0250] In summary, these research results indicate that NB21-mFc alleviates diet-induced obesity mainly by promoting LGR4-dependent thermogenic capacity.

[0251] The present invention not only provides a new perspective for understanding the mechanism of action of LGR4 in obesity, but also opens up a new path for the development of novel therapeutic drugs targeting LGR4. Compared with traditional small molecule drugs, nanobodies have better targeting, lower off-target risk and potentially longer in vivo half-life, and are expected to become a safe and effective anti-obesity treatment.

[0252] All documents mentioned in the present invention are cited herein by reference as if each document was individually cited by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A nanobody targeting LGR4, characterized in that: The VHH chain of the Nanobody comprises a heavy chain variable region as shown in SEQ ID No: 7, and the complementarity determining regions CDR1, CDR2 and CDR3 comprised in the heavy chain variable region are defined by Chothia, Abm, Kabat, or IMGT rules.

2. The Nanobody according to claim 1, characterized in that The CDR1, CDR2 and CDR3 are selected from the following group: (a) CDR determined based on IMGT rules: The amino acid sequence of CDR1 is as shown in SEQ ID No: 1, The amino acid sequence of CDR2 is as shown in SEQ ID No: 2, and The amino acid sequence of CDR3 is as shown in SEQ ID No: 3; or (b) CDRs determined based on Kabat rules: The amino acid sequence of CDR1 is as shown in SEQ ID No: 4, The amino acid sequence of CDR2 is as shown in SEQ ID No: 5, and CDR3 having amino acids as shown in SEQ ID No:6; Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the LGR4 binding affinity.

3. An antibody targeting LGR4, characterized in that: Comprising the Nanobody targeting LGR4 as claimed in claim 1.

4. An isolated polynucleotide, characterized in that The polynucleotide encodes a nanobody targeting LGR4 as claimed in claim 1.

5. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 5, or the polynucleotide according to claim 4 is integrated into its genome.

7. An immunoconjugate, characterized in that: The immunoconjugate comprises: (a) a Nanobody targeting LGR4 as claimed in claim 1; and (b) a coupling moiety selected from the group consisting of fluorescein, a small molecule compound, PEG, a radioisotope, a contrast agent, a fatty acid chain, a protein fragment, a colored microsphere, a fluorescent microsphere, a polymer microsphere, a magnetic microsphere, agarose, dextran, cellulose, a filter membrane, or a combination thereof.

8. A fusion protein, characterized in that The fusion protein contains: (a) the Nanobody of claim 1 or 2; (b) Optional polypeptide molecules or fragments having therapeutic functions.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (ii) the Nanobody targeting LGR4 according to claim 1 or 2, the antibody targeting LGR4 according to claim 3, the immunoconjugate according to claim 7, or the fusion protein according to claim 8; and (ii) a pharmaceutically acceptable carrier.

10. Use of the nanobody according to claim 1 or 2, the antibody according to claim 3, the immunoconjugate according to claim 7, or the fusion protein according to claim 8, characterized in that: Used for preparing anti-obesity drugs.

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