A nanobody NB21 targeting LGR4 and its application in anti-obesity therapy

Abstract

The present application provides a kind of nanobody NB21 targeted to LGR4 and its application in anti-obesity treatment.Specifically, the present application discloses a nanobody NB21 targeted to LGR4 and its derivative protein, gene sequence for coding them, expression vector and expression system for producing them.In addition, it is verified by in vitro and in vivo experiments that the nanobody NB21 targeted to block LGR4 has high affinity, high specificity, plays a biological function of promoting thermogenesis and reducing fat, and provides a new strategy for obesity treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to a nanobody NB21 targeting LGR4 and its application in anti-obesity treatment. Background Technology

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

[0003] (1) It is smaller in size, only one-tenth the size of ordinary antibodies, and has stronger penetrating power in animal tissues. It can pass through human brain tissue and reach the high-density tumor interior, thereby treating certain tumors or brain diseases.

[0004] (2) Stable efficacy: Nanobodies do not decompose naturally in the body for a longer time than ordinary antibodies, which means a longer duration of efficacy. Moreover, they are adaptable to a wider temperature range and can still function at temperatures as high as 90°C, while traditional antibodies would be inactivated under such conditions. They are also very stable even at extreme pH values ​​and retain their effectiveness even after passing through the human stomach.

[0005] (3) They have good antigen specificity and are easy to genetically modify; nanobodies can recognize unique antigenic epitopes, have a wider range of antigen binding capabilities than ordinary antibodies, and are easy to artificially modify to obtain antibodies against different pathogens; nanobodies can be easily synthesized in microorganisms, can be expressed in large quantities in microorganisms such as bacteriophages, Escherichia coli, and yeast, and are easy to produce on a large scale.

[0006] With the rising incidence of obesity, various obesity-related complications, such as diabetes, cardiovascular disease, and cancer, are also increasing. From lifestyle interventions to bariatric surgery, various methods have been used to prevent and manage obesity, but their effectiveness is low or the risks are high. Discovering new targets is crucial for weight loss treatment. Currently, G protein-coupled receptors (GPCRs) are a key area for weight loss drug development. For example, currently marketed GLP-1R agonists, GIPR agonists, and GCGR agonists all target GPCRs.

[0007] In 2013, the Genetic Study of Severe Obesity in Chinese Adolescents (GOCY) cohort first discovered an activating LGR4 mutation (A750T) in the leucine repeat-containing G protein-coupled receptor 4 (LGR4, also known as GPR48), significantly increasing the risk of obesity. Almost simultaneously, a study in Iceland showed that over 300 individuals carrying a rare loss-of-function LGR4 mutation (p.R126X) exhibited weight loss. Animal studies have shown that knocking out the LGR4 gene increases energy expenditure by promoting the transformation of preadipocytes from white adipocytes to brown adipocytes, potentially alleviating obesity caused by a high-fat diet and genetic defects. LGR4 is a new member of the GPCR superfamily and plays a crucial role in maintaining endocrine and metabolic homeostasis; these findings and insights have gained widespread acceptance. 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 of the presence of two overly smooth, curved β-sheets on its concave surface, lacking drug action pockets.

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

[0010] The purpose of this invention is to provide a safer, more efficient and easier-to-prepare nanobody targeting LGR4.

[0011] Another object of the present invention is to provide applications of nanobodies targeting LGR4, particularly for anti-obesity purposes.

[0012] In a first aspect of the invention, a nanobody targeting LGR4 is provided, wherein the complementarity-determining region (CDR) of the VHH chain of the nanobody is selected from the group consisting of:

[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 from any one or more of the amino acid sequences in CDR1, CDR2, or CDR3, and is an amino acid sequence that has 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 contains an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95% (e.g., 96%, 97%, 98%, or even more preferably at least 99%) sequence identity with any of the SEQ ID NO:1-3.

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

[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 able to enhance mitochondrial respiration and thermogenic activity in brown adipocytes.

[0024] In another preferred embodiment, the LGR4 is an LGR4 of a human or non-human mammal.

[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 as described in SEQ ID No. 7.

[0028] In a second aspect, the present invention provides an antibody targeting LGR4, said antibody targeting LGR4 comprising one or more LGR4-targeting nanobodies 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 monospecific antibodies, bispecific antibodies, and multispecific antibodies (such as trispecific antibodies).

[0031] In a third aspect, the present invention provides a polynucleotide encoding 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.

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

[0033] Nucleic acid molecules have:

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

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

[0036] (iii) A nucleotide sequence that encodes the same protein as (i) or (ii), but differs 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 which has the same or similar function as the nucleotide sequence shown in (i), (ii) or (iii).

[0038] In a fourth aspect, the present invention provides an expression vector containing the polynucleotide 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, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0040] In a fifth aspect, the present invention provides a host cell containing the expression vector described in the fourth aspect of the present invention, or having the polynucleotide 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 embodiment, the mammalian cells include (but are not limited to) HEK293F cells and CHO cells.

[0044] In a sixth aspect, the present invention provides an immunoconjugate comprising:

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

[0046] (b) The coupling motif selected from the group consisting of: fluorescein, small molecule compounds, PEG, radioisotopes, contrast agents, fatty acid chains, protein fragments, or combinations thereof.

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

[0048] In another preferred embodiment, the coupling portion is a chemical marker and a biological marker.

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

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

[0051] In another preferred embodiment, the small molecule compound includes, but is not limited to, drugs or toxins that have a proven or potential therapeutic or adjuvant therapeutic effect on tumors or autoimmune diseases.

[0052] In another preferred embodiment, the radioactive isotope includes:

[0053] (i) a diagnostic isotope 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 combinations thereof; and / or

[0054] (ii) Therapeutic isotopes, wherein the therapeutic isotopes are 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 combinations 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 used for MRI or CT.

[0057] In another preferred embodiment, the protein fragments include, but are not limited to, antibodies Fc, biotin, avidin, HRP, antibodies, enzymes, cytokines, and other bioactive proteins or polypeptides.

[0058] In another preferred embodiment, the coupling portion is a detectable marker.

[0059] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0060] In a seventh aspect of the invention, a fusion protein is provided, the 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) Optional polypeptide molecules or fragments with therapeutic functions.

[0063] In another preferred embodiment, the therapeutic polypeptide molecule or fragment 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 therapeutic polypeptide molecule or fragment includes, but is not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analog, albumin, antibody fragment, and cytokines.

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

[0066] In another preferred embodiment, the fusion protein further comprises a tag sequence that assists in expression and / or purification.

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

[0068] In another preferred embodiment, the fusion protein further comprises portions 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, luminescent enzyme, maltose-binding protein, glutathione transferase, toxin protein, and antibody Fc fragment.

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

[0070] An eighth aspect of the present invention provides a pharmaceutical composition comprising:

[0071] (ii) The LGR4-targeting nanobody as described in the first aspect of the present invention, the LGR4-targeting antibody 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;

[0072] (ii) Pharmaceutically acceptable carriers.

[0073] In a ninth aspect of the present invention, a nanoantibody complex is provided, the complex comprising:

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

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

[0076] In a tenth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: a nanobody targeting LGR4 as described in the first aspect of the invention, an antibody targeting LGR4 as described in the second aspect of the invention, an immunoconjugate as described in the sixth aspect of the invention, or a fusion protein as described in the seventh aspect of the invention, or combinations thereof, for (a) the preparation of a medicament for treating obesity and related diseases; and / or (b) the preparation of reagents, affinity media products, detection plates, or kits for detecting LGR4.

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

[0078] In another preferred embodiment, the simple obesity is selected from the group consisting of: hyperphagia-type obesity, absorptive-type obesity, and basal metabolic rate-decreased obesity.

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

[0080] In another preferred embodiment, the reagent is used to detect LGR4 or fragments thereof in a sample.

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

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

[0083] In another preferred embodiment, the kit includes an immunochromatographic kit, an enzyme-linked immunosorbent assay (ELISA) kit, an immunoturbidimetric assay kit, and chemical, electrochemical, and bioluminescent kits.

[0084] In an eleventh aspect, the present invention provides a method for in vitro detection of LGR4 or fragments thereof in a sample, the method comprising the steps of:

[0085] (1) In vitro, the sample is contacted with the LGR4-targeting nanobody as described in the first aspect of the present invention, the LGR4-targeting antibody as described in the second aspect of the present invention, and the immunoconjugate as described in the sixth aspect of the present invention.

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

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

[0088] According to a thirteenth aspect of the present invention, a kit is provided, the kit comprising:

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

[0090] (2) A second container containing a secondary antibody against 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.

[0091] A fourteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, characterized in that the method comprises:

[0092] (a) Culturing the host cells described in the fifth aspect of the present invention under suitable expression conditions;

[0093] (b) Isolating a recombinant polypeptide from the culture, said recombinant polypeptide being a nanobody targeting LGR4 as described in the first aspect of the invention, an antibody targeting LGR4 as described in the second aspect of the invention, or a fusion protein as described in the seventh aspect of the invention.

[0094] The fifteenth aspect of the present invention provides a method for treating obesity, characterized in that the method comprises: administering to a desired subject 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, a fusion protein as described in the seventh aspect of the present invention, a pharmaceutical composition as described in the eighth aspect of the present invention, or a combination thereof.

[0095] In another preferred embodiment, the method further includes administering other drugs or treatments to the subject in need for combined treatment.

[0096] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

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

[0098] Figure 2 The cryo-electron microscopy structures of LGR4 and its complex with RSPO2(FU) are shown: a. Structure of the LGR4-MB52 complex, showing the cryo-electron microscopy image (left) and atomic model (right); b. Structure of the RSPO2(FU)-LGR4-MB52 complex, showing the cryo-electron microscopy image (left) and atomic model (right); c. Interface between LGR4 and RSPO2(FU); d. Cryo-electron density map of the interaction interface between LGR4 and RSPO2(FU). Interacting residues are represented by rods. LGR4 is cyan; RSPO2(FU) is purple; MB52 is gray.

[0099] Figure 3 The cryo-electron microscopy structure and analysis of the LGR4-NB21 complex are shown: a. Structure of the NB21-LGR4 complex, showing the cryo-electron microscopy image (left) and atomic model (right), with NB21 indicated in pink; b. Interface between LGR4 and NB21; c. Cryo-electron 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 The study demonstrated that NB21-mFc inhibits the Wnt / β-catenin signaling pathway and promotes the transformation of white adipocytes into brown adipocytes in vitro.

[0101] (ac) sWAT-SVFs from WT mice, co-cultured with hRSPO1 (FU) (0.1 μg / ml) and / or NB21-mFc (0.1 μM) for 48 hours; a. Immunoblotting analysis of non-phosphorylated (active) and total β-catenin (left) and its quantification (right) (n=3), HSP90 was used to normalize the total protein loading; b. Representative images of immunofluorescence staining of β-catenin (green) and DAPI (blue) (top) and quantitative analysis of the average fluorescence intensity of β-catenin in the cell nucleus (bottom), 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] (dg) qPCR analysis of induced brown 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 components; ANOVA, analysis of variance.

[0103] Figure 5 The results showed that NB21-mFc increases energy expenditure and promotes heat production under cold stimulation:

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

[0105] (ek) Eight-week-old female C57BL / 6J mice were fed HFD and subjected to chronic cold stimulation (5°C) with intraperitoneal injection of PBS or NB21-mFc (0.2 mg / kg) daily for 7 days (n=6); e, schematic diagram of the treatment protocol; f, i, representative images of H&E staining and UCP1 (green) and Perilipin (red) immunofluorescence staining in vWAT(f) and sWAT(i), scale bar, 100 μm; g, j, qPCR analysis of heat production-related genes and mitochondrial respiratory chain complex genes in vWAT(g) and sWAT(j); h, k, immunoblotting 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 The study showed that NB21-mFc promotes adipose browning in an LGR4-dependent manner:

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

[0109] (ej) 8-week-old female WT and Lgr4 m / m Mice were fed HFD and treated with PBS or NB21-mFc (0.2 mg / kg) intraperitoneally every two days for 6 weeks (WT group n=6, Lgr4). m / m Group n=3); e, Schematic diagram of the treatment protocol; fg, Body weight gain (f) and tissue weight (g) of vWAT and sWAT after 6 weeks of treatment for the two genotypes; h, Representative images of H&E staining and UCP1 (green) and Perilipin (red) immunofluorescence staining of vWAT, scale bar, 100 μm; i, qPCR analysis of heat production-related genes and mitochondrial respiratory chain complex genes in vWAT; j, Immunoblot analysis (left) and quantification (right) of mitochondrial respiratory chain complex (UQCRC2, MTCO1 and NDUFB8) and heat production gene (UCP1) in vWAT, HSP90 was used to normalize the total protein loading.

[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

[0111] Through extensive and in-depth research and screening, the inventors have developed a nanobody, NB21, that targets LGR4. This nanobody specifically binds to LGR4 and blocks its interaction with RSPO1 / 2, thereby inhibiting the activation of the Wnt signaling pathway and enhancing mitochondrial respiration and thermogenesis in brown (and brown-like) adipocytes. Furthermore, in vitro and in vivo experiments have verified that the NB21 nanobody possesses high affinity, high specificity, and anti-obesity biological functions, providing a novel strategy for anti-obesity therapy.

[0112] the term

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

[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 “substantially consisting of”.

[0115] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to 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" (sdAb, or nanobody) have the same meaning and are used interchangeably. A single-domain antibody (VHH) is constructed by cloning the variable region of the antibody heavy chain, consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0118] Nanobodies / single-domain antibodies are novel small-molecule antibody fragments cloned from the variable heavy chain region (VHH) of natural camel heavy-chain antibodies. Nanobodies (Nb) possess excellent biological properties, with a molecular weight of 12-15 kDa, about one-tenth the size of a complete antibody. They exhibit excellent tissue penetration, high specificity, and good water solubility. Due to their unique structural properties, they combine the advantages of traditional antibodies and small-molecule drugs, almost perfectly overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and demanding storage conditions. They are gradually becoming an emerging force in next-generation antibody therapy, showing broad application prospects in immunodiagnosis and treatment.

[0119] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are closely packed together through 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, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0120] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the nanobodies or fragments thereof targeting the novel coronavirus.

[0121] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0122] As used in this article, 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 aforementioned heavy chain variable region and heavy chain constant region.

[0125] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to LGR4, such as proteins or peptides 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 has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0127] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame 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 a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

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

[0129] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0130] As used herein, the terms “fragment,” “derivative,” and “analyte” 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 may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved 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 substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6xHis tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0131] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stress conditions, and polypeptides or proteins obtained using antiserum of the antibody of the present invention.

[0132] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, 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 of the antibody of the present invention.

[0133] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0134] Table A

[0135]

[0136]

[0137] Nanobodies targeting LGR4

[0138] As used herein, the terms “nanobody of the present invention”, “nanobody of the present invention targeting LGR4”, 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, wherein the complementarity-determining region (CDR) of the VHH chain of the nanobody is selected from the following group:

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

[0141] Amino acids such as CDR1 as shown in SEQ ID No:1,

[0142] Amino acids such as CDR2 as shown in SEQ ID No:2, and

[0143] Amino acids such as CDR3 as shown in SEQ ID No:3; or

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

[0145] Amino acids such as CDR1, as shown in SEQ ID No:4,

[0146] Amino acids such as CDR2 as shown in SEQ ID No:5, and

[0147] Amino acids such as CDR3, as shown in SEQ ID No:6;

[0148] (c) A sequence having LGR4 binding affinity by adding, deleting, modifying and / or substituting at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid from any of the above amino acid sequences.

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

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

[0151] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more 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 more of the sequences in SEQ ID NO:1, 2 and 3 above, or sequences having binding affinity at the N-terminus of LGR4 after addition, deletion, modification and / or substitution of at least one amino acid, are located in the CDR region of the heavy chain variable region (VH).

[0153] In a preferred embodiment of the present invention, any one or more of the sequences in SEQ ID NO:4, 5 and 6 above, or sequences having binding affinity for the C-terminus of LGR4 after addition, deletion, modification and / or substitution of at least one amino acid, are located in the CDR region of the heavy chain variable region (VH).

[0154] In this invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0155] In this 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 region and no mutations or only one or two conserved mutations in the CDR region, and still retain the N-terminal or C-terminal specific binding and affinity of LGR4.

[0157] Antibody preparation

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

[0159] Any suitable form of LGR4 can be used as an immunogen (antigen) to generate non-human antibodies specific to LGR4 and screen for the biological activity of said antibodies. The triggering immunogen can be recombinant LGR4 or a fragment thereof. 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 a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA). The DNA encoding the immunogen can be expressed using suitable genetic vectors, including but not limited to: adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, proteotropic and nonviral vectors.

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

[0161] The antibodies of this invention can be selected from any type of immunoglobulin of any species, including IgG and IgE. Preferred antibodies are IgG antibodies, such as the IgG1 subtype. The optimization of the essential constant domain sequence to produce the desired biological activity can be easily achieved by screening antibodies using the biological assays described in the examples below.

[0162] Similarly, any type of light chain can be used in the compounds and methods described herein. Specifically, κ, λ chains, or variations thereof, can be used in the compounds and methods of this invention.

[0163] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, 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 using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0165] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0166] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0167] The host cell can be a prokaryotic cell, such as a bacterial cell; 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, and HEK-293 cells.

[0168] The steps of transforming host cells with recombinant DNA as described in this invention can be performed using techniques well known in the art. The obtained transformants can be cultured using conventional methods, and the transformants express the polypeptide encoded by the gene of this invention. Depending on the host cell used, the cells are cultured in a conventional culture medium under suitable conditions.

[0169] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using 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, which are well known to those skilled in the art.

[0170] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies 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 containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0173] The pharmaceutical compositions of the present invention can be directly used to bind to LGR4 protein molecules, and therefore can be used to treat tumors or cancer. Furthermore, other therapeutic agents can be used simultaneously.

[0174] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described single-domain antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the antibodies of the present invention, or their active fragments, or their fusion proteins, can also be used with other therapeutic agents.

[0175] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically 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 between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

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

[0177] 1. This invention develops a specific nanobody NB21 targeting LGR4 and elucidates its role in inhibiting the Wnt signaling pathway by blocking the binding of LGR4 to RSPO1 / 2.

[0178] 2. This invention unexpectedly discovered the biological effects of NB21 in enhancing thermogenesis and anti-obesity in brown adipocytes.

[0179] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0180] Example 1. Construction and screening of nanobody phage display libraries

[0181] (1) Animal immunization: The LGR4 antigen and Freund's adjuvant were mixed in a 1:1 ratio and then injected subcutaneously into the back of adult alpacas at a dose of 1 mg / time. A total of 4 immunizations were performed with an interval of 2 weeks between immunizations.

[0182] (2) Extract total RNA: Take 10 ml of peripheral blood from the immunized animal in step (1), separate the peripheral blood of the immunized animal by density gradient centrifugation, take 1 ml of peripheral blood from the immunized animal, extract total RNA, and adjust the RNA concentration to 1 μg / μL.

[0183] (3) Obtaining the antibody variable region gene: Using the RNA obtained in step (2) as a template, reverse transcription was performed on cDNA. Antibody variable region gene amplification: The cDNA obtained from reverse transcription was used as a template for polymerase chain reaction. Amplification was performed in two rounds. The primer sequences for the first round of polymerase chain reaction are as follows:

[0184] SEQ ID No.9: GTCCTGGCTGCTCTTCTACAAGG

[0185] SEQ ID No.10:GGTACGTGCTGTTGAACTGTTCC

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

[0187] The primer sequences for the second round of polymerase chain reaction are 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℃ for 5 minutes; 95℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds, 15 cycles; nanobody fragments were recovered by agarose gel excision at 72℃ for 7 minutes.

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

[0192] SEQ ID No.13: TTATGCTTCCGGCTCGTATG

[0193] SEQ ID No.14: CCACAGACAGCCCTCATAG

[0194] (5) Phage screening of nanobodies: Take an appropriate amount of the bacterial library from step (4) and inoculate it into a culture medium containing tetracycline and ampicillin. Add helper phage M13KO7 and infect for 30 minutes. Add kanamycin and isopropyl-β-D-thiogalactoside, thiogalactoside, isopropyl-β-D-thiogalactoside, isopropyl-β-D-thiogalactoside, and galactose. Precipitate with polyethylene glycol 8000 / sodium chloride and dissolve in phosphate buffer to obtain the phage display library. Coat with LGR4 antigen, block with 3% bovine serum albumin, and wash with phosphate buffer. Add 100 μL of phage, incubate at room temperature, wash with phosphate buffer, and the eluted phage is transfected with TG1 and then enters the next round of screening.

[0195] After three rounds of screening, positive clones were verified using enzyme-linked immunosorbent assay (ELISA). 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] QVQLQESGGGSVQAGGSLRLSCTASGYTYSKYCMGWFRQVPGKEREG VAGITTGGLSPYYADSVKGRFTISRDNIKNTLYLQMNSLKPEDTAMYYCAAS RLSCSALDFKDFRNFVYWGQGTQVTVSS(SEQ ID No.7)

[0198] The encoding nucleic acid sequence of 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] Example 2: Expression, purification, and affinity determination of LGR4 antibody

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

[0212] (2) Antibody purification: Ni-NTA was used for purification. The treated sample was subjected to column chromatography. The specific operation was as follows: After induction, the bacterial cells were lysed and the supernatant was collected. The bacterial lysate was diluted by an equal number of times and loaded onto the column at a flow rate of 10 column volumes / hour. The flow-through was collected. Impurities were washed away with 15 column volumes of HEPES (pH 7.4), sodium chloride, 5% glycerol, and imidazole. Elution was carried out with 5 column volumes of HEPES (pH 7.4), sodium chloride, 5% glycerol, and imidazole. The eluent was collected.

[0213] (3) Antibody affinity assay: The StrepAvidin sensor was immersed in PBS buffer for 2 minutes to equilibrate and 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, the sensor was rinsed with washing 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. Curve fitting was performed using a bimolecular model (1:1) in Octet software to analyze the binding and dissociation curves at each concentration, and the binding rate constant, dissociation rate constant, and affinity constant were calculated.

[0214] The results showed that NB21 was screened based on the established LGR4-targeting nanobody phage display library. This antibody showed a significant inhibitory effect on LGR4 in the TOPFlash experiment. Figure 1 a). The binding kinetics were assessed using the biolayer interference method, revealing a binding affinity of 1.12 nM and the ability to competitively block the binding of RSPO1 / 2. Figure 1 b). Furthermore, NB21 exhibits a high affinity for mouse LGR4, with a KD of 0.82 ± 0.01 nM. Figure 1 cd).

[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 FreeStyle™ 293 medium.

[0217] (2) LGR4 purification: 1 μM NB21 was added to form a complex. After lysis, 1% lauryl maltose neopentyl glycol and 0.1% cholesterol hemisuccinate were added to the same buffer and dissolved at 4°C for 2 hours. The supernatant was collected and applied to an anti-dykdddddk affinity column. The column was washed with the same buffer, gradually reducing the detergent concentration to 0.02% LMNG and 0.002% CHS, and finally eluted 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. Size exclusion chromatography was used. TM 6. Perform final purification.

[0218] (3) LGR4 frozen sample preparation: In order to prepare the cryogenic electron microscope grid, 3 μl of purified complex at a concentration of 2-3 mg / mL was applied to freshly discharged 300 mesh R1.2 / R1.31.3 ultrafoil gold grid or ANTcryo™ Au300-1.2 / 1.3, frozen in liquid ethane using Vitrorok IV (Fischer Scientific, FEI), and cooled with liquid nitrogen.

[0219] (4) LGR4 cryo-electron microscopy data collection: 3,885 images of LGR4-NB21 were collected on a Titan Krios equipped with a K3 detector.

[0220] (5) LGR4 Cryo-Electron Microscopy Data Processing: This dataset was imported into cryoSPARC v3.3.2 (Structural Biotechnology, Canada). Image stacks were aligned using the Patch Motion Correction module. The contrast transfer function (CTF) parameters for each unweighted micrograph 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 4A for subsequent processing. A manual selector was used, followed by several rounds of 2D classification to generate a specific template for subsequent automatic selection rounds. Two rounds of 2D classification extraction were performed on the automatically selected particles. 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 produce a model and high-quality particles. Global CTF refinement and local CTF refinement were then performed on these particles to generate the final image.

[0221] For the NB21-LGR4 complex, approximately 1,836,475 particles were selected. After two-dimensional classification, particles with good characteristics were selected for five-class three-dimensional classification. The four classes of particles were combined and retained, and then subjected to non-uniform refinement and CTF refinement to obtain the NB21-LGR4 complex image, with a final resolution of [resolution missing].

[0222] Structural analysis shows that NB21 can block the binding of RSPO1 to LGR4. Figure 2 The extracellular domain (ECD) of LGR4 exhibits a unique horseshoe shape and contains 17 LRR (leucine-enriched repeat) domains. The binding mechanism of NB21 to LGR4's d is as follows ( Figure 3 a, b), the K31 side chain of NB21 forms two salt bridges with D137 and D161 of LGR4 (which are also binding residues of RSPO1 / 2). Figure 3 c). Furthermore, K31 also undergoes a σ-π interaction with the W159 aromatic ring of LGR4. Simultaneously, the phenolic hydroxyl group at 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). Additionally, 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] Stacking 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 inhibits the interaction between LGR4 and RSPO1 / 2, which is crucial for RSPO1 / 2 to bridge the gap between LGR4 and ZNRF3 / RNF43 to enhance Wnt signaling. Therefore, NB21, by disrupting this association, could theoretically impair Wnt signaling. Furthermore, these binding residues are completely identical in human and mouse LGR4, 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 with mouse IgG2) was designed and expressed, and applied in in vitro and in vivo functional experiments.

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

[0227] (2) Isolation of SVFs and Differentiation of Brown Adipocytes: Mouse stromal vascular fractions (SVFs) were isolated from sWATs of 6-8 week old mice and subsequently induced to differentiate into brown adipocytes. The induction period was from day 0 to 2, followed by the addition of growth medium containing insulin, T3, and rosiglitazone on days 3-4 according to experimental requirements. To assess the effect of NB21 on the browning process, NB21 was administered twice, at day -2 and day 0, before and during the induction program, respectively. In the antagonism assay, recombinant human RSPO1 (4645-RS, R&D, USA) or RSPO2 (3266-RS, R&D, USA) protein was co-administered with NB21-mFc.

[0228] (3) Immunofluorescence staining: SVFs were cultured in 8-well glass slides and treated with hRSPO1 (0.1 μg / ml) and / or NB21-mFc (0.1 μM) for 48 h, followed by fixation with 4% paraformaldehyde. SVFs were incubated with blocking solution to reduce non-specific antibody binding, and then incubated overnight at 4 °C with non-phosphorylated (active) β-catenin (Ser33 / 37 / Thr41) primary antibody. After 48 h, SVFs were incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody at room temperature for 120 min. They were then mounted on slides containing 4',6-diamidinyl-2-phenylindole (DAPI) and imaged using confocal microscopy.

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

[0230] (5) Protein preparation and Western blot (WB) assay: Total protein was extracted from SVFs and adipose tissue using a cold radioimmunoprecipitation reagent (RIPA) lysis buffer supplemented with a protease inhibitor mixture, and the concentration of extracted protein was determined. Protein samples were separated by SDS-PAGE and then transferred to 0.45 μm PVDF membranes. The PVDF membranes were blocked with 5% bovine serum albumin (BSA) solution for 90 min and then incubated overnight at 4 °C with primary antibody. The membranes were then incubated at room temperature with HRP-conjugated secondary antibody for 90 min. 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 in Seahorse XF96 V3 PS cell culture microplates and coated with polylysine. Cells were induced to differentiate into brown adipocytes at 37°C for 3 days after treatment with exogenous RSPO1, RSPO2, or NB21 for -2 to 2 days, followed by assessment of oxygen consumption rate (OCR) at 37°C. During the measurement, 1 μM oligomycin (for detecting coupled respiration), 2 μM 4-phenylhydrazine carbamate (FCCP; for assessing uncoupled respiration), and 0.5 μM rotenone / antimycin (for measuring non-mitochondrial respiration) were injected sequentially.

[0232] Germline-enhanced germline activating mutations in human RSPO1 and LGR4 partially suppress the browning ability of white adipocytes via the classical Wnt pathway, thereby promoting obesity. To assess the potential role of NB21 in this process, stromal vascular components (SVFs) of subcutaneous white adipose tissue (sWAT) were treated with human RSPO1 (hRSPO1) and / or NB21-mFc. The results showed that hRSPO1 treatment significantly increased the levels of activated (non-phosphorylated) and total β-catenin, while this effect was significantly attenuated upon the addition of NB21-mFc. Figure 4 a). Simultaneously, NB21-mFc inhibited hRSPO1-induced accumulation of nuclear β-catenin ( Figure 4 b) and weakened its promoting effect on the transcription of Axin2 (a classic Wnt pathway activation marker) and its downstream target genes (such as Wisp2, Tcf7l2, Nkd1, CyclinD1, c-Myc, and Id2). Figure 4 c).

[0233] Furthermore, RSPO2(FU) also activates the Wnt signaling pathway and inhibits the browning process in brown adipocytes. NB21, by blocking the shared binding site of RSPO1 / 2 on LGR4, dose-dependently inhibited RSPO1 or RSPO2(FU)-induced Wnt pathway activation and the expression of pyrogen genes in brown adipocytes. Figure 4 dg).

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

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

[0236] (1) Mouse Culture: Male and female mice were used in the study and were treated as described in the illustration. 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 daily. For chronic cold stimulation, 8-week-old female C57BL / 6J mice were fed HFD and received intraperitoneal injections of PBS or NB21-mFc (0.1 mg / kg) daily for 7 days in cages 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 experiments, mice were fed 60 kcal% HFD. Male ob / ob mice obtained from Jackson Laboratory, 8 weeks old, were fed a normal 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: Body composition, including fat and lean mass, was measured using an Echo MRI-100H component analyzer. To measure energy expenditure (EE), mice were housed individually in a comprehensive laboratory animal monitoring system (Promethion GAFR, Sable Systems International, USA) at either 22°C or 4°C to assess their food intake, O2 consumption, CO2 production, and physical activity. Respiratory exchange ratio (RER) and EE were calculated based on O2 and CO2 data. EE, O2 consumption, and CO2 production were assessed using analysis of covariance (ANCOVA) with body weight as a covariate. Baseline EE data were collected after 24 hours of acclimatization to room temperature (22°C), followed by data collection over the next 4 hours after the cage temperature was lowered to 4°C.

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

[0239] (4) H&E staining: Adipose 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. The tissue sections were scanned using the Tissue FAXS system.

[0240] (5) Immunofluorescence staining of tissues: Paraffin sections of mouse fat samples were serially stained with anti-UCP1 and anti-Perilipin antibodies. CF-488-Tyramide and CF-594-Tyramide were used as tyramine conversion reagents. Antigen retrieval was performed using 1 mmol pH 9.0 Tris-EDTA solution. Samples were blocked with DAPI. Full slide scanning was performed using the TissueFAXS Plus automated collection system.

[0241] 5.1NB21-mFc enhances heat generation

[0242] Next, the ability of NB21-mFc to promote thermogenesis in vivo was assessed. Seven days after intraperitoneal injection of NB21-mFc ( Figure 5 a) ANCOVA analysis was performed with body weight as a covariate. The results showed that total energy expenditure (EE) was significantly increased in NB21-mFc-treated mice. Figure 5 b) There were no significant differences in cumulative food intake, physical activity, and respiratory exchange rate (RER) between the two groups.

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

[0244] The above results indicate that NB21-mFc can enhance thermogenesis and energy consumption in mouse adipose tissue.

[0245] 5.2 The promoting effect of NB21-mFc on browning is dependent on LGR4

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

[0247] Through changes in the expression of pyrogen genes and mitochondrial respiratory genes ( Figure 6 (ac) and changes in oxygen consumption capacity ( Figure 6 d) This indicates that the neutralizing effect of NB21-mFc on the inhibition of adipose browning by the RSPO1-LGR4 complex is observed in LGR4-deficient brown adipocytes (Lgr4). m / m These results indicate that NB21-mFc enhances adipose-derived 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), this phenomenon is mainly attributed to the reduction of vWAT and sWAT. Figure 6 g), and accompanied by a decrease in adipocyte volume and stronger staining of the browning marker (UCP1). Figure 6 h). Consistent with in vitro results, NB21-mFc enhanced the expression of pyrogen and mitochondrial respiratory genes in vWAT and sWAT. Figure 6 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 these browning-promoting and anti-obesity effects in Lgr4m / m littermates. Figure 6 ej).

[0250] In summary, these findings suggest that NB21-mFc primarily alleviates diet-induced obesity by promoting LGR4-dependent thermogenesis.

[0251] This invention not only provides a new perspective on understanding the mechanism of action of LGR4 in obesity, but also opens up new avenues for developing novel therapeutic drugs based on the LGR4 target. 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 this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

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, which comprises complementarity determining regions CDR1, CDR2 and CDR3 defined by Chothia, Abm, Kabat, or IMGT rules.

2. The Nanobody of claim 1, wherein The CDR1, CDR2 and CDR3 are selected from the group consisting of: (a) CDRs determined based on IMGT rules: a CDR1 of amino acids as shown in SEQ ID No: 1, a CDR2 of amino acids as shown in SEQ ID No: 2, and a CDR3 of amino acids as shown in SEQ ID No: 3; or (b) CDRs determined based on Kabat rules: a CDR1 of amino acids as shown in SEQ ID No: 4, a CDR2 of amino acids as shown in SEQ ID No: 5, and a CDR3 of amino acids as shown in SEQ ID No:

6.

3. The nanobody of claim 1, wherein The nanobody is capable of specifically binding to LGR4 and blocking its interaction with RSPO1 / 2.

4. The nanobody of claim 1, wherein The antibody is a bivalent or multivalent antibody.

5. An isolated polynucleotide, comprising, The polynucleotide encodes a LGR4-targeting nanobody as claimed in claim 1 or 4.

6. The polynucleotide of claim 5, wherein The polynucleotide comprises: (1) a nucleotide sequence as shown in SEQ ID No. 8; or (2) a complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID No. 8; (3) a nucleotide sequence encoding the same protein as the nucleotide sequence of (1) or (2), but differs from the nucleotide sequence of (1) or (2) due to the degeneracy of the genetic code.

7. An expression vector comprising the nucleic acid of claim 1. The expression vector contains the polynucleotide as claimed in claim 5.

8. A host cell, characterized in that, The host cell contains the expression vector as claimed in claim 7, or has integrated into its genome the polynucleotide as claimed in claim 5.

9. A fusion protein, characterized in that, The fusion protein contains: (a) the nanobody as claimed in claim 1 or 2; and (b) a tag sequence to facilitate expression and / or purification, or an antibody Fc fragment.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (ii) the LGR4-targeting nanobody as claimed in claim 1 or 2, the LGR4-targeting antibody as claimed in claim 4, or the fusion protein as claimed in claim 9; and (ii) a pharmaceutically acceptable carrier.

11. Use of a Nanobody according to claim 1 or 2, an antibody according to claim 4 or a fusion protein according to claim 9, characterized in that, for the preparation of an anti-obesity medicament.

12. Use of an active ingredient, characterized in that The active ingredient is selected from the group consisting of the LGR4-targeting nanobody as claimed in claim 1, the LGR4-targeting antibody as claimed in claim 4, or the fusion protein as claimed in claim 9, or a combination thereof, (a) for the preparation of a medicament for the treatment of obesity; and / or (b) for the preparation of a reagent for the detection of LGR4, an affinity medium product, a detection plate or a kit.

13. The use according to claim 12, characterized in that, The obesity is selected from the group consisting of simple obesity, obesity syndrome, secondary obesity.

14. A method of detecting LGR4 or a fragment thereof in a sample in vitro which is neither diagnostic nor therapeutic, characterized in that, The method comprises the steps of: (1) contacting, in vitro, the sample with the LGR4-targeting nanobody as claimed in claim 1, or the LGR4-targeting antibody as claimed in claim 4; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of LGR4 or a fragment thereof in the sample.

15. A method of producing a recombinant polypeptide, comprising: The method comprises: (a) culturing the host cell of claim 8 of the present application under conditions suitable for expression; (b) isolating from the culture the recombinant polypeptide, which is the LGR4-targeting nanobody of claim 1, the LGR4-targeting antibody of claim 4, or the fusion protein of claim 9.

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