Calcium-sensitive receptor nanobodies with positive allosteric regulation effects and their applications

Abstract

This invention belongs to the field of biotechnology, specifically relating to calcium-sensitive receptor nanobodies with positive allosteric regulation effects and their applications. These nanobodies can specifically recognize and bind to CaSR with good affinity, and can serve as positive allosteric regulators of CaSR. They can be used to prepare products for the diagnosis, prevention, and / or treatment of CaSR-related diseases, or for detecting the presence or level of CaSR in samples.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to calcium-sensitive receptor nanobodies with positive allosteric regulatory effects and their applications. Background Technology

[0002] Calcium-sensing receptors (CaSRs) are receptors for calcium ions (Ca) in the human body. 2+ CaSRs are biological receptors for various nutrients, including calcium ions (C1N1 and C2N1), located on the cell membrane surface and belonging to the C group of G protein-coupled receptors (GPCRs). Studies have confirmed that CaSRs are widely distributed in various tissues and organs of the human body, typically existing as homodimers on the cell membrane surface to perform physiological functions. (The text abruptly shifts to a seemingly unrelated topic about blood Ca2N1 and its role in nutrient absorption.) 2+ Ligands bind to the extracellular Venus flytrap domain (VFT domain) of CaSR, activating and inducing conformational changes in the CaSR homodimer, further triggering the coupling of the seven-transmembrane domain (7TM domain) to intracellular G protein signaling pathways and downstream cellular physiological activities. CaSR primarily couples to the intracellular Gq / 11 class G protein signaling pathway, inducing the accumulation of phosphatidylinositol and Ca2+ in the endoplasmic reticulum. 2+ Release and other cellular physiological behaviors. Studies have shown that CaSR can also couple to the Gi / o pathway, inhibiting the accumulation of cyclic adenosine monophosphate (cAMP) and activating the mitogen-activated protein kinase (MAPK)-extracellular regulated protein kinase (ERK1 / 2) pathway. 2+ It plays an important role in physiological activities such as muscle contraction, cell secretion, and blood clotting. Blood calcium... 2+ The concentration is usually in a steady state. When the blood calcium concentration is too high or too low, the body regulates the blood calcium concentration through specific feedback mechanisms to maintain a balance with calcium levels. 2+ All related physiological functions proceed normally. CaSR is mainly distributed on the surface of parathyroid cells, and its most important physiological function is to maintain the body's calcium levels by controlling the secretion of parathyroid hormone (PTH). 2+ Homeostasis is crucial for calcium metabolism and it plays a vital role in various physiological processes, including bone metabolism. When PTH levels rise, it activates parathyroid hormone receptors (PTHRs) expressed in bones and kidneys, thereby directly or indirectly promoting calcium uptake by organs such as bones, kidneys, and the gastrointestinal tract. 2+ The reabsorption of calcium leads to an increase in blood calcium concentration. Correspondingly, the secretion of PTH in parathyroid cells is affected by calcium... 2+ Concentration feedback regulation occurs when elevated blood calcium levels activate CaSR on the surface of parathyroid cells, thereby inducing the accumulation of phosphatidylinositol and endoplasmic reticulum calcium. 2+This involves cellular physiological behaviors such as release, thereby inhibiting PTH secretion and stabilizing blood calcium levels. Besides regulating parathyroid gland secretion and maintaining the body's calcium levels... 2+ In a homeostatic state, CaSR is also widely distributed in different tissues and organs of the body, playing different physiological functions.

[0003] Because CaSR plays an important role in many physiological activities, its dysfunction can lead to problems related to Ca. 2+ Various function-related diseases. For example, certain gain-of-function genetic mutations can lead to CaSR-Ca... 2+ Excessive sensitivity can lead to familial hypocalciciuric hypercalcemia type 1 (FHH1), characterized by lifelong slightly elevated serum calcium, low urinary calcium, and elevated parathyroid hormone levels; or it can cause neonatal severe hyperparathyroidism (NSHPT), characterized by significantly elevated serum calcium and parathyroid hormone levels. Conversely, certain loss-of-function genetic mutations can cause CaSR to be more sensitive to calcium. 2+ Insufficient sensitivity leads to autosomal dominant hypocalcemia type 1 (ADH1), manifesting as epilepsy, convulsions, and seizures. In severe cases, it is accompanied by Bartter syndrome type V (BSV), exhibiting severe symptoms such as hypokalemia, hyperkalemia, and renal hypoplasia. As related research continues to deepen, CaSR has become a key indicator of calcium deficiency. 2+ Important drug targets for functional diseases.

[0004] Because CaSR plays a crucial role in the pathogenesis of various diseases, it has become a research hotspot. Developing specific drugs targeting CaSR, especially allosteric modulators, to regulate CaSR activity at disease sites will help further explore the activation mechanism of CaSR and its correlation with various diseases. To date, four positive allosteric modulators (PAMs) targeting CaSR have been approved for marketing, all of which are peptides or compounds. No antibody drugs targeting CaSR are currently in clinical trials. Summary of the Invention

[0005] The first aspect of the present invention aims to provide CaSR nanobodies or antigen-binding fragments thereof.

[0006] A second aspect of the present invention aims to provide a CaSR heavy chain antibody or an antigen-binding fragment thereof.

[0007] A third aspect of the present invention aims to provide a chimeric antigen receptor.

[0008] The fourth aspect of this invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0009] The fifth aspect of this invention aims to provide isolated nucleic acid molecules.

[0010] The sixth aspect of this invention is to provide a carrier.

[0011] The seventh aspect of the present invention is to provide a cell.

[0012] The object of the eighth aspect of the present invention is to provide a method for preparing a nanobody or antigen-binding fragment thereof according to the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof according to the second aspect, a chimeric antigen receptor according to the third aspect, or a multispecific antibody or antigen-binding fragment thereof according to the fourth aspect.

[0013] The object of the ninth aspect of the present invention is to provide a coupling.

[0014] The object of the tenth aspect of this invention is to provide a pharmaceutical composition.

[0015] The object of the eleventh aspect of this invention is to provide a diagnostic or therapeutic reagent kit.

[0016] The object of the twelfth aspect of the present invention is to provide the use of the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the carrier of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] A first aspect of the present invention provides a CaSR nanobody or an antigen-binding fragment thereof, said CaSR nanobody or antigen-binding fragment comprising:

[0019] The heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3, having the amino acid sequence shown in SEQ ID NO: 12 or 17.

[0020] CaSR nanobody or antigen-binding fragment thereof, wherein the CaSR nanobody or antigen-binding fragment thereof includes a heavy chain variable region, the heavy chain variable region comprising:

[0021] a1) CDR-H1 having the amino acid sequence shown in SEQ ID NO:9, CDR-H2 having the amino acid sequence shown in SEQ ID NO:10, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:11; or

[0022] a2) CDR-H1 having the amino acid sequence shown in SEQ ID NO:14, CDR-H2 having the amino acid sequence shown in SEQ ID NO:15, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:16.

[0023] In some embodiments, the heavy chain variable region of the CaSR nanobody or its antigen-binding fragment further includes the framework region of the heavy chain variable region.

[0024] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0025] In some embodiments, the CaSR nanobody or its antigen-binding fragment includes a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:12 or 17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0026] A second aspect of the present invention provides a CaSR heavy chain antibody or an antigen-binding fragment thereof, comprising an immunoglobulin Fc domain and a nanobody or an antigen-binding fragment thereof of the first aspect of the present invention.

[0027] In some embodiments, the immunoglobulin Fc domain includes the Fc domain of an immunoglobulin derived from a mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, or goose, or a mutant thereof.

[0028] A third aspect of the invention provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof of the first aspect of the invention, or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the invention.

[0029] A fourth aspect of the present invention provides a multispecific antibody or an antigen-binding fragment thereof comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof of the first aspect of the present invention or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the present invention.

[0030] A fifth aspect of the invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding nanobodies or antigen-binding fragments thereof of the first aspect of the invention, heavy chain antibodies or antigen-binding fragments thereof of the second aspect, chimeric antigen receptors of the third aspect, or multispecific antibodies or antigen-binding fragments thereof of the fourth aspect.

[0031] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0032] In some embodiments, the nucleotide sequence encoding the nanobody or its antigen-binding fragment of the first aspect of the invention comprises: SEQ ID NO: 13 or 18, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0033] A sixth aspect of the present invention provides a carrier comprising the nucleic acid molecule of the fifth aspect of the present invention.

[0034] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic cell expression vectors and / or prokaryotic cell expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0035] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0036] A seventh aspect of the present invention provides a cell comprising a nanobody or antigen-binding fragment thereof of the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present invention, a chimeric antigen receptor of the third aspect of the present invention, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a nucleic acid molecule of the fifth aspect of the present invention, or a carrier of the sixth aspect of the present invention.

[0037] In some embodiments, the cells do not involve reproductive material.

[0038] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the nanobody or its antigen-binding fragment, heavy chain antibody or its antigen-binding fragment, chimeric antigen receptor or multispecific antibody or its antigen-binding fragment disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0039] In some embodiments, the cells may be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, dendritic cells (DCs), and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptors described above (i.e., modified immune cells).

[0040] The eighth aspect of the present invention provides a method for preparing the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, obtained by culturing the cells of the seventh aspect of the present invention.

[0041] A ninth aspect of the invention provides a conjugate comprising a nanobody or an antigen-binding fragment thereof of the first aspect of the invention, or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the invention; and a conjugation portion.

[0042] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0043] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the nanobodies or their antigen-binding fragments of the present invention, or heavy chain antibodies or their antigen-binding fragments, of different lengths via linkers of varying lengths to reduce potential steric hindrance.

[0044] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0045] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of CaSR-related diseases.

[0046] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0047] A tenth aspect of the present invention provides a pharmaceutical composition comprising: a nanobody or antigen-binding fragment thereof of the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present invention, a chimeric antigen receptor thereof of the third aspect of the present invention, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a nucleic acid molecule thereof of the fifth aspect of the present invention, a carrier thereof of the sixth aspect of the present invention, a cell thereof of the seventh aspect of the present invention, or a conjugate thereof of the ninth aspect of the present invention; and a pharmaceutically acceptable carrier.

[0048] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0049] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug that can prevent and / or treat CaSR-related diseases.

[0050] In some embodiments, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0051] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0052] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0053] The eleventh aspect of the present invention provides a diagnostic or therapeutic kit comprising: a nanobody or antigen-binding fragment thereof of the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present invention, a chimeric antigen receptor thereof of the third aspect of the present invention, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a nucleic acid molecule thereof of the fifth aspect of the present invention, a carrier thereof of the sixth aspect of the present invention, a cell thereof of the seventh aspect of the present invention, a conjugate thereof of the ninth aspect of the present invention, or a pharmaceutical composition thereof of the tenth aspect of the present invention.

[0054] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0055] In some implementations, the kit can be used to diagnose CaSR-related diseases.

[0056] In some embodiments, the kit can be used to prevent and / or treat CaSR-related diseases.

[0057] The twelfth aspect of the present invention provides the use of the nanobody or antigen-binding fragment thereof of the first aspect, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the carrier of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect in any one of c1)-c4):

[0058] c1) Prepare products for diagnosing CaSR-related diseases;

[0059] c2) Prepare drugs for the prevention and / or treatment of CaSR-related diseases;

[0060] c3) Prepare a product to detect the presence or level of CaSR in the sample;

[0061] c4) Preparation of positive allosteric modifiers for CaSR.

[0062] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0063] In some embodiments, the body fluid includes at least one of blood and lymph.

[0064] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0065] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0066] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0067] In some implementations, the subject of the test includes humans.

[0068] In some embodiments, the product comprises at least one of reagents, kits, test strips, chips, devices, and systems.

[0069] In this invention, the CaSR is a human CaSR (preferably whose amino acid sequence contains SEQ ID NO: 1).

[0070] In this invention, the CaSR-related diseases are selected from the group consisting of: familial hypocalcemia with hypercalcemia (FHH), autosomal dominant hypocalcemia (ADH), severe neonatal hyperparathyroidism (NSHPT), primary hyperparathyroidism (PHPT), severe secondary hyperparathyroidism in patients undergoing dialysis due to renal failure, triple hyperparathyroidism, persistent or recurrent hyperparathyroidism, hyperparathyroidism occurring after kidney transplantation, lithium-induced hyperparathyroidism, hypoparathyroidism, kidney stones, hypomagnesemia, hypermagnesemia, calcium allergy, osteoporosis, CaSR dysfunction caused by activation or inactivation of autoantibodies, hypocalcemia, hypercalcemia, and hypomagnesemia-related diseases.

[0071] The beneficial effects of this invention are:

[0072] This invention provides CaSR nanobodies or antigen-binding fragments thereof, which can specifically recognize and bind to CaSR and have good affinity for it, and can be used as positive allosteric modulators of CaSR. They can be used to prepare products for the diagnosis, prevention and / or treatment of CaSR-related diseases, or for the detection of the presence or level of CaSR in samples. Attached Figure Description

[0073] Figure 1 A schematic diagram showing the results of the affinity test between the nanobody NB-34F7 and the antigen is presented.

[0074] Figure 2 A schematic diagram showing the results of the affinity test between the nanobody NB-53A12 and the antigen is presented.

[0075] Figure 3 The positive allosteric regulatory effect of nanobody NB-34F7 on CaSR activation was demonstrated.

[0076] Figure 4 The positive allosteric regulatory effect of nanobody NB-53A12 on CaSR activation was demonstrated. Detailed Implementation

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0078] Definitions

[0079] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0080] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0081] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0082] K D values dissociation constant (K) D K is a specific type of equilibrium constant used to measure the tendency of a larger component to separate (dissociate) from another smaller component. It is the reciprocal of the association constant and is measured in mol / L (M) or nmol / L (nM). D The smaller the value, the stronger the binding ability of the two substances.

[0083] Nanobody Naturally occurring antibodies lacking the light chain, found in the peripheral blood of camels, contain only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Unlike artificially engineered single-chain antibody fragments, they do not readily adhere to each other or aggregate. Individually cloned and expressed VHH structures exhibit structural stability and antigen-binding activity comparable to the original heavy chain antibody, representing the smallest known unit capable of binding to target antigens. VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only about 15 kDa, hence the name nanobody (Nb). Compared to traditional animals like mice and rabbits that can only recognize flat peptides on the surface of antigens, the immune system in camels can recognize the complex spatial structures on the antigen surface, producing highly specific and high-affinity nanobodies.

[0084] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of this invention utilizes antibodies produced by the alpaca's immune system, known as "nanobodies." Nanobodies are tiny antibody fragments isolated from immunoglobulins in animals such as camels. They possess the same antigen-binding ability and structural stability as intact antibodies and are the smallest existing units capable of binding target antigens, with a relative molecular mass of only about 15 kDa. Compared to traditional methods where mice and rabbits can only recognize flat polypeptides on the surface of antigens, the immune systems in animals like alpacas can recognize the complex spatial structures on the surface of antigens, enabling the production of highly specific and high-affinity nanobodies.

[0085] According to the technical solution of the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below.

[0086] Table 1

[0087] Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gin Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gin Val Ile; Leu Ile Leu; Val

[0088] Furthermore, due to the degeneracy of bases, bases in polynucleotide sequences can be substituted without altering the activity or function of the polynucleotide sequence, as shown in Table 2 below.

[0089] Table 2

[0090]

[0091]

[0092] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0093] Example 1. Preparation of antigen

[0094] 1.1 The DNA vector plasmid (pRK5) containing nucleotides encoding human CaSR was co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher);

[0095] 1.2 After transfection, cells were cultured for approximately 24 hours to allow CaSR to be fully expressed on the cell membrane surface;

[0096] 1.3 Collect approximately 4*10^8 cells, which are then subjected to ultrasonic disruption, homogenization, and ultracentrifugation. The cell membrane extract is then obtained as a suitable antigen.

[0097] The antigen was divided into four equal portions for use in the subsequent four alpaca immunizations.

[0098] The advantage of this invention is that CaSR can be correctly expressed and exists on the cell membrane surface, maintaining the correct assembly, folding and conformational state, especially maintaining the complex spatial structure of the surface.

[0099] The amino acid sequence of human CaSR is as follows:

[0100]

[0101] The nucleotide sequence encoding human CaSR is as follows:

[0102]

[0103]

[0104]

[0105] Example 2. Alpaca Immunization

[0106] In this embodiment, the antigen prepared in Example 1 is used to immunize alpacas. The specific steps are as follows:

[0107] (1) The alpaca was immunized a total of 4 times. The antigen was injected subcutaneously into the animal. The first immunization was recorded as day 1, and the subsequent immunizations were on day 11, day 21 and day 31 respectively.

[0108] (2) On day 30, before the fourth immunization injection, about 200 mL of alpaca peripheral blood was collected from the vein;

[0109] (3) On day 45, that is, 14 days after the fourth immunization, about 200 mL of alpaca peripheral blood was collected.

[0110] Compared to traditional immunization techniques using animal antibodies from mice, rabbits, etc., the technical advantage of this invention lies in the collection of a large amount of peripheral blood from alpacas, which is beneficial for subsequent screening to obtain highly diverse nanobodies.

[0111] Example 3. Construction of an alpaca nanobody library

[0112] Using two batches of alpaca peripheral blood collected in Example 2 as raw materials, a highly diverse nanobody library was constructed. The processing methods for the two batches of alpaca peripheral blood were the same, and the specific steps are as follows:

[0113] (1) Lymphocytes were isolated from peripheral blood of alpaca veins using density gradient centrifugation and other methods;

[0114] (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA;

[0115] (3) Using appropriate DNA primers (see Table 3 below), the above cDNA was used as a template to amplify the VHH fragments of alpaca immunoglobulins IgG2 and IgG3 by polymerase chain reaction (PCR), which are the DNA fragments of nanobodies.

[0116] Table 3. Primers used to construct nanobody libraries

[0117]

[0118]

[0119] (4) Link the DNA of VHH to the phage surface display selection vector phen1 to form a VHH-His-pIII fusion protein expression vector plasmid library; where pIII is a protein present on the flagella on the surface of the phage, and His is a histidine tag (6 consecutive histidines, i.e. His-His-His-His-His-His).

[0120] (5) The DNA ligation product was transformed into TG1 competent Escherichia coli by electroporation. After culturing, all colonies were collected to obtain the alpaca nanobody library.

[0121] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this invention can preserve all nanobody fragments (i.e., libraries) of alpacas for a long time, which can continuously support the subsequent screening and development of nanobodies.

[0122] Example 4. Displaying screening-specific nanobodies on the surface of bacteriophages

[0123] This embodiment uses the nanobody library obtained in Example 3 as the source, and antigen-specific nanobodies are obtained through phage surface display screening. The specific steps are as follows:

[0124] (1) Take an appropriate amount of frozen nanobody library, inoculate it into LB medium containing host Escherichia coli TG1, and after culturing, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture under appropriate conditions.

[0125] (2) The bacteriophages amplified in the bacterial culture supernatant were extracted by PEG-NaC method and frozen in an ultra-low temperature freezer at -80℃ for later use.

[0126] (3) The DNA vector plasmid (pRK5) containing nucleotides encoding human CaSR was co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher);

[0127] (4) Negative screening: Prepare about 5*10^7 HEK293T tool cells that have not been transfected with any exogenous DNA vector plasmids, thaw the phage, incubate the phage and tool cells together for 2 hours, centrifuge and retain the supernatant;

[0128] (5) Positive screening: Collect approximately 5*10^7 HEK293T cells overexpressing CaSR obtained in (3) and incubate them together with the supernatant obtained after centrifugation in (4) for 2 hours.

[0129] (6) Washing: Discard the phages, then wash the antigen 3-5 times with PBS buffer to wash away the phages that are not specifically bound to the antigen, and retain the phages that are specifically bound to the antigen.

[0130] (7) Elution: The phages that specifically bind to the antigen are treated with an acidic glycine solution to dissociate the phages from the antigen and retain them.

[0131] At this point, bacteriophages expressing specific nanobodies have been obtained, and these bacteriophages can be used for the following technical operations:

[0132] (8) Transformation into a specific nanobody library: The phage is reinfected into TG1 competent E. coli, but no helper phage is added. After the phage infection is complete, the specific nanobody exists in the E. coli in the form of DNA plasmids. Collecting all these E. coli results in an antigen-specific nanobody library. This library can be used as raw material to return to step (1) for the next round of phage surface display screening.

[0133] (9) Conversion into monoclonal nanobody colonies: Take a small amount of the phage obtained in step (7) (approximately 0.5%), dilute it, and re-infect TG1 competent Escherichia coli, but do not add helper phages. After the phage infection is complete, spread these E. coli evenly on bacterial culture dishes and culture to obtain monoclonal colonies containing nanobody DNA plasmids. Use these monoclonal colonies as raw materials to identify positive monoclonal nanobodies.

[0134] Example 5. Identification of positive monoclonal nanobodies and sequencing of nanobodies

[0135] This embodiment utilizes the bacterial culture dish with monoclonal colonies obtained in step (9) of Example 4 to identify positive monoclonal nanobodies. The specific steps are as follows:

[0136] (1) Pick single colonies and culture them in microplates;

[0137] (2) Adding IPTG (isopropyl-β-D-thiogalactoside) to induce the expression of VHH-pIII (i.e., the fusion protein containing nanobodies);

[0138] (3) The DNA vector plasmid (pRK5) containing nucleotides encoding human CaSR was co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher);

[0139] (4) Collect the bacterial culture supernatant containing nanobodies obtained in (2) and incubate it with the cells obtained in (3) for 2 hours. Use about 2*10^5 CaSR-overexpressing cells in each well of the 96-well plate. After incubation, centrifuge and discard the supernatant.

[0140] (5) Incubate the cells obtained above with the His tag-specific mouse monoclonal antibody labeled with Alexa Flour 647 (brand: Research & Development, IC0501R) for 2 hours;

[0141] (6) Centrifuge, discard the supernatant, and resuspend the cells with PBS buffer. Resuspend each well of the 96-well plate with 100 μL of PBS solution.

[0142] (7) The distribution of Alexa Flour 647 fluorescence intensity on the cell surface was detected by flow cytometry to determine whether the nanobody could bind to CaSR on the cell surface.

[0143] For monoclonal nanobodies (NB-34F7, NB-53A12) that can specifically recognize and bind to cell surface antigens, the DNA plasmids of the TG1 strain expressing the relevant monoclonal nanobodies were cultured overnight at 37°C and then Sanger sequenced to obtain the nucleotide sequence of the nanobodies. After translation, the complete amino acid sequence of the nanobodies was obtained, as shown in Table 4-5.

[0144] Table 4: Amino acid and nucleotide sequences of NB-34F7

[0145]

[0146]

[0147] Table 5: Amino acid and nucleotide sequences of NB-53A12

[0148]

[0149] Example 6: Recombinant Expression and Purification of Small-Batch Monoclonal Nanobodies

[0150] (1) In Example 5, monoclonal nanobodies that can specifically recognize and bind to antigens were obtained. The DNA plasmids encoding the above nanobodies (NB-34F7, NB-53A12) were transformed into BL21(DE3) competent cells. The nanobodies were recombinantly expressed and purified by Escherichia coli, and the batch production capacity was about a few milligrams.

[0151] (2) Flow cytometry analysis method: different concentrations of nanobodies were incubated, and the affinity between the nanobodies and the antigen was measured based on the binding ability of the nanobodies to HEK293T cells expressing human CaSR (preparation method is the same as in Example 1).

[0152] Test results as follows Figures 1-2 As shown, the affinity values ​​K of monoclonal nanobodies NB-34F7 and NB-53A12 are... D The values ​​are 23.19 nM and 29.50 nM, respectively.

[0153] (3) Functional identification of nanobodies

[0154] Upon activation of CaSR on the cell membrane, the Gq protein trimer coupled with CaSR dissociates. The Gq protein further activates phosphatidylinositol-specific phospholipase C (PLC) on the inner side of the plasma membrane. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) near the inner side of the plasma membrane into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These two hydrolysates act as second messengers in cellular signal transduction. IP3 binds to the IP3 ligand-gated channel on the endoplasmic reticulum membrane, opening calcium ion channels and releasing calcium ions from the endoplasmic reticulum into the cell, causing a rapid increase in intracellular calcium ion concentration. IP3 is further converted into inositol 1,4-bisphosphate (IP2) and inositol 1-phosphate (IP1). The accumulation of IP1 can be detected using the IP-one HTFR assay (HTRF IP-OneGq Detection Kit, Revvity#62IPAPEB) to characterize CaSR activation. In the presence of lithium chloride (LiCl), IP1 degradation is inhibited and it accumulates in signaling cells. In IP-One HTRF assays, IP1 with a d2 fluorescent receptor can compete with naturally produced IP1 for binding to IP1-specific monoclonal antibodies with fluorescent donors of cavitation compounds. Increased production of unlabeled IP1 (natural IP1) leads to disruption of TR-FRET and attenuation of HTRF signal intensity.

[0155] The steps of the IP-one HTFR experiment are as follows:

[0156] 1) HEK293T cells expressing human CaSR (prepared in the same way as in Example 1) were separated into 96-well cell culture plates with a black, opaque background as required;

[0157] 2) Dissolve and dispense IP1-d2 and Ab-K according to the instructions, and store at -20℃ away from light;

[0158] 3) Based on the experimental dosage, dilute 5× drug stimulation buffer (SB) with ultrapure water to 1× for later use;

[0159] 4) Prepare 1×SB (+NB-34F7 / +NB-53A12) and 1×SB (-NB-34F7 / -NB-53A12) containing stimulating drugs using 1×SB: 1×SB (+NB-34F7 / +NB-53A12) containing stimulating drugs refers to 1×SB containing nanobody (NB-34F7 / NB-53A12) and CaCl2, with a final concentration of 100 nM for the nanobody (NB-34F7 / NB-53A12) and 8 final concentrations of CaCl2 (starting at 0.02 M and 2-fold dilution); 1×SB (-NB-34F7 / -NB-53A12) refers to 1×SB containing CaCl2, with 8 final concentrations of CaCl2 (starting at 0.02 M and 2-fold dilution).

[0160] 5) Discard the culture medium in the 96-well cell culture plate and add 70 μL / well of 1×SB(+NB-34F7 / +NB-53A12) or 1×SB(-NB-34F7 / -NB-53A12) containing the stimulating drug.

[0161] 6) Place the 96-well cell culture plate in a cell culture incubator and stimulate for 30 minutes;

[0162] 7) Prepare appropriate amounts of IP1-d2 and Ab-K in lysis buffer (LB) at a ratio of 1:19, add them to the cells, first add IP1-d2, then add Ab-K, 15 μL / well;

[0163] 8) Incubate at room temperature in the dark for 1 hour;

[0164] 9) Use the PHERA Star multi-functional microplate reader for detection. Excite with 337nm wavelength and read the emission values ​​at 665nm and 615nm wavelengths respectively.

[0165] 10) Export the values ​​and process the data, then use GraphPadPrism to create the graph.

[0166] The results are as follows Figures 3-4 As shown, the pEC50 values ​​of calcium ions are as follows: 2.568 (-NB-34F7), 2.744 (+NB-34F7), 2.571 (-NB-53A12), and 2.735 (+NB-53A12). Based on the above results, it can be seen that the nanobodies NB-34F7 and NB-53A12 can promote the activation of CaSR by calcium ions, and act as positive allosteric regulators of CaSR.

[0167] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A CaSR nanobody or its antigen-binding fragment, wherein the CaSR nanobody or its antigen-binding fragment comprises: CDR-H1, CDR-H2, and CDR-H3 are included in the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 12 or 17.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The CaSR nanobody or its antigen-binding fragment includes a heavy chain variable region, the heavy chain variable region comprising: a1) CDR-H1 of the amino acid sequence shown in SEQ ID NO: 9, CDR-H2 of the amino acid sequence shown in SEQ ID NO: 10, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 11; or a2) CDR-H1 with the amino acid sequence shown in SEQ ID NO: 14, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 15, and CDR-H3 with the amino acid sequence shown in SEQ ID NO:

16.

3. The nanobody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain variable region of the CaSR nanobody or its antigen-binding fragment also includes the framework region of the heavy chain variable region.

4. The nanobody or its antigen-binding fragment according to claim 3, characterized in that, The frame region of the heavy chain variable region includes the frame region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

5. The nanobody or its antigen-binding fragment according to claim 4, characterized in that, The CaSR nanobody or its antigen-binding fragment includes a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:12 or 17.

6. A CaSR heavy chain antibody or an antigen-binding fragment thereof, comprising an immunoglobulin Fc domain and a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-5.

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a nanobody or an antigen-binding fragment thereof as claimed in any one of claims 1-5 or a heavy chain antibody or an antigen-binding fragment thereof as claimed in claim 6.

8. A carrier comprising the nucleic acid molecule of claim 7.

9. A cell comprising the nanobody or antigen-binding fragment thereof as described in any one of claims 1-5, the heavy chain antibody or antigen-binding fragment thereof as described in claim 6, the nucleic acid molecule as described in claim 7, or the carrier as described in claim 8.

10. A method for preparing the nanobody or its antigen-binding fragment according to any one of claims 1-5 or the heavy chain antibody or its antigen-binding fragment according to claim 6, wherein the nanobody is obtained by culturing the cells according to claim 9.

11. A conjugate comprising a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-5, or a heavy chain antibody or an antigen-binding fragment thereof as described in claim 6; and a conjugation portion.

12. The coupling according to claim 11, characterized in that, The coupling component includes a detectable marker or therapeutic agent.

13. The coupling according to claim 12, characterized in that, The detectable markers include enzymes, radionuclides, luminescent substances, and / or biotin; and / or The therapeutic agents include drugs for the prevention and / or treatment of CaSR-related diseases; The CaSR-related diseases were selected from the following groups: familial hypocalcemia with hypercalcemia (FHH), autosomal dominant hypocalcemia (ADH), severe neonatal hyperparathyroidism (NSHPT), primary hyperparathyroidism (PHPT), severe secondary hyperparathyroidism in patients undergoing dialysis for renal failure, triple hyperparathyroidism, persistent or recurrent hyperparathyroidism, hyperparathyroidism after kidney transplantation, lithium-induced hyperparathyroidism, hypoparathyroidism, kidney stones, hypomagnesemia, hypermagnesemia, calcium hypersensitivity, osteoporosis, CaSR dysfunction caused by activation or inactivation of autoantibodies, hypocalcemia, and hypercalcemia.

14. The coupling according to claim 13, characterized in that, The detectable markers include fluorescent dyes.

15. The coupling according to claim 13, characterized in that, The detectable markers include colored substances.

16. A pharmaceutical composition comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-5, the heavy chain antibody or its antigen-binding fragment according to claim 6, the nucleic acid molecule according to claim 7, the carrier according to claim 8, the cell according to claim 9, or the conjugate according to any one of claims 11-15; and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition also includes additional pharmaceutically active agents.

18. Diagnostic or therapeutic reagent kits, comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-5, the heavy chain antibody or its antigen-binding fragment according to claim 6, the nucleic acid molecule according to claim 7, the carrier according to claim 8, the cell according to claim 9, the conjugate according to any one of claims 11-15, or the pharmaceutical composition according to any one of claims 16-17.

19. The diagnostic or therapeutic kit according to claim 18, characterized in that, The kit also includes instructions and / or a delivery device.

20. The use of the nanobody or antigen-binding fragment thereof according to any one of claims 1-5, the heavy chain antibody or antigen-binding fragment thereof according to claim 6, the nucleic acid molecule according to claim 7, the carrier according to claim 8, the cell according to claim 9, the conjugate according to any one of claims 11-15, or the pharmaceutical composition according to any one of claims 16-17 in any one of c1)-c3): c1) Prepare products for diagnosing CaSR-related diseases; c2) Prepare drugs for the prevention and / or treatment of CaSR-related diseases; c3) Prepare a product to detect the presence or level of CaSR in the sample; The CaSR-related diseases were selected from the following groups: familial hypocalcemia with hypercalcemia (FHH), autosomal dominant hypocalcemia (ADH), severe neonatal hyperparathyroidism (NSHPT), primary hyperparathyroidism (PHPT), severe secondary hyperparathyroidism in patients undergoing dialysis for renal failure, triple hyperparathyroidism, persistent or recurrent hyperparathyroidism, hyperparathyroidism after kidney transplantation, lithium-induced hyperparathyroidism, hypoparathyroidism, kidney stones, hypomagnesemia, hypermagnesemia, calcium hypersensitivity, osteoporosis, CaSR dysfunction caused by activation or inactivation of autoantibodies, hypocalcemia, and hypercalcemia.

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