Nano antibody as well as preparation method and application thereof

Abstract

The invention discloses a nano antibody and a preparation method and application thereof, the nano antibody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the amino acid sequence of the heavy chain variable region of the nano antibody comprises SEQ ID NO: 3. The nano antibody provided by the invention is determined as a competitive OTR inhibitor with extracellular treatment potential; in-vitro and in-vivo experiments prove that the nano antibody has a curative effect equivalent to that of atosiban which is widely used clinically, and a new direction is provided for developing effective anti-premature delivery medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a nanobody and its preparation method and application. Background Art

[0002] Oxytocin receptor (OTR) is a G protein-coupled receptor (GPCR) that is widely expressed in various tissues such as the uterus, mammary gland, brain, and heart. At the same time, the activation of OTR is closely related to reproductive processes such as childbirth and lactation. The OT-OTR system coordinates uterine contractions during childbirth and promotes lactation during breastfeeding, reflecting its important role in reproductive physiology. Emerging translational neuroscience research has shown that targeted activation of OTR has important therapeutic potential for a series of mental health disorders. These disorders include autism spectrum disorder, Asperger's syndrome, schizophrenia, and social anxiety disorder. By modulating social and cognitive functions, activation of OTR may provide new treatment approaches for these diseases. On the other hand, OTR antagonists have demonstrated clinical utility in various therapeutic settings. They have shown effectiveness in the treatment of male sexual dysfunction, assisted reproductive technology, and the management of spontaneous preterm birth. The endogenous agonist oxytocin is commonly used for induction of labor, and the OTR antagonist atosiban has been specifically approved for the clinical treatment of preterm birth.

[0003] Preterm birth is characterized by delivery before 37 weeks of gestation and poses a significant risk to maternal and neonatal health. Preterm neonates are at increased risk of developing respiratory distress syndrome, intraventricular hemorrhage, and long-term developmental disorders, and in severe cases, it may lead to maternal and neonatal death. In recent years, the preterm birth rate has been on the rise, from about 9.8% in 2000 to 10.6% in 2023. There is an urgent need for drugs to treat preterm birth. To address these key issues, targeting G protein-coupled receptor (GPCR)-mediated fetal resolution has become a key focus in the development of drug interventions aimed at reducing the incidence and complications of preterm birth. To date, GPCR drugs used clinically for the treatment of preterm birth include the β2-adrenergic receptor agonist ritodrine and the oxytocin receptor antagonist atosiban. Compared with ritodrine, atosiban has higher safety and specificity for both the mother and the fetus, but the widespread application of atosiban is still limited by high costs and the need for continuous intravenous administration, and such small molecule drugs tend to have a tendency to cross the placental barrier, thereby causing toxicity and fetal growth retardation.

[0004] Nanobodies (Nb), also known as single-domain antibodies (sdAbs), are a promising class of therapeutic agents that are derived from the unique heavy-chain antibodies found in camels. Nanobodies consist only of the variable domain (VHH) of the heavy-chain antibody and have the advantages of small size (12 - 15 kDa), strong tissue penetration, high stability, and simple preparation compared to traditional antibodies. Therefore, nanobodies have great potential in the field of biopharmaceutical research and development. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art.

[0006] To this end, the present invention provides a nanobody, which is identified as a competitive OTR inhibitor with extracellular therapeutic potential; the present invention has confirmed through in vitro and in vivo experiments that this nanobody has an efficacy equivalent to that of atosiban, a drug widely used clinically, providing a new direction for the development of effective anti-preterm labor drugs.

[0007] The present invention also provides a recombinant protein.

[0008] The present invention also provides a biomaterial related to the above-mentioned nanobody or the above-mentioned recombinant protein.

[0009] The present invention also provides a conjugate.

[0010] The present invention also provides a solid-phase carrier.

[0011] The present invention also provides an application.

[0012] The present invention also provides a product.

[0013] The present invention also provides a preparation method of the above-mentioned nanobody or the above-mentioned recombinant protein.

[0014] According to a first aspect of the present invention, there is provided a nanobody, the nanobody comprising a heavy-chain variable region, the heavy-chain variable region comprising CDR1, CDR2, and CDR3;

[0015] The amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

[0016] In some embodiments of the present invention, the amino acid sequence of the heavy-chain variable region of the nanobody comprises:

[0017] a1) SEQ ID NO:3; or

[0018] a2) An amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids to SEQ ID NO: 3 and having the same function as the protein shown in SEQ ID NO: 3; or

[0019] a3) An amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology with SEQ ID NO: 3 and having the same function as the protein shown in SEQ ID NO: 3.

[0020] According to the second aspect of the present invention, there is provided a recombinant protein comprising: the nanobody as described in the first aspect of the present invention and optionally a tag sequence for assisting expression and / or purification.

[0021] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag.

[0022] According to the third aspect of the present invention, there is provided a biological material related to the nanobody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect of the present invention, the biological material comprising at least one of h1) to h8):

[0023] h1) A nucleic acid molecule encoding the nanobody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect of the present invention;

[0024] h2) An expression cassette comprising the nucleic acid molecule of h1);

[0025] h3) A vector comprising the nucleic acid molecule of h1);

[0026] h4) A vector comprising the expression cassette of h2);

[0027] h5) A transgenic cell line comprising the nucleic acid molecule of h1);

[0028] h6) A transgenic cell line comprising the expression cassette of h2);

[0029] h7) A transgenic cell line comprising the vector of h3);

[0030] h8) A transgenic cell line comprising the vector of h4).

[0031] In some embodiments of the present invention, the transgenic cell line does not contain propagation materials.

[0032] In some embodiments of the present invention, the nucleic acid molecule encoding the nanobody described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the nanobody described in the first aspect of the present invention.

[0033] In some preferred embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the nanobody described in the first aspect of the present invention comprises:

[0034] a211) the nucleotide sequence shown in SEQ ID NO: 10; or

[0035] a212) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or several nucleotides to SEQ ID NO: 10 and having the same function as the nucleic acid molecule shown in SEQ ID NO: 10; or

[0036] a213) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homology with SEQ ID NO: 10 and having the same function as the nucleic acid molecule shown in SEQ ID NO: 10.

[0037] According to the fourth aspect of the present invention, a conjugate is provided, comprising: at least one of the nanobody described in the first aspect of the present invention and the recombinant protein described in the second aspect of the present invention;

[0038] and a coupling moiety, the coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, and a virus coat protein.

[0039] In some embodiments of the present invention, the detectable label is a fluorescent or luminescent label.

[0040] In some preferred embodiments of the present invention, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase, and alkaline phosphatase.

[0041] According to the fifth aspect of the present invention, a solid-phase carrier is provided, the surface of which is conjugated with the nanobody described in the first aspect of the present invention and / or the recombinant protein described in the second aspect of the present invention.

[0042] According to the sixth aspect of the present invention, there is provided the use of the nanobody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect, the biomaterial as described in the third aspect, the conjugate as described in the fourth aspect, and / or the solid-phase carrier as described in the fifth aspect in the preparation of a product.

[0043] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a kit, and a detection chip.

[0044] In some preferred embodiments of the present invention, the drug has the function of treating and / or preventing a disease or disorder for which it is known or can be shown that inhibiting oxytocin produces a beneficial effect.

[0045] In some more preferred embodiments of the present invention, the disease or disorder is selected from sexual dysfunction, hypoactive sexual desire disorder, sexual arousal disorder, orgasm disorder, dyspareunia, premature ejaculation, pre-term labor, delivery complications, appetite and eating disorders, benign prostatic hyperplasia, premature birth, dysmenorrhea, congestive heart failure, arterial hypertension, cirrhosis, renal hypertension, ocular hypertension, obsessive-compulsive disorder, and neuropsychiatric diseases.

[0046] In some more preferred embodiments of the present invention, the disease or disorder is premature birth.

[0047] According to the seventh aspect of the present invention, there is provided a product comprising at least one of k1) to k4):

[0048] k1) the nanobody as described in the first aspect of the present invention;

[0049] k2) the recombinant protein as described in the second aspect of the present invention;

[0050] k3) the conjugate as described in the fourth aspect of the present invention;

[0051] k4) the solid-phase carrier as described in the fifth aspect of the present invention.

[0052] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a test plate, a kit, and a detection chip.

[0053] In some preferred embodiments of the present invention, the drug has the function of treating and / or preventing a disease or disorder for which it is known or can be shown that inhibiting oxytocin produces a beneficial effect.

[0054] In some more preferred embodiments of the present invention, the disease or disorder is selected from sexual dysfunction, hypoactive sexual desire disorder, sexual arousal disorder, orgasm disorder, dyspareunia, premature ejaculation, prepartum delivery, delivery complications, appetite and eating disorders, benign prostatic hyperplasia, premature birth, dysmenorrhea, congestive heart failure, arterial hypertension, cirrhosis, renal hypertension, ocular hypertension, obsessive-compulsive disorder and neuropsychiatric diseases.

[0055] According to the eighth aspect of the present invention, there is provided a method for preparing the nanobody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect, which is obtained by culturing the transgenic cell line as described in the third aspect of the present invention.

[0056] The present invention has at least the following beneficial effects:

[0057] The nanobody Nb2G5 provided by the present invention can specifically bind to OTR and maintain the inactive conformation of OTR, inhibit the ligand function of OTR, thereby playing a role in treating and preventing premature birth; its inhibitory effect is equivalent to that of atosiban, a commonly used OTR inhibitor in current clinical practice. However, compared with atosiban, the nanobody Nb2G5 has superior tissue penetration and a longer half-life, can approach and bind to inaccessible epitopes, also has strong stability, is easy to genetically manipulate and can be directly produced in microbial systems; the nanobody Nb2G5 can not only specifically bind to OTR, but also stabilize the inactive state of OTR, and it has a unique molecular mechanism different from traditional small molecule antagonists, providing new ideas and technical support for the development of effective drugs for treating premature birth. Description of the Drawings

[0058] The following further describes the present invention in conjunction with the drawings and examples, wherein:

[0059] Figure 1 It is a schematic flow chart of screening nanobodies using a yeast synthetic nanobody display library in Example 1 of the present invention;

[0060] Figure 2 It is a schematic flow chart of screening nanobodies using a yeast synthetic nanobody display library in Example 1 of the present invention;

[0061] Figure 3 It is an SDS-PAGE electrophoresis result diagram of the nanobody Nb2G5 expressed in Example 3 of the present invention;

[0062] Figure 4This is the structural diagram of the complex of Nb2G5 and OTR observed by cryo-electron microscopy in the experimental examples of the present invention; among them, a is the overall pattern diagram of Nb2G5 binding to OTR, b is the structural analysis of the binding of CDR1 of Nb2G5 to OTR, c is the structural analysis of the binding of CDR3 of Nb2G5 to OTR, d is the structural analysis of the binding of the C’-sheet of Nb2G5 to OTR, e and f are the structural comparison analysis of Nb2G5 for the active state and inactive state of OTR, and g - i are the molecular mechanism comparison of the binding of Nb2G5 to OTR and the binding of oxytocin / atosiban to OTR;

[0063] Figure 5 This is the test result diagram of the inhibitory activity of the nanobody Nb2G5 with alanine substituted for TPY105 in the experimental examples of the present invention;

[0064] Figure 6 This is the test result diagram of the glosensor cAMP test of the nanobody Nb2G5 and atosiban in the experimental examples of the present invention;

[0065] Figure 7 This is the test result diagram of the glosensor cAMP test of the nanobody Nb2B1 and atosiban in the experimental examples of the present invention;

[0066] Figure 8 This is the test result diagram of the glosensor cAMP test of the nanobody Nb2B11 and atosiban in the experimental examples of the present invention;

[0067] Figure 9 This is the test result diagram of the glosensor cAMP test of the nanobody Nb2H4 and atosiban in the experimental examples of the present invention;

[0068] Figure 10 This is the test result diagram of verifying the inhibition of uterine smooth muscle cell contraction by Nb2G5 through cell experiments in the experimental examples of the present invention; among them, a is the schematic diagram of the mechanism by which the inhibition of OTR promotes smooth muscle relaxation and thus inhibits premature birth, b is the detection of the inhibitory effects of atosiban and Nb2G5 on V1aR (Nb2G5 on the left, atosiban on the right), and c and d are the detection of the inhibitory effects of Nb2G5 and atosiban on uterine smooth muscle contraction. Detailed implementation manners

[0069] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0070] Example 1 Screening of Nanobodies

[0071] In this example, nanobodies with extracellular binding activity to OTR were screened based on a yeast synthetic nanobody display library. The flow chart of the screening process is as shown in Figure 1 and Figure 2 shown below. The specific screening method is as follows:

[0072] 1) Yeast library cells with an original library capacity of 5×10 8 were washed in screening buffer A (formulation: 20 mM HEPES [pH 7.5], 100 mM NaCl, 2 mM CaCl 2 , 0.05% dodecyl maltoside-neopentyl glycol, 0.005% CHS, 0.1% BSA, 0.2% maltose), and incubated with 1 μM M1-FLAG-FITC and 10% (v / v) anti-FITC magnetic beads (Miltenyi) in a 5 mL system at 4°C for 40 min; the reaction system was centrifuged at 3500 g for 3 min, resuspended in 5 mL of screening buffer A, and added to an LD column (Miltenyi) to remove yeast cells expressing nanobodies that bind to M1-FLAG-FITC or anti-FITC magnetic beads; after removing non-specific binding using the LD column, the yeast cells were incubated with 1 μM FLAG-OTR 5 and 100 nM M1-FLAG-FITC in a 5 mL screening buffer A system for 1 h; centrifuged at 3500 g for 3 min, resuspended the cells in 5 mL of screening buffer A containing 10% (v / v) anti-FITC magnetic beads (Miltenyi), and incubated at 4°C for 20 min; then, centrifuged at 3500 g for 3 min, washed, resuspended in screening buffer A, and added to an LS column (Miltenyi), and the yeast cells were eluted from the column to obtain MACS1 in Figure 2 .

[0073] 2) Based on MACS1, amplification culture was carried out, and the second round of MACS magnetic bead sorting was performed according to the same procedure as in step 1), but the reagents were adjusted as follows: anti-FITC magnetic beads and anti-AlexaFluor647 magnetic beads were respectively combined with M1-FLAG-FITC (200 nM) and M1-FLAG-AlexaFluor647 (1 μM) to deplete the yeast pool; after pre-clearing, the yeast cells were stained with 500 nM OTR 5 and 50 nM M1-FLAG-FITC / 647, and then the yeast cells were recovered to obtain MACS2 in Figure 2 for flow cytometry sorting and amplification.

[0074] 3) At the flow cytometry sorting stage, the MACS2 yeast cells obtained in step 2) are resuspended or washed with screening buffer A, and stained with 100 nM FLAG-OTR 5 -Clip3 and 20 nM M1-FLAG-AlexaFluor488 in screening buffer A at 4 °C for 30 min; the cells are washed, and 1×10 7 cells are sorted on an Aria SORP cell sorter to screen out the cells that have bound OTR and FLAG-AlexaFluor488, obtaining Figure 2 FACS1 in

[0075] The above-mentioned FLAG-OTR 5 The amino acid sequence of the OTR 5 -Clip3 part is: MKTIIALSYIFCLVF ADYKDDDDAMEGALAANWSAEAANASAAPPGAEGNRTAGPPRRNEALARVEVAVLCLILLLALSGNACVLLALHTTRQKHSRLFFFMKHLSIADLVVAVFQVLPQLLWDITFRFYGPDLLCRLVKYLQVVGMFASTYLLLLMSLDRCLAICQPLRSLRRRTARLAVLATWLGCLVASAPQVHIFSLREVADGVFDCWAVFIQPWGPKAYITWITLAVYIVPVIVLAACYGLIAFKIWQNALGKELAAAFRKEMSLEEYKELLNALAERVGASEEAIEKAKKLLETYGAKKTPEEILIGRISAACYILADGAKRLGEPVAILTLSFKNNLSPEDLELLKKAGEEDELAGEAYKGLEEGSKAGEATMRTVKMTFIIVLAFIVCWTPFFFVQMWSVWDANAPKEASAFIIVMLLASLNSCCKPWIYMLFMGHLFHELVQRFLCCSASYLKGRRLGETSASKKSNSSSFVLSHRSSSQRSCSQPSTA (SEQ ID NO:1),

[0076] The amino acid sequence of the Clip3 portion is as follows: ALGKELAAAFRKEMSLEEYKELLNALAERVGA SEEAIEKAKKLLETYGAKKTPEEILIGRISAACYILADGAKRLGEPVAILTLSFKNNLSP EDLELLKKAGEEDELAGEAYKGLEEGSKAGEATMR (SEQ ID NO:2).

[0077] 4) Amplify the cells sorted in step 3) using a lower concentration of 50 nM FLAG-OTR 5 -Clip3 for final FACS sorting to obtain Figure 2 FACS2 in, i.e., yeast cells carrying nanobodies with high affinity for OTR; after each round of selection, analyze the nanobody expression, FLAG-OTR binding, and non-specific binding of the population by small-scale analysis of the staining reaction.

[0078] Example 2 Yeast Monoclonal Staining and Sequencing

[0079] In this example, the yeast cells (FACS2) carrying nanobodies with high affinity for OTR screened in Example 1 were stained and subjected to monoclonal sequencing. The specific experimental methods are as follows:

[0080] 1) Randomly select monoclonal colonies on an agarose gel - trp (tryptophan - deficient medium) plate for yeast cell staining. Culture in the - trp plate for 36 - 48 h to induce nanobody expression. Use an equal amount of 5×10 6 yeast cells for staining for each monoclonal colony; centrifuge the yeast cells and then wash twice with screening buffer B (the composition of screening buffer B is: 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.01% L - MNG, 0.002% CHS, 2 mM CaCl 2 , 0.1% (w / v) bovine serum albumin, 5 mM maltose), and stain with 50 nM FLAG-OTR 5 / OTR 5 -Clip3 and 20 nM M1 - FLAG - AlexaFluor488 and 1 mg / mL anti - HA - AlexaFluor647 (in a volume of 50 μL) at 4°C for 25 min. After washing, resuspend in screening buffer B; analyze the binding of OTR in the cells using a CytoFLEX flow cytometer, and distinguish intracellular and extracellular nanobodies based on the different staining results of OTR 5 and OTR 5 -Clip3.

[0081] 2) To analyze the sequence changes during the entire screening process, the nanobody sequences were directly amplified from yeast cells by PCR using primers P209 and P210, and Sanger sequencing was performed to analyze the enrichment degree of each monoclonal.

[0082] The amino acid sequences of the nanobodies obtained by sequencing are as follows:

[0083] Nb2G5: QVQLQESGGGLVQAGGSLRLSCAASGSISPGYWMGWYRQAPGKEREF VASIDLGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARIQYAYD YLGTHGYWGQGTQVTVSS (SEQ ID NO:3),

[0084] Nb2B1: QVQLQESGGGLVQAGGSLRLSCAASGSISDDAVMGWYRQAPGKERELV ATISYGAITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAAYYRLET QYWRFLFYYWGQGTQVTVSS (SEQ ID NO:4),

[0085] Nb2B11: QVQLQESGGGLVQAGGSLRLSCAASGSISGITDMGWYRQAPGKEREFV ASITYGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVASSRGNSY SLLGGYILTYWGQGTQVTVSS (SEQ IDNO:5),

[0086] Nb2H4: QVQLQESGGGLVQAGGSLRLSCAASGNIFIKYAMGWYRQAPGKERELV ASINDGTTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASVIYYLRD FFATWYIHYYWGQGTQVTVSS (SEQ ID NO:6).

[0087] Among them, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the nanobody Nb2G5 are shown in Table 1.

[0088] Table 1 CDR1, CDR2, and CDR3 of the heavy chain variable region of the nanobody Nb2G5

[0089]

[0090] Example 3 Nanobody Expression

[0091] Based on the monoclonal fragments screened and sequenced in Example 2, this example performs the expression of nanobodies Nb2G5, Nb2B1, Nb2B11, and Nb2H4. The specific experimental method is as follows: Clone the nanobody monoclonal fragments with a C-terminal 6×His-tag into the pcDNA3.4 vector. Transiently transfect the plasmid and the transfection reagent PEI (1 mg / mL) into Expi293F cells at a mass ratio of 1:3. After culturing and expressing for 30 - 48 h, collect the supernatant. Add 5 mM CaCl 2 , 10 mM MgCl 2 , 10 mM HEPES (pH = 7.5), and 2 mM NiCl 2 for chelation and then centrifuge. Add 1 - 2 mL of nickel chelating resin to the supernatant for nickel purification. Use the method of alternating high and low salt washing for washing (high salt buffer: 500 mM NaCl, 20 mM HEPES (pH = 7.5), 20 mM IMD; low salt buffer: 100 mM NaCl, 20 mM HEPES (pH = 7.5), 20 mM IMD). Finally, elute the protein with the eluent (100 mM NaCl, 20 mM HEPES (pH = 7.5, 200 mM IMD); Concentrate the protein to a volume of 1 ml using a 10 kDa concentrator tube. Subsequently, perform Superdex 75 Increase molecular sieve purification. The nanobody elutes at 14 - 16 mL. Collect the eluate here and run an SDS-PAGE gel to confirm the presence of the protein. The obtained electrophoresis results are as Figure 3 shown; Filter the protein with a 0.22 μM sterile membrane and set it aside for use.

[0092] Among them, the nucleotide sequence of the monoclonal fragment encoding the nanobody Nb2G5 is: 5’-CAGGTGCAGCTG CAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTCCGGGTTACTGGATGGGCTGGTATCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTTGCCAGTATTGATCTGGGTGGTACTACCTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCTCGTATCCAGTACGCTTACGACTACCTGGGTACTCATGGTTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGATCCCGTCTGGAGGAAGAGTTGCGTCGACGGCTGACAGAGCCGCACCACCACCACCACCACTGA-3’ (SEQ ID NO:10).

[0093] Experimental Example

[0094] In this experimental example, the molecular mechanism of the binding of the nanobody Nb2G5 prepared in Example 3 to OTR was explored through experiments, and the inhibitory effect of this nanobody on the function of OTR was further verified through cell experiments. The specific experimental methods and results are as follows:

[0095] 1. Verifying the OTR binding ability and inhibitory effect of the nanobody Nb2G5 by structural biology methods

[0096] To verify the molecular mechanism of the binding of OTR to the nanobody Nb2G5, in this experimental example, the cryo-electron microscopy structure of the complex of the nanobody Nb2G5 and OTR was detected by cryo-electron microscopy. After collecting sufficient electron microscopy images, particle extraction and analysis were carried out. The results obtained are as Figure 4 shown.

[0097] In the two-dimensional classification of the data set, the features of both the nanobody Nb2G5 and the fusion protein Clip3 can be clearly distinguished. The nanobody Nb2G5 and the fusion protein Clip3 are located on both sides of the receptor, successfully resolving the near-atomic resolution structure of these nanobody-receptor complexes, achieving resolution ( Figure 4a); These structural analyses indicate that the nanobody Nb2G5 resembles a lid covering the extracellular region of the OTR, effectively occupying the orthosteric pocket of the receptor, while the curved scaffold region supports the extracellular domain of the receptor;

[0098] The interaction surface can be divided into three regions: CDR1 of the nanobody interacts with ECL3 of the receptor through polar interactions involving SER27, SER29, and GLU307 ( Figure 4 b); A large hydrophobic pocket accommodates a series of hydrophobic residues from the nanobody, including LEU106, TRY105, TRY103, and TRY101, which interact with receptor residues such as ILE204, PHE292, PHE291, MET315, TRP99, and PHE103 to stabilize the interface. Figure 4 c); The C’-sheet forms β-strand interactions with ECL2. Figure 4 d); The most crucial interactions occur within the orthosteric pocket.

[0099] Nb2G5 stabilizes the receptor in an inactive conformation. Different from atosiban, Nb2G5 induces a slight deflection of ECL2 towards the receptor, possibly due to its interaction with the scaffold region. Figure 4 e and Figure 4 f). Notably, ECL2 undergoes significant conformational changes during receptor activation and adopts a position away from the receptor in the active state.

[0100] This experimental example also compared the structures of Nb2G5, atosiban, or oxytocin after binding to the OTR ( Figure 4 g~ Figure 4 i). Although Nb2G5 is an inhibitor, it mimics the binding mode of oxytocin; in the core region, the backbone of Nb2G5 is consistent with that of oxytocin, and residues such as TPY105, LEU106, and THR108 of Nb2G5 correspond to TPY2, ILE3, and GLN4 of oxytocin. Pharmacological experiments have shown that substituting TPY105 with alanine can significantly reduce the inhibitory activity of Nb2G5. Figure 5 ). In addition, Nb2G5 occupies the magnesium-binding pocket of the receptor, which is crucial for activation. The structural similarity between Nb2G5 and oxytocin endows it with strong binding ability to the receptor, but the conflict between the active ECL2 conformation of the receptor and the scaffold region of Nb2G5 limits activation, stabilizing the receptor in an inactive state. Moreover, the disruption of the magnesium-binding pocket further stabilizes the inactive conformation.

[0101] In summary, Nb2G5 "grabs" the receptor like a palm, stabilizes ECL2, and maintains the receptor in an inactivated state.

[0102] 2. Verification by cell experiments

[0103] 1) Inhibitory effect of the nanobody provided in Example 3 on OTR function:

[0104] The classical GPCR function experiment, the glosensor cAMP experiment, was used for inhibitory analysis. The specific experimental methods and results are as follows:

[0105] Considering that OTR bound to Gq protein does not activate adenylate cyclase and thus does not induce cAMP production, a chimeric Gsq protein was designed by replacing the 15 amino acids at the C-terminus of the Gs protein sequence with those of Gq. HEK293T cells were seeded in six-well plates and reached a cell confluence of 50% - 80% before transfection. A plasmid mixture containing 250 ng OTR, 250 ng Gsq chimera, and 2000 ng pGloSensor TM -22FcAMP plasmid (Promega) was prepared in 250 μL of Opti-MEM (Gibco), and HEK293T cells were transfected using polyethyleneimine (PEI). After 24 hours of transfection, the cells were harvested, resuspended in HBSS (Gibco) supplemented with 20 mM HEPES (pH 7.5) and 150 μg / mL fluorescein, and transferred to 96-well plates. The cells were incubated at 37 °C for 1 h and then at room temperature for another 1 h to equilibrate the GloSensor. Baseline luminescence was measured before adding different concentrations of nanobodies and ligands (10 μL each), and then luminescence counts were recorded every minute. Data analysis was performed using GraphPad Prism version 10.3, and the results are as Figures 6 to 9 shown.

[0106] As Figures 6 to 9 can be seen, the nanobody Nb2G5 inhibits the activation of oxytocin on OTR due to competitive binding within the orthosteric pocket. The clinical drug atosiban and Nb2G5 have inhibitory effects on the oxytocin-dependent signaling pathway (IC50 values are 74 nM and 78 nM, respectively); compared with atosiban, Nb2G5 shows a similar inhibitory efficiency, while the inhibitory effects of Nb2B1, Nb2B11, and Nb2H4 are all inferior to atosiban, indicating that Nb2G5 is a more preferred nanobody with OTR inhibitory effect.

[0107] It is worth noting that for the possible side effects caused by the off-target V1aR of atosiban in clinical practice ( Figure 10a), The nanobody Nb2G5 exhibits excellent receptor specificity in this regard. In previous literature, it has been reported that atosiban also has a similar inhibitory effect on V1AR, and the GloSensor functional experiment in this test example also verified such a similar result. From the experimental results, the inhibitory effect of atosiban on OTR and V1AR only differs by 5-fold, which may be the reason for its side effects of causing maternal hypotension or pulmonary edema. However, the nanobody Nb2G5 only inhibits OTR and does not act on V1aR Figure 10 b). The excellent specificity gives the nanobody a natural advantage different from existing drugs in the development of preterm labor drugs targeting the OTR receptor.

[0108] 2) Inhibitory effect of Nb2G5 on uterine smooth muscle contraction:

[0109] To evaluate the effect of Nb2G5 on the contraction of human uterine smooth muscle (hUSM), the cell (collagen) contraction experiment of human uterine smooth muscle cells (hUSMCs) was carried out in this test example. The specific experimental procedure is as follows:

[0110] Approved by the institutional ethics committee and with the informed consent of all participants, uterine smooth muscle tissue was collected from the lower uterine segment incision of pregnant women undergoing cesarean section. The living tissue was immediately placed in sterile physiological saline containing 100 U / mL penicillin and 100 μg / mL streptomycin to prevent contamination. Uterine smooth muscle cells (USMCs) were isolated by the explant method; the tissue samples were minced and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with double antibodies and 10% fetal bovine serum (FBS). The medium was changed every two days until the primary culture reached confluence. Subsequently, cells between the third and sixth generations were used for the experiment, and there were no significant differences in the experimental results either between individual passages or between cells from different uteri.

[0111] The USMCs were seeded at 4×10 per well 4The density of the cells was resuspended in serum-free medium and 5 mg / mL rat tail type I collagen (Corning) was added at a ratio of 1:5. The mixture was plated in a 12-well plate and incubated at 37 °C for 1 h to polymerize the collagen / cell suspension. After polymerization, the gel was gently peeled off the surface of the plate and DMEM containing 10% fetal bovine serum (FBS) was added. The treatment groups included DMEM alone with 10% FBS, DMEM with 10% FBS and 0.1 mM nanobody Nb2G5, and DMEM with 10% FBS and 0.1 mM atosiban. Untreated cells showed basal contraction and the gel area decreased over time. Gel images were captured using a CanoScan 9000F scanner at baseline (0 h), 24 h, and 36 h. The gel area was quantified using ImageJ software (NIH). The measured areas at 24 h and 36 h were expressed as a percentage of the initial area at 0 h, and the results are shown as Figure 10 c and Figure 10 d.

[0112] As shown by Figure 10 c and Figure 10 d, Nb2G5 significantly inhibited oxytocin (OT)-mediated contraction of hUSMCs; for the highest dose of Nb2G5, its effect on inhibiting uterine strip contraction was even comparable to that of atosiban. The above results indicate that Nb2G5 can effectively inhibit the contractility of uterine smooth muscle.

[0113] In summary, in vitro and in vivo experiments showed that the nanobody Nb2G5 provided by the present invention has an efficacy comparable to that of atosiban, a drug widely used clinically. Pharmacological experiments and structural studies revealed the unique molecular mechanism of Nb2G5, which is different from traditional small molecule antagonists. The present invention expands the pharmacological potential of antibody drugs and provides new tools and prospects for the development of effective anti-preterm labor drugs.

[0114] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A nanobody, characterized in that The Nanobody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2 and CDR3; The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the Nanobody comprises: a1) SEQ ID NO: 3; or a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 3 and which has the same function as the protein shown in SEQ ID NO: 3; or a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 60%, 50%, 40% or 30% homologous to SEQ ID NO:1 and has the same function as the protein shown in SEQ ID NO:

3.

3. A recombinant protein comprising: the Nanobody according to claim 1 or 2; and Optional tag sequence to facilitate expression and / or purification.

4. A biomaterial associated with the Nanobody of claim 1 or 2, or the recombinant protein of claim 3, wherein the biomaterial comprises at least one of h1) to h8): h1) a nucleic acid molecule encoding the Nanobody according to claim 1 or 2 or the recombinant protein according to claim 3; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) A transgenic cell line comprising the vector described in h4).

5. A conjugate comprising: at least one of the Nanobody according to claim 1 or 2 and the recombinant protein according to claim 3; and a coupling moiety, the coupling moiety comprising at least one of a detectable label, a drug, a toxin, an electron-dense label, biotin / avidin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, and a viral coat protein; Preferably, the detectable marker is a fluorescent or luminescent marker; Preferably, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase.

6. A solid phase carrier, the surface of which is coupled with the nanobody according to claim 1 or 2 and / or the recombinant protein according to claim 3.

7. Use of at least one of (1) to (5) in the preparation of a product; (1) The Nanobody according to claim 1 or 2; (2) The recombinant protein according to claim 3; (3) The biomaterial according to claim 4; (4) The conjugate according to claim 5; (5) The solid phase carrier according to claim 6; Preferably, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the medicament has the function of treating and / or preventing a disease or condition for which it is known or can be shown that inhibition of oxytocin will produce a beneficial effect; More preferably, the disease or disorder is selected from sexual dysfunction, hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder, dyspareunia, premature ejaculation, pre-natal delivery, delivery complications, appetite and eating disorders, benign prostatic hyperplasia, premature delivery, dysmenorrhea, congestive heart failure, arterial hypertension, cirrhosis, renal hypertension, ocular hypertension, obsessive-compulsive disorder and neuropsychiatric disease.

8. A product, comprising at least one of k1) to k4): k1) The Nanobody of claim 1 or 2; k2) The recombinant protein according to claim 3; k3) the conjugate according to claim 5; k4) The solid phase carrier according to claim 6.

9. The product according to claim 8, characterized in that: The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the medicament has the function of treating and / or preventing a disease or condition for which it is known or can be shown that inhibition of oxytocin will produce a beneficial effect; More preferably, the disease or disorder is selected from sexual dysfunction, hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder, dyspareunia, premature ejaculation, pre-natal delivery, delivery complications, appetite and eating disorders, benign prostatic hyperplasia, premature delivery, dysmenorrhea, congestive heart failure, arterial hypertension, cirrhosis, renal hypertension, ocular hypertension, obsessive-compulsive disorder and neuropsychiatric disease.

10. A method for preparing the antibody according to claim 1 or 2 or the recombinant protein according to claim 3, which is obtained by culturing the transgenic cell line according to claim 4.

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