Monoclonal antibodies against human PIEZO2 and uses thereof

Abstract

The invention relates to a monoclonal antibody aiming at human PIEZO2 and application of the monoclonal antibody. The monoclonal antibody, or an antigen-binding fragment thereof, comprises: (a) a heavy chain CDR1 comprising SEQ ID NO: 5, a heavy chain CDR2 comprising SEQ ID NO: 6, and a heavy chain CDR3 comprising SEQ ID NO: 7; and (b) a light chain CDR1 comprising SEQ ID NO: 8, a light chain CDR2 comprising SEQ ID NO: 9, and a light chain CDR3 comprising SEQ ID NO: 10. The inventor prepares the monoclonal antibody aiming at PIEZO2 for the first time. The antibody has specific PIEZO2 high affinity, can efficiently and specifically recognize PIEZO2, and does not recognize a homologous protein PIEZO1 of the PIEZO2.

Description

Technical Field

[0001] The present invention relates to monoclonal antibodies against human PIEZO2 and uses thereof. More specifically, it relates to monoclonal antibodies against human PIEZO2 or antigen-binding fragments thereof, nucleic acid molecules encoding the same, cells and hybridomas producing the monoclonal antibodies or nucleic acid molecules, and uses of the monoclonal antibodies, nucleic acid molecules, cells or hybridomas in the preparation of analgesics. Background Art

[0002] In 2010, Ardem Patapoutian's team first identified the cation channels that sense mechanical force in humans: PIEZO1 and PIEZO2. Subsequently, scientists discovered that PIEZO1 / 2 plays an important role in many physiological functions. PIEZO2 is specifically expressed in sensory neurons, and mouse experiments have shown that it mediates gentle touch perception. PIEZO2-mediated touch sensation has also been confirmed in humans, and it mediates pathological touch hypersensitivity. However, the expression of PIEZO1 is completely opposite, very extensive, and its functions involve many aspects. It plays an important role in the development of blood vessels and lymphatic vessels, red blood cell volume regulation, bone formation and remodeling, brain structure and function, innate immune response, adipogenesis and adipose inflammation, pancreatitis caused by stress, digestive tract motility, colitis, skin wound healing and tumor development.

[0003] As one of the Nobel Prize star molecules, PIEZO2 is a direct molecular receptor for touch and has been shown to mediate tactile hypersensitivity. In addition, PIEZO2 is specifically expressed in sensory neurons and some specialized mechanosensory epithelial cells, with a high distribution specificity, highlighting that it is a very promising new analgesic drug target with low potential side effects. The development of drugs and antibodies targeting PIEZO2 is in its infancy, and no related antibody drugs have been approved clinically. In addition, the antibodies for scientific research purposes against PIEZO2 sold on the market are all polyclonal antibodies, and their specificity at the cellular tissue level is unclear (see below).

[0004] List of Piezo2 antibodies for scientific research use on the market:

[0005] alomone labs:APC-090

[0006] Thermo Fisher:PA5-111032PA5-53405 PA5-72976 PA5-72975

[0007] Abcam:ab244349

[0008] Novus Biologicals:NBP1-78624 NBP1-78538 NBP2-61130 NBP2-58161 NBP1-89893

[0009] Proteintech:26205-1-AP

[0010] Sigma-Aldrich:HPA031974HPA031975HPA015986SAB2100761SAB2108846SAB2108847 SAB2108848 ZRB1468

[0011] Therefore, it is currently expected to obtain monoclonal antibodies against PIEZO2. Considering the high structural similarity between PIEZO1 and PIEZO2 and the 42% sequence conservation, when designing antibody drugs against PIEZO2, it is necessary to fully consider its specificity for PIEZO2 to avoid the side effects caused by targeting PIEZO1. Summary of the invention

[0012] Based on the resolved cryo-electron microscopy three-dimensional structure of PIEZO2 and the study of its mechanical gating mechanism, the inventor's research group prepared PIEZO2 monoclonal antibodies through hybridoma technology and obtained a new monoclonal antibody against human PIEZO2. This antibody has a specific high affinity for PIEZO2, can efficiently and specifically recognize PIEZO2 but not its homologous protein PIEZO1, and has the effect of enhancing the functional activity of PIEZO2.

[0013] The present invention is achieved by having the following features.

[0014] In one aspect, the present invention provides a monoclonal antibody against human PIEZO2, or an antigen-binding fragment thereof, comprising:

[0015] (a) a heavy chain CDR1 comprising SEQ ID NO: 5, a heavy chain CDR2 comprising SEQ ID NO: 6, and a heavy chain CDR3 comprising SEQ ID NO: 7; and

[0016] (b) a light chain CDR1 comprising SEQ ID NO:8, a light chain CDR2 comprising SEQ ID NO:9, and a light chain CDR3 comprising SEQ ID NO:10.

[0017] In one embodiment, the antibody comprises: a heavy chain variable region having at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1; and a light chain variable region having at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. Preferably, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2.

[0018] In one embodiment, the antibody comprises: a heavy chain having at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11; and a light chain having at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. Preferably, the amino acid sequence of the heavy chain is SEQ ID NO: 11 and the amino acid sequence of the light chain is SEQ ID NO: 12.

[0019] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0020] In one embodiment, the nucleic acid molecule comprises: a nucleic acid sequence of SEQ ID NO: 3 encoding the heavy chain variable region of the monoclonal antibody against human PIEZO2; and a nucleic acid sequence of SEQ ID NO: 4 encoding the light chain variable region of the monoclonal antibody against human PIEZO2.

[0021] In one embodiment, the nucleic acid molecule comprises: a nucleic acid sequence of SEQ ID NO: 13 encoding the heavy chain of the monoclonal antibody against human PIEZO2; and a nucleic acid sequence of SEQ ID NO: 14 encoding the light chain of the monoclonal antibody against human PIEZO2.

[0022] In yet another aspect, the present invention provides a cell that produces the above monoclonal antibody, or contains the above nucleic acid molecule.

[0023] In yet another aspect, the present invention provides a pharmaceutical composition comprising the above monoclonal antibody.

[0024] In another aspect, the present invention provides the use of the above-mentioned monoclonal antibody, the above-mentioned nucleic acid molecule, the above-mentioned cell, and the above-mentioned pharmaceutical composition in the preparation of a drug for diagnosing, preventing or treating the following diseases: analgesia; tactile abnormalities; proprioception abnormalities; visceral abnormalities; Angelman syndrome; Gordon syndrome; Madden-Walker syndrome; scoliosis; and distal arthrogryposis syndrome.

[0025] In yet another aspect, the present invention provides a kit comprising the above monoclonal antibody, the above nucleic acid molecule and / or the above cell.

[0026] In another aspect, the present invention provides a method for diagnosing or treating the following diseases: analgesia; tactile abnormalities; proprioception abnormalities; visceral abnormalities; Angelman syndrome; Gordon syndrome; Madden-Walker syndrome; scoliosis; distal arthrogryposis syndrome, the method comprising administering a therapeutically effective amount of the above-mentioned monoclonal antibody, the above-mentioned nucleic acid molecule and / or the above-mentioned cell to a subject in need thereof.

[0027] The sequence involved in the present invention is as follows:

[0028] SEQ ID NO: 1, heavy chain variable region:

[0029] DVRLQESGPGLVKPSQSLSLTCTVTGYSITSDHAWNWIRQFPGNKLEWMGYISYIGNTTYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCTRGTNGASWGQGTLVTVSA

[0030] SEQ ID NO: 2, light chain variable region:

[0031] DVVMIQTPLSLPVSLGDQVSLSCRSSQSLVHSNGNTYLHWCLQKPGQSPKLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELK

[0032] SEQ ID NO: 3, nucleic acid sequence encoding the heavy chain variable region:

[0033] GATGTGCGACTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATCATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATCAGCTACATTGGTAACA CTACTTACAATCCATCTCTCAAAAGTCGAATCTCTATTACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAAGACACAGCCACATATTTCTGTACAAGAGGAACCAACGGCGCTTCCTGGGGCCAAGGGACTCTGGTCACTGTCTCGGCT

[0034] SEQ ID NO:4, nucleic acid sequence encoding the light chain variable region:

[0035] GATGTTGTGATGATCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGTCTCCCTCTCTTGCAGATCTAGTCAGAGCCTTGTACAGTAATGGAAACACCTATTTACATTGGTGCCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGCT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0036] SEQ ID NO: 5, amino acid sequence of heavy chain CDR1: SDHAWN

[0037] SEQ ID NO: 6, amino acid sequence of heavy chain CDR2: YISYIGNTTYNPSLKS

[0038] SEQ ID NO: 7, amino acid sequence of heavy chain CDR3: TRGTNGAS

[0039] SEQ ID NO: 8, amino acid sequence of light chain CDR1: RSSQSLVHSNGNTYLH

[0040] SEQ ID NO: 9, amino acid sequence of light chain CDR2: KASNRFS

[0041] SEQ ID NO: 10, amino acid sequence of light chain CDR3: SQSTHVPLT

[0042] SEQ ID NO: 11, amino acid sequence of the heavy chain:

[0043] DVRLQESGPGLVKPSQSLSLTCTVTGYSITSDHAWNWIRQFPGNKLEWMGYISYIGNTTYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCTRGTNGASWGQGT LVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0044] SEQ ID NO: 12, amino acid sequence of light chain:

[0045] DVVMIQTPLSLPVSLGDQVSLSCRSSQSLVHSNGNTYLHWCLQKPGQSPKLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKL ELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0046] SEQ ID NO: 13, heavy chain encoding nucleic acid sequence:

[0047]

[0048] SEQ ID NO: 14, nucleic acid sequence encoding the light chain:

[0049] GATGTTGTGATGATCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGTCTCCCTCTCTTGCAGATCTAGTCAGAGCCTTGTACAGTAATGGAAACACCTATTTACATTGGTGCCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAA AGCTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGG AGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTC CTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0050] Beneficial Effects

[0051] The inventors prepared a monoclonal antibody against PIEZO2 for the first time. The antibody has a specific high affinity for PIEZO2, can efficiently and specifically recognize PIEZO2, but not its homologous protein PIEZO1, and has the effect of enhancing the functional activity of PIEZO2. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Shows the flow chart of hybridoma production of monoclonal antibodies.

[0053] Figure 2Displays the typing and identification of monoclonal antibodies. Different types of specific secondary antibodies are used to identify the type of primary antibody, including IgG1, IgG2a, IgG2b, IgG3, and IgM.

[0054] Figure 3 Shows the purification of monoclonal antibodies. All 9 monoclonal antibodies were successfully prepared and purified. The above are 4 representative antibody purification images.

[0055] Figure 4 The monoclonal antibodies' ability to recognize PIEZO2-Cap is shown. All nine monoclonal antibodies were successfully prepared and purified, and their specific concentrations are listed in the left figure. The right figure is a dot-blotting experiment, where 5ug of PIEZO2-Cap antigen was diluted 10 times layer by layer and then recognized by antibodies. It was found that most antibodies could recognize antigen proteins in the range of 5-50ng.

[0056] Figure 5 The monoclonal antibodies show their ability to recognize full-length Piezo2. 293T-wt represents HEK293T cells, which do not express PIEZO2, and 293T-OE represents cells that overexpress PIEZO2 through transfection. It can be seen that 4 monoclonal antibodies (1G11, 2E1, 5H8, 9E12) can specifically recognize Piezo2, among which 2E1 has the strongest recognition ability.

[0057] Figure 6 The monoclonal antibody 2E1 specifically recognizes PIEZO2 and distinguishes PIEZO1. 293T cells do not express PIEZO2 by themselves, but are transfected to heterologously overexpress fluorescently tagged PIEZO2 (upper panel) or PIEZO1 (lower panel). 2E1 can specifically recognize PIEZO2 but not PIEZO1.

[0058] Figure 7 The monoclonal antibody 2E1 is shown to specifically recognize PIEZO2 expressed in the dorsal root ganglion. In the upper figure, the mouse expresses the PIEZO2-EGFP fusion protein. The green channel is the specific signal of EGFP, representing the expression distribution of PIEZO2, and the red channel is the signal of 2E1. The high degree of fusion between red and green indicates that 2E1 specifically recognizes PIEZO2 in the dorsal root ganglion tissue. In the lower figure, the Western blotting detection results of the 2E1 antibody against PIEZO2 in DRG lysates from mice with conditional knockout of PIEZO2 in DRG, wild mice in the control group, and mice with conditional overexpression of PIEZO2 in DRG.

[0059] Figure 8It was shown that the 2E1 antibody did not change the magnitude of the PIEZO2-mediated mechanosensitive current, but slowed down its inactivation rate, indicating that it has the activity of enhancing the function of the PIEZO2 channel. DETAILED DESCRIPTION

[0060] Example 1 Preparation, screening and expression of monoclonal monomers using hybridoma technology

[0061] The inventors used the classic B cell hybridoma fusion screening strategy, the process is as follows Figure 1 The steps are as follows:

[0062] (I) Mouse immunization: PIEZO2 extracellular domain CED was used as an antigen for immunization. In order to enhance the immunogenicity of the antigen, Freund's complete adjuvant and Freund's incomplete adjuvant were used. The antigen CED solution and adjuvant were mixed in equal volumes and whipped into a chyme. A drop was placed on the water surface and it was not easy to spread immediately and was in the form of small drops, indicating that the oil-in-water state had been reached. The following immunization scheme was used: 50 μg of CED antigen was used for the first immunization, and Freund's complete adjuvant was added for multiple intraperitoneal injections (1 ml, 0.2 ml / point). Two weeks later, the second immunization was performed, with the same dose as above, and Freund's incomplete adjuvant was added for a single intraperitoneal injection (the dose should not exceed 0.5 ml). Two weeks later, the third immunization was performed, with the same dose as above, and Freund's incomplete adjuvant was added for a single intraperitoneal injection (the dose should not exceed 0.5 ml). After 5-7 days, blood was collected from the tail vein to measure its titer and detect the immune effect. Finally, after 2 weeks, a booster immunization was performed, with a dose of 100 μg, no adjuvant, and 100 ul was injected into the tail vein. After 3 days, the spleen was ready for fusion.

[0063] (ii) Myeloma cells

[0064] The myeloma cell line and the immunized mice belong to the same strain, which ensures a high hybrid fusion rate and facilitates the inoculation of hybridoma cells in the peritoneal cavity of the same strain of mice to produce a large amount of monoclonal antibodies. The myeloma cell line used is: SP2 / 0. DMEM medium is used for the culture of myeloma cells, the concentration of fetal bovine serum is 20%, the maximum density of cells shall not exceed 1000000 / ml, and they are passaged every 2-3 days at a ratio of 1:5. The doubling time of SP2 / 0 cells is about 20 hours. They grow slightly attached to the wall. Cells can be suspended by gently blowing or tapping the bottle wall with an elbow dropper. Generally, myeloma cells should be revived two weeks before preparing for fusion. To ensure the sensitivity of the cells to HAT, 8-AG (8-azaguanine) should be screened once every 3-6 months to prevent cell mutations. Ensuring that myeloma cells are in the logarithmic growth phase, have good morphology, and a live cell count higher than 95% is also the key to determining cell fusion.

[0065] (III) Immune spleen cells

[0066] Immune splenocytes refer to B lymphoblasts in the spleen in an immune state. Generally, the spleen is taken 3 days after the last booster immunization and prepared into a cell suspension. Since the proportion of B lymphoblasts is larger at this time, the success rate of fusion is higher.

[0067] Preparation of spleen cell suspension: Take out the spleen under sterile conditions, wash it once with incomplete culture medium, place it on a stainless steel mesh in a flat dish, grind it into a cell suspension with a syringe needle, pass it through a 60-mesh cell mesh, and count it. Generally, the volume of the spleen after immunization is about twice that of a normal mouse spleen, and the number of cells is about 2×108.

[0068] (IV) Cell fusion process

[0069] (1) Take logarithmically growing myeloma cells SP2 / 0, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, suspend the cells in incomplete culture medium and count them, take the required number of cells, and wash them twice with incomplete culture medium (DMEM without serum and double antibody).

[0070] (2) At the same time, prepare immune spleen cell suspension and wash twice with incomplete culture medium.

[0071] (3) Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, washed once with incomplete culture medium in a 50 ml plastic centrifuge tube, and centrifuged at 1200 rpm for 8 minutes.

[0072] (4) Discard the supernatant and use a dropper to remove any remaining liquid to avoid affecting the concentration of PEG 4000 to be added later.

[0073] (5) Gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly.

[0074] (6) Fusion at room temperature:

[0075] ① Add preheated 1 ml 45% PEG (molecular weight 4000) + 5% DMSO within 30 seconds while stirring.

[0076] ②The effect lasts for 90 seconds, and can be extended to 120 seconds in winter when the room temperature is lower.

[0077] ③ Add preheated incomplete culture medium to terminate the PEG action, and add 1ml, 2ml, 3ml, 4ml, 5ml and 10ml every 2 minutes.

[0078] (7) Centrifugation, 1000 rpm, 5 min. No brake mode to terminate the centrifugation.

[0079] (8) Discard the supernatant and gently suspend the cell culture medium with about 6 ml of 10% fetal bovine serum (DMSO). Be careful not to blow vigorously to avoid breaking up the fused cells.

[0080] (9) Add complete culture medium according to the number of 96-well culture plates used, 20 ml per 96-well plate.

[0081] (10) Add the fused cell suspension to a 96-well plate, 200 μl / well, and culture in a sterile box at 37°C and 5% CO2. One 96-well plate contains approximately 1×107 spleen cells.

[0082] (V) HAT selection of hybridomas

[0083] After fusion for 24 hours, add HAT selection culture medium. Both HT and HAT have commercial reagents stored at 50×. When used, add 1 ml to 50 ml of 20% fetal bovine serum complete culture medium. Generally, HAT selection culture medium is selected to maintain culture for two weeks, then switch to HT culture medium, and then maintain culture for another two weeks, and then switch to general culture medium.

[0084] (VI) ELISA screening of antigen-specific positive wells

[0085] Screening of hybridoma cells Among the hybrid cell lines obtained by selective culture, only a few can secrete specific antibodies against the immunogen. Generally, when the hybridoma cells cover 1 / 3 of the bottom of the well, the specific antibodies can be detected and the required hybridoma cell lines can be screened out. The method for detecting antibodies should be different according to the nature of the antigen and the type of antibody. Different screening methods should be selected, generally based on the principle of rapid, simple, specific and sensitive methods. The inventor used ELISA to detect the affinity of the supernatant secreted by hybridoma cells to the antigen CED. This method is a routine method in general laboratories, so the specific experimental process is not introduced here.

[0086] (VII) Cloning of antigen-specific positive wells

[0087] Cloning generally refers to cloning the antibody-positive wells. After HAT screening, the hybridoma clones cannot guarantee that there is only one clone in one well. In actual work, there may be several or even more clones, which may include antibody-secreting cells, antibody-non-secreting cells; the required antibody (specific antibody)-secreting cells and other irrelevant antibody-secreting cells. In order to separate these cells from each other, cloning is required. The principle of cloning is that hybrid clones that test antibody positive should be cloned as early as possible, otherwise the antibody-secreting cells will be inhibited by the antibody-non-secreting cells, because the growth rate of antibody-non-secreting cells is faster than that of antibody-secreting cells, and the competition between the two will cause the antibody-secreting cells to be lost.

[0088] The inventors used limiting dilution method for cloning:

[0089] ①Prepare feeder cell suspension (same as preparation before fusion)

[0090] ② Count the cells in the positive wells and adjust the cell number to 1-5×10 3 / ml

[0091] ③ Take 130 cells and put them into 13 ml of complete culture medium containing feeder cells, that is, 10 cells / ml, and add 100 μl / well to a 96-well plate, with 1 cell per well.

[0092] ④ After 4-5 days of culture, small cell clones can be seen under the microscope, and culture medium is added to 200μl / well.

[0093] ⑤ On the 8th to 9th day, cell clones can be seen with the naked eye and antibody testing should be performed in time.

[0094] In summary, after fusion screening, antigen ELISA was used for further screening, and finally 9 candidate monoclonal antibodies were obtained, and these 9 antibodies were typed and identified, such as Figure 2 As shown, antibody 2E1 is IgG2B. (in the red box)

[0095] Next, the antibody was purified using a protein A / G column. The routine procedure was to first intraperitoneally inject 0.5 ml of Freund's incomplete adjuvant into Balb / c mice, and then intraperitoneally inject 10 6 Hybridoma cells can produce ascites 7-10 days after inoculation. Closely observe the health status of the animals and the signs of ascites. When ascites is as much as possible and the mice are close to death, kill the mice and use a dropper to suck the ascites into a test tube. Generally, 1-10ml of ascites can be obtained from one mouse. Ascites can also be extracted with a syringe and can be collected repeatedly several times. The content of monoclonal antibodies in ascites can reach 5-20mg / ml. Figure 3 The following are 4 representative purification profiles. The first half is the binding phase, where the antibody binds to ProteinA / G with affinity, and the peak in the second half is the elution phase, where the antibody is eluted from the column using an acidic buffer to obtain a monoclonal monomer.

[0096] Example 2 Functional identification of monoclonal antibodies

[0097] After obtaining 9 candidate antibodies, the inventors conducted biochemical identification on them through dot-blotting experiments, such as Figure 4 As shown, it was confirmed that all 9 antibodies could recognize PIEZO2-Cap protein at the level of 5-50 ng.

[0098] The above data prove that the antibody can recognize the Cap recombinant protein expressed by prokaryotic bacteria, but the ability to recognize the real human PIEZO2 needs further exploration. The inventors first used Western hybridization to explore the antibody's ability to recognize the full-length PIEZO2 protein in cells. Take the sample in the protein purification step, mix it with 6 times the reducing loading buffer at a volume ratio of 5:1, and after 100°C metal bath for 10 minutes, centrifuge to remove the precipitate. Use 8% concentration gel, electrophoresis 120V, 90min. Use transfer solution (25mMTris, 192mM glycine, 20% methanol) membrane for wet transfer on ice, 100V, 120min, and transfer the protein to the PVDF membrane. After the transfer is completed, block with 5% skim milk configured with TBST (TBS, 0.1% Tween-20) for 1h, incubate with the antibody to be detected for 1h, and wash the membrane 3 times with TBST, 5min each time. Then incubate with sheep anti-mouse secondary antibody with HRP for 1h. The membrane was washed three times with 1TBST, 5 min each time, and then subjected to exposure and development analysis. Figure 5 It was shown that 2E1 had the ability to recognize the full-length PIEZO protein and could clearly identify the PIEZO2 protein (above 310KD) at an exposure time of 0.5s.

[0099] Figure 5 In the figure, 293T-wt represents HEK293T cells, which do not express PIEZO2, and 293T-OE represents cells that overexpress PIEZO2 through transfection. It can be seen that four monoclonal antibodies (1G11, 2E1, 5H8, 9E12) can specifically recognize Piezo2, among which 2E1 has the strongest recognition ability.

[0100] Furthermore, the inventors identified the specificity of the 2E1 antibody and its selectivity for PIEZO1 / PIEZO2 by cell staining. Figure 6 As can be seen (upper half), the fusion fluorescent label of 2E1 antibody and PIEZO2 overlaps well in space, indicating that 2E1 can recognize PIEZO2 protein efficiently and specifically.

[0101] In addition, the inventors also performed tissue staining on the dorsal root ganglion of sensory neurons, and found that 2E1 could recognize the PIEZO2-EGFP fusion protein expressed in DRG. Western blotting experiments showed that 2E1 could recognize PIEZO2 endogenously expressed in the DRG of wild mice and PIEZO2 overexpressed in the DRG of PIEZO2 transgenic overexpressing mice, while no PIEZO2 protein band was detected in the DRG-specific knockout mice of PIEZO2, proving that 2E1 could specifically recognize PIEZO2 expressed in DRG. Figure 7 ).

[0102] Functionally, the electrophysiological silent clamp technique was used to record the current induced by mechanical stimulation mediated by PIEZO2 expressed in HEK293T cells. After the addition of 2E1, the magnitude of the current did not change, but the inactivation rate of the current slowed down, proving that the antibody can enhance the ion channel activity of PIEZO2 ( Figure 8 ).

[0103] The amino acid sequence of the 2E1 antibody was analyzed, and its heavy chain amino acid sequence was SEQ ID NO: 11, the light chain amino acid sequence was SEQ ID NO: 12, and it had a heavy chain variable region with an amino acid sequence of SEQ ID NO: 1, a light chain variable region with an amino acid sequence of SEQ ID NO: 2, and had a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 6, and a heavy chain CDR3 of SEQ ID NO: 7; and a light chain CDR3 of SEQ ID NO: 8.

[0104] CDR1, light chain CDR2 of SEQ ID NO:9 and light chain CDR3 of SEQ ID NO:10.

[0105] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

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Claims

1. A monoclonal antibody against human PIEZO2, or an antigen-binding fragment thereof, comprising: (a) a heavy chain CDR1 comprising SEQ ID NO: 5, a heavy chain CDR2 comprising SEQ ID NO: 6, and a heavy chain CDR3 comprising SEQ ID NO: 7; and (b) a light chain CDR1 comprising SEQ ID NO:8, a light chain CDR2 comprising SEQ ID NO:9, and a light chain CDR3 comprising SEQ ID NO:

10.

2. The monoclonal antibody according to claim 1, wherein The antibodies include: A heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1; and A light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

2.

3. The monoclonal antibody according to claim 1, wherein The antibodies include: A heavy chain having at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11; and A light chain having at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

12.

4. An isolated nucleic acid molecule encoding the monoclonal antibody of any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, comprising: The nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody against human PIEZO2 is SEQ ID NO: 3; and The nucleic acid sequence encoding the light chain variable region of the monoclonal antibody against human PIEZO2 is SEQ ID NO:

4.

6. The nucleic acid molecule according to claim 4, comprising: The nucleic acid sequence encoding the heavy chain of the monoclonal antibody against human PIEZO2 is SEQ ID NO: 13; and The nucleic acid sequence encoding the light chain of the monoclonal antibody against human PIEZO2 is SEQ ID NO:

14.

7. A cell which produces the monoclonal antibody according to any one of claims 1 to 3, or comprises the nucleic acid molecule according to any one of claims 4 to 6.

8. A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 3.

9. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to any one of claims 4 to 6, the cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for diagnosing, preventing or treating the following diseases: analgesia; tactile abnormalities; proprioception abnormalities; visceral abnormalities; Angelman syndrome; Gordon syndrome; Madden-Walker syndrome; scoliosis; distal arthrogryposis syndrome. 10 . A kit comprising the monoclonal antibody according to claim 1 , the nucleic acid molecule according to claim 4 , and / or the cell according to claim 7 .

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