Method for preparing functional p2x1 membrane protein monoclonal antibody and use thereof

Abstract

Disclosed in the present invention are a method for preparing a functional P2X1 membrane protein monoclonal antibody and the use thereof. The method comprises: immunizing mice by means of preparing a human P2X1 protein having a natural spatial structure and activity; preparing eight functional P2X1 monoclonal antibodies by using a monoclonal antibody preparation technique, and selecting two clones with a relatively good functionality to perform gene sequencing so as to obtain functional variable region sequences; and selecting the clone with the best function, and replacing a murine Fc fragment thereof with a human Fc fragment to prepare one semi-humanized chimeric genetically engineered antibody. Function verification shows that the engineered antibody exhibits comparable functional activity to the original murine antibody, thus indicating that there is a specific correlation between the sequence of the variable region F(ab)2 of the engineered antibody and the functional activity of the engineered antibody. The functional P2X1 monoclonal antibody has the effects of inhibiting calcium ion influx in P2X1-overexpressing cells, inhibiting cell dysfunction caused by P2X1 overactivation, restoring the chemotactic function of neutrophil granulocytes inhibited by endotoxin, improving the survival rate of septic mice, inhibiting the division and proliferation of tumor cells, and inhibiting tumor growth in tumor-bearing mice.

Description

Preparation method and application of functional P2X1 membrane protein monoclonal antibody TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a natural active and spatial structure P2X1 protein antigen and a preparation method and application of a functional P2X1 monoclonal antibody. BACKGROUND

[0002] P2X1 belongs to the ATP purinergic receptor family. P2X1 exists in platelets, neutrophils, and macrophages, and is also expressed in smooth muscle cells and some tumor cells. P2X1 acts as a ligand-gated membrane calcium channel to mediate relatively high calcium ion permeability. In neutrophils, it is involved in LPS-induced acute systemic inflammation, and P2X1 plays an important role in LPS-induced lethal endotoxemia by participating in the migration of neutrophils in small venules. P2X1 is involved in apoptosis, and increases intracellular calcium ion concentration to cause programmed cell death. P2X1 binds to ATP to mediate synaptic transmission between neurons and synaptic transmission from neurons to smooth muscle, for example, it is responsible for sympathetic vasoconstriction of small arteries and vas deferens; mouse studies have shown that this receptor is essential for normal male reproductive function. The p2x1 receptor expressed in blood vessels can be activated by ATP and released sympathetic neurotransmitter norepinephrine, causing smooth muscle depolarization and contraction. The expression of P2X1 receptor in platelets of patients with acute coronary syndrome (ACS) and the increase in intracellular calcium concentration after activation of P2X1 receptor have a regulatory effect on the function of platelets in ACS.

[0003] In summary, P2X1 receptor has extensive and important physiological functions, and the development of functional antibodies of P2X1 receptor for regulating abnormal physiological functions caused by P2X1 receptor target has important significance. Changes in intracellular free calcium ion concentration through the action of calmodulin is one of the important key events in cell cycle regulation. Tumor cells are a type of cells with uncontrolled division and proliferation, and P2X1 as an ATP ligand-gated calcium channel affects the cell cycle by manipulating intracellular free calcium ion concentration, and its abnormal function is one of the important reasons for uncontrolled division and proliferation of tumor cells. In addition, studies have shown that P2X1 is involved in apoptosis and increases intracellular calcium ion concentration to cause programmed cell death. Therefore, the development of a functional P2X1 monoclonal antibody with anti-tumor effect has important application value. To prepare a functional P2X1 monoclonal antibody, a human P2X1 protein with natural spatial structure and activity must be prepared as an immunizing antigen, but P2X1 belongs to an ATP-gated ion channel, which is composed of three homologous subunits, each subunit has two transmembrane fragments, and the structure is complex, and its function is dynamic structure.

[0004] Therefore, it is difficult to prepare human P2X1 protein with natural spatial structure and activity. Currently, the antibodies of P2X1 on the market are mostly obtained by immunization of a synthetic small polypeptide as an antigen, and can only be used as detection antibodies, and cannot have physiological functional activity. Currently, no functional P2X1 antibody is available on the market. SUMMARY

[0005] In view of the above problems, the purpose of the present application is to provide a preparation method of a functional P2X1 membrane protein monoclonal antibody and its application, wherein human P2X1 protein with natural spatial structure and activity is used as an immunizing antigen, and active antigen screening and functional verification are used to successfully prepare a functional P2X1 monoclonal antibody with the function of regulating the concentration of free calcium ions in cells.

[0006] The specific technical solutions are as follows:

[0007] A preparation method of a functional P2X1 membrane protein monoclonal antibody, comprising the following steps:

[0008] 1) preparing human P2X1 protein with natural spatial structure and activity;

[0009] 2) using mouse hybridoma monoclonal antibody preparation technology to prepare a functional P2X1 monoclonal antibody;

[0010] The specific operation method of step 1) is as follows: hP2X1mFc.copGFP / 293 stable transfection cells with high expression of human P2X1 protein with a mouse IgG Fc segment purification tag are amplified and collected, homogenized in a Tris hydrochloride buffer under ice bath, centrifuged, and the supernatant is removed. The wet weight of the membrane fragment precipitate is weighed, and the wet weight of the membrane fragment precipitate is not less than 100 grams. Tris hydrochloride buffer is added to prepare a membrane suspension. In the membrane suspension, 5% (W / V) of polystyrene-maleic acid copolymer freeze-dried powder is added and mixed uniformly. After oscillation and centrifugation, the supernatant is collected and filtered through a 0.45 μm filter membrane. The filtrate after filtration is passed through a Protein A column, and the column is washed with PBS buffer. Then, the column is equilibrated with 2 times the column volume of EK enzyme digestion buffer, and finally, an equal column volume of EK enzyme digestion buffer is added. The EK enzyme is shaken at room temperature for 12 hours. Then, the column is eluted by gravity flow, and the eluate is collected. The eluate is replaced with PBS buffer using a 10 kD ultrafiltration tube, and concentrated by 100 times to obtain soluble polystyrene-maleic acid ring-enclosed human P2X1 protein nanoparticles with natural spatial structure and activity on the membrane lipid bilayer.

[0011] Further, the specific operation process of step 2) is that the human P2X1 protein with natural spatial structure and activity prepared in step 1) is used as an antigen to immunize Balb / c mice with Quick Antibody water-soluble adjuvant; and the human P2X1 protein with natural spatial structure and activity is used as a coating antigen, and an enzyme-labeled goat anti-mouse IgG F(ab)2 is used as a secondary antibody, and the serum titer of the immunized mice is detected by ELISA, and only the immunized mice with OD value greater than or equal to 1 at a dilution of 204800 times can be used for subsequent hybridoma preparation; the serum titer of the mice reaching the requirement is used to prepare hybridoma from the spleen; the human P2X1 protein with natural spatial structure and activity is also used as a coating antigen, and an enzyme-labeled goat anti-mouse IgG F(ab)2 is used as a secondary antibody, and positive clones are screened by ELISA; the clones with OD value greater than 1 are positive clones, and the clones are obtained, and the clones with larger OD value are selected by half optimization, and the hybridoma cell culture solution is diluted by 200 times in turn, and the same ELISA is used for determination, and the clones with OD greater than 0.5 at a dilution of 200 times are candidate positive clones, so as to obtain the functional P2X1 monoclonal antibody.

[0012] The application of the functional P2X1 monoclonal antibody prepared by the above preparation method comprises the following steps: the functional P2X1 monoclonal antibody is verified and screened by P2X1 cell intracellular free calcium ion determination experiment, and the screened clones are required to restore the abnormal free calcium concentration in P2X1 overexpressed cells to more than 95% of the normal group of cells, so as to obtain two P2X1 monoclonal antibodies with better functions, and it is proved by neutrophil chemotaxis experiment that they can restore the neutrophil chemotaxis ability inhibited by LPS stimulation to the normal group level; and then the influence of the P2X1 monoclonal antibody on the survival rate of sepsis model mice, the influence on the division and proliferation of cultured tumor cells, the influence on the growth of tumor in tumor-bearing mice, etc. are determined, the best clone is selected, the mouse Fc segment of the clone is replaced by a human Fc segment, and a semi-humanized chimeric genetic engineering antibody is prepared by a transgenic cell method.

[0013] Further, the two P2X1 monoclonal antibodies with better functions obtained are marked as P007 and P008, and each comprises three complementarity determining regions of a heavy chain variable region and three complementarity determining regions of a light chain variable region. The three complementarity determining regions of the heavy chain variable region of the P007 clone comprise the following amino acid sequences:

[0014] 7H-CDR1: GYSFTGYS,

[0015] 7H-CDR2: VSCYNGAT,

[0016] 7H-CDR3: VRGDDYDRAGAY;

[0017] The P007 clone heavy chain variable region includes three complementarity determining regions with the amino acid sequences as follows:

[0018] 7L-CDR1: ESVDNSGISF,

[0019] 7L-CDR2: GAS,

[0020] 7L-CDR3: QQSKEVPP;

[0021] The P008 clone heavy chain variable region includes three complementarity determining regions with the amino acid sequences as follows:

[0022] 8H-CDR1: SYNMH,

[0023] 8H-CDR2: AIYPGNGDTSYNQKFKG,

[0024] 8H-CDR3: RYRHGDGAHFDY;

[0025] The P008 clone light chain variable region includes three complementarity determining regions with the amino acid sequences as follows:

[0026] 8L-CDR1: RSSQSLVHSNGNTYLH,

[0027] 8L-CDR2: KVSNRFS,

[0028] 8L-CDR3: SQSTHVPWT.

[0029] Further, the amino acid sequence of the heavy chain variable region of the P007 antibody is SEQ ID NO. 1, the nucleotide sequence is SEQ ID NO. 2, the amino acid sequence of the light chain variable region of the P007 antibody is SEQ ID NO. 5, the nucleotide sequence is SEQ ID NO. 6, the amino acid sequence of the heavy chain variable region of the P008 antibody is SEQ ID NO. 3, the nucleotide sequence is SEQ ID NO. 4, the amino acid sequence of the light chain variable region of the P008 antibody is SEQ ID NO. 7, the nucleotide sequence is SEQ ID NO. 8, the amino acid sequence of the heavy chain of the semi-humanized chimeric genetically engineered antibody hP007 is SEQ ID NO. 9, and the nucleotide sequence is SEQ ID NO. 10.

[0030] The present application has the following advantages:

[0031] The functional P2X1 monoclonal antibody prepared by the application has the effects of inhibiting P2X1 overexpressed cell calcium ion influx, inhibiting cell function abnormalities caused by P2X1 overexpression or overactivation, restoring the chemotactic function of neutrophils inhibited by bacterial endotoxin, improving the survival rate of sepsis model mice, inhibiting the division and proliferation of tumor cells, and inhibiting the growth of tumors in tumor-bearing mice. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a plasmid map of hP2X1 mFc.copGFP / 293 stable transfection cells and their transfected plasmids;

[0033] Figure 2 is a PAGE, WB and transmission electron microscopy of the P2X1 protein nanoparticles prepared by purification;

[0034] Figure 3 is the serum titer of the mouse immune to the naturally active human P2X1 protein after immunization;

[0035] Figure 4 is the screening of hybridoma cells against the naturally active human P2X1 protein;

[0036] Figure 5 is the titer of the supernatant of the hybridoma subclone strain culture solution against the naturally active human P2X1 protein;

[0037] Figure 6 is the effect of the antibody on LPS-induced abnormal calcium ion influx of neutrophils;

[0038] Figure 7 is the effect of P007 and P008 antibodies on LPS-induced neutrophil chemotaxis;

[0039] Figure 8 is the effect of P007 on the survival rate of LPS-induced WT C57BL / 6 mouse sepsis model;

[0040] Figure 9 is the effect of P007 and P008 on the division and proliferation of cultured tumor cells determined by CCK8 method;

[0041] Figure 10 is the effect of P007 on the division and proliferation of cultured tumor cells;

[0042] Figure 11 is the effect of P007 on the tumor formation of mice inoculated with human lung cancer cells H460;

[0043] Figure 12 is the effect of P007 on the tumor formation of mice inoculated with human melanoma cells;

[0044] Figure 13 is the effect of P007 and hP007 on LPS-induced abnormal calcium ion influx of neutrophils. DETAILED DESCRIPTION

[0045] The application will be described in detail below in conjunction with the drawings and specific examples of the specification:

[0046] The application discloses a preparation method of a functional P2X1 membrane protein monoclonal antibody and application thereof, and those skilled in the art can refer to the content of the application, and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and are regarded as being included in the application. The preparation method and application of the application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation method and application described in the application without departing from the content and range of the application, to realize and apply the technical solution of the application.

[0047] Part of the terms and abbreviations are explained:

[0048] P2X1 or P2RX1: purinergic receptor P2X, ligand-gated ion channel 1

[0049] PCR: Polymerase Chain Reaction

[0050] ELISA: enzyme-linked immunosorbent assay

[0051] CD: Chemotaxis distance

[0052] CCR: Chemo cell ratio

[0053] CI: Chemo index

[0054] CFS: Chemotaxis function score

[0055] In the following examples, the molecular biology experimental techniques, including PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis and the like, are generally operated according to conventional methods, and specific reference can be made to Molecular Cloning Experiments Guide (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang et al, 2002, Beijing: Science Press) or the conditions suggested by the manufacturer. The raw reagents and materials used in the following examples can be commercially available. Among them:

[0056] hP2X1mFc.copGFP / 293 stable cells (as shown in Figure 1, A is a fluorescence micrograph of the stable cells, and B is a plasmid map of the transfected stable cells) and styrene-maleic acid copolymer freeze-dried powder were purchased from Hangzhou Maierde Biotechnology Co., Ltd.; Quick Antibody water-soluble adjuvant was purchased from Biodragon; 4-5-week-old BALB / C mice and 6-8-week-old NOD-SCID mice were purchased from the Experimental Animal Center of Zhejiang Chinese Medical University, and 6-8-week-old NSG immunodeficient mice were purchased from Nanjing Model Animal Research Center; human lung cancer cells H460 and human melanoma cells A375 (purchased from China Typical Culture Collection Center).

[0057] Example 1: Preparation of naturally spatially structured and active human P2X1 protein

[0058] 1.1 The hP2X1mFc.copGFP / 293 cells were collected, homogenized in Tris hydrochloride buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% (v / v) glycerol) in an ice bath; centrifuged at 100,000 x g at 4°C for 30 min, the supernatant was removed, and the wet weight of the membrane fragment precipitate was measured, a total of 125 g; and the membrane suspension was prepared by adding Tris hydrochloride buffer at 30 mg / ml;

[0059] 1.2 In the above membrane suspension, 5% (W / V) of styrene-maleic acid copolymer freeze-dried powder was added and mixed evenly; 37°C, 250 rpm shaking bed for 1 h; 4°C, 100000g centrifugation for 30 min, collect the supernatant and pass through a 0.45 μm filter membrane;

[0060] 1.3 The above collected supernatant was passed through a Protein A column, the column was washed with PBS buffer (pH 7.4) and then equilibrated with 2 column volumes of EK enzyme digestion buffer (50 mM Tris-HCl pH 8.0, 50% (v / v) glycerol), and finally 10 μL of EK enzyme was mixed with the filler and shaken, and the enzyme digestion was carried out at room temperature for 12 h;

[0061] 1.4 The flow-through liquid was collected and replaced with PBS buffer (pH 7.4) by using 10 kD ultrafiltration tube (millipore) and concentrated 100 times to obtain the soluble human P2X1 protein nanoparticles with natural spatial structure and activity on the membrane lipid bilayer of styrene-maleic acid ring. The concentration was determined and the purity was detected by SDS-PAGE electrophoresis (≧95%, A in Fig. 2), WB identification (B in Fig. 2) and transmission electron microscope observation of the nanoparticles (C in Fig. 2). About 10 mg of the human P2X1 protein with a purity of ≧95% was obtained and stored in aliquots of 1 mg per aliquot for later use.

[0062] Example 2: Preparation of hP2X1 monoclonal antibody

[0063] 2.1 Seven Balb / c mice were immunized with the human P2X1 protein with natural spatial structure and activity prepared in Example 1 as an antigen by using Quick Antibody water-soluble adjuvant. The human P2X1 protein with natural spatial structure and activity was used as a coating antigen, and the enzyme-labeled goat anti-mouse IgG F(ab)2 was used as a secondary antibody. The serum titer of the immunized mice was detected by ELISA. The immunized mice whose serum dilution of 204800 times still had OD value greater than or equal to 1 were used for subsequent hybridoma preparation. There was only one mouse that met the requirements, as shown in Fig. 3.

[0064] 2.2 The mouse whose serum titer met the requirements was used to prepare hybridoma from the spleen. The human P2X1 protein with natural spatial structure and activity was used as a coating antigen, and the enzyme-labeled goat anti-mouse IgG F(ab)2 was used as a secondary antibody. The positive clones were screened by ELISA. The clones with OD value greater than 1 were positive clones. There were 17 clones in four 96-well plates, and the OD values were 1.154, 1.342, 1.375, 1.397, 1.489, 1.500, 1.617, 1.618, 1.629, 1.691, 1.725, 1.929, 1.979, 2.065, 2.373, 2.614, 3.5, respectively, as shown in Fig. 4. Eight clones with larger OD values were selected by half-preference, and were identified as P001, P002, P003, P004, P005, P006, P007 and P008, respectively. The hybridoma cell culture liquids of the eight clones were diluted by half successively, and were detected by the same ELISA method. The clones with OD greater than 0.5 when diluted 200 times were candidate positive clones. The eight clones all met the requirements, as shown in Fig. 5.

[0065] Example 3: In vitro functional verification screening

[0066] 3.1 Intracellular free calcium ion determination experiment

[0067] The positive clones screened by ELISA method are verified by high expression P2X1 intracellular free calcium ion assay experiment, and the experimental steps are as follows:

[0068] 1) Peripheral blood neutrophil separation kit was used to purify neutrophils, and grouping was performed, ① control group; ② LPS lipopolysaccharide stimulation group (1 μg / ml of LPS stimulation for one hour, then washed); ③ LPS stimulation after antibody intervention group (1 μg / ml of LPS stimulation for one hour, then washed, 1 μg / ml of antibody intervention for one hour), the antibody intervention group includes P001, P002, P003, P004, P005, P006, P007, P008, a total of 8 antibody groups. The treated cells were washed with PBS twice, and the cell concentration was adjusted to 1×10 7 individuals / mL.

[0069] 2) According to the operation steps of Rhod-2 AM calcium ion fluorescent probe kit, Rhod-2, AM working solution was added to each group of cells, and incubated at 37℃ for 2h; discard the working solution, and wash with HHBS buffer to remove residual probe; continue to incubate at room temperature for 30min; finally, flow detection, 5000 cells were analyzed in each group, and the percentage of positive fluorescent cells was detected by calcium ion fluorescent probe as an index, the result of blank control group was 20.00%, and the result of LPS model group was 80.00%; a total of 2 functional clones with ≤25.00% were obtained, which were labeled as P007 and P008, and the results were 20.17% and 23.33%, respectively. The results are shown in Figure 6.

[0070] The positive clones screened above are verified by neutrophil chemotaxis experiment, and the experimental steps are as follows:

[0071] 1) Peripheral blood neutrophil separation kit was used to purify neutrophils, and grouping was performed, ① control group; ② LPS lipopolysaccharide stimulation group (1 μg / ml of LPS stimulation for one hour, then washed); ③ LPS stimulation after P007 antibody intervention group (1 μg / ml of LPS stimulation for one hour, then washed, 1 μg / ml of antibody intervention for one hour).

[0072] 2) The treated cells were washed with PBS twice, and the cell concentration was adjusted to 1×10 7 individuals / mL, 10 μL of cells were added to the agarose chemotaxis model hole in each group, and placed in the incubator for chemotaxis for 2h.

[0073] 3) The cells after chemotaxis were taken pictures under microscope and uploaded to CCAP system for analysis of chemotaxis function. The analysis indexes included CD (Chemotaxis distance) (A in FIG. 7), CFS (Chemotaxis function score) (B in FIG. 7), CR (Chemo cell ratio) (C in FIG. 7), and CI (Chemo index) (D in FIG. 7).

[0074] The results showed that the CFS value of the normal control group was 16; the CFS value of the LPS inhibition model control group was 6, which was extremely significantly different from that of the normal control group, i.e. ****P<0.0001; P007 and P008 could restore the chemotaxis ability of neutrophils inhibited by LPS to the level of the normal group, and the CFS values were 16 and 15.8, respectively, which were extremely significantly different from those of the LPS inhibition model control group, i.e. P<0.0001, and were not different from those of the normal control group (ns), P>0.05, as shown in B in FIG. 7.

[0075] Example 4: Effect of functional antibody on survival rate of sepsis model mice

[0076] 4.1 Put 50 ml centrifuge tubes, micropipettes, disposable sterile rubber bulb pipettes and other consumables into the clean bench in advance, and sterilize under ultraviolet light for 30 min. Weigh an appropriate amount of LPS powder at a concentration of 1 mg / ml and dissolve it with sterile normal saline, and mix it repeatedly with a dropper until no visible clumps are present.

[0077] 4.2 Observe the effect of P007 on the survival rate of 6-8 week old WT male Balb / C mice by LPS intraperitoneal injection method for severe modeling (LPS concentration 10 mg / kg). The experiment is divided into 5 groups, 6 mice in each group, namely normal control group (Control), model group (Model), 500 μg / kg P007 group, 1 mg / kg P007 group and 1.5 mg / kg P007 group. The drug is administered by intraperitoneal injection, and the administration time is 2 h before LPS intraperitoneal injection modeling, 1 h after modeling and 2.5 h after modeling. Observe every 6 h after modeling, record the symptoms and death number of each group of mice, and draw the survival rate curve after 5 days of data statistics.

[0078] 4.3 The sepsis model of Balb / C mice was induced and established by LPS intraperitoneal injection for severe modeling, and the results are shown in FIG. 8: At the end of 5 days of observation, the symptoms of the model group of Balb / C mice were severe, and all died within 48 h. Compared with the model group, the survival rate of the P007 group was significantly improved, and there was a dose-effect relationship.

[0079] Example 5: Effect of functional antibodies on the division and proliferation of tumor cells in vitro

[0080] The effect of the positive clones P007 and P008 screened in Example 3 on the division and proliferation of cultured tumor cells was determined by CCK8 method experiment. The CCK8 method experiment procedure is as follows:

[0081] 5.1. Cell culture

[0082] Human malignant melanoma cells A375 were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody at 37°C in a saturated humidity incubator containing 5% CO2.

[0083] 5.2 Cell plating

[0084] (1) Take the A375 cells in the logarithmic growth phase and count the cells.

[0085] (2) Seed the cells into a 96-well plate at a density of 10,000 cells per well and continue to culture. The experimental groups are: blank control group (Tumor NC); add 1 μg / ml concentration of antibodies

[0086] (3) After the cells adhere, add the test antibodies to the drug groups. The experimental groups are: blank control group (Tumor NC); add 1 μg / ml concentration of antibodies, including Tumor+P007 and Tumor+P008; each group has 10 replicate wells. CCK8 detection was performed after 24 hours.

[0087] 5.3 CCK8 detection experiment procedure

[0088] (1) At the detection time point, add 1 / 10 volume of CCK8 to each well of the cells in each group and incubate in a 37°C, 5% CO2 incubator for 3 hours.

[0089] (2) Select 450 nm wavelength and measure the light absorption value of each well on an enzyme-linked immunoassay instrument and record the results.

[0090] The results show that the two P2X1 monoclonal antibodies P007 and P008 both have the effect of inhibiting the division and proliferation of tumor cells. The survival rate of tumor cells after 24 hours of action is 6.8% and 8.2% of the normal control group (Tumor NC), respectively, which reaches a very significant difference compared with the normal control group, i.e. P≦0.0001, as shown in Figure 9. Under a microscope, the tumor cells are significantly reduced compared with the normal control group at 1 μg / ml of P007 antibody, which has a significant killing effect on the tumor and this killing has a time-dependent effect, with a tumor cell killing rate of more than 90% after 24 hours of action, as shown in Figure 10.

[0091] Example 6: Effect on tumor formation of mice inoculated with human lung cancer cells H460

[0092] 1) Extract 20ml of healthy person's blood, use Solerbao human mononuclear cell sorting kit, sort out human primary mononuclear cells (PBMCs) by density gradient centrifugation. Transfer to serum-free RPMI-1640 medium for 2h, and then transfer to 1640 complete medium containing 10% FBS and 1% double antibody after adaptation. Change the liquid every 24h. At the same time, H460 cells are expanded and cultured.

[0093] 2) PBMCs primary cell culture to stable state, H460 expansion to sufficient quantity. Collect PBMCs and H460 by centrifugal sedimentation, resuspend each with 200ul PBS, and estimate the cell number by plate counting. Dilute PBMCs to 2x10 4 cells / ul, dilute H460 cells to 1x10 4 cells / ul, take 1ml of each cell resuspension and mix well for injection.

[0094] 3) Establishment of immunoheterograft tumor model: 40 NOD-SCID mice aged 6-8 weeks were ordered from Zhejiang Chinese Medicine University Experimental Animal Center and fed in SPF environment. The pre-suspended cell suspension was injected subcutaneously at the back of each mouse at 5x10 5 H460 cells / 50ul + 1x10 6 personal PBMCs / 50ul; tumor bodies appeared after one week of injection, and the tumor volume was measured every three days thereafter. The tumor volume was calculated by the formula: (length x width 2 ) / 2. The model was considered successful when the tumor volume was >50mm 3 .

[0095] 4) Animal grouping and drug treatment: after cell inoculation, NOD-SCID mice were randomly divided into 4 groups, 10 mice in each group, and given intraperitoneal injection of drugs: 200ul PBS blank; 0.92mg / kg Durvalumab positive drug control group; 0.92mg / kg P2X1 monoclonal antibody P007 group; 0.46mg / kg Durvalumab + 0.46mg / kg P007 positive drug and P007 combination group, all drugs were diluted with PBS to 200ul suspension for intraperitoneal injection. Intraperitoneal injection of drugs was performed on the 1st, 4th, 7th, and 10th days after cell inoculation, and the body weight and tumor size of each mouse were measured. When the tumor volume was >=2000mm 3 , the mice were euthanized by CO2.

[0096] 5) The results show that the 0.92 mg / kg P2X1 monoclonal antibody P007 group completely inhibited the growth of NOD-SCID mice inoculated with human lung cancer cells H460, with an inhibition rate of 100%, better than the 0.92 mg / kg Durvalumab positive drug control group, with a very significant difference; and the tumor tissue inside the 0.92 mg / kg P2X1 monoclonal antibody P007 group and the combined group showed a cavity. The experimental results are shown in Figure 11.

[0097] Example 7: Effect on tumor formation of mice inoculated with human melanoma cells

[0098] 7.1 Construction of tumor-bearing mouse model by xenotransplantation

[0099] A375 cells were routinely expanded in DMEM medium containing 10% FBS, 1% double antibody. NSG immunodeficient mice (6-8 weeks old, 6-7 per group) purchased from Nanjing Model Animal Center were raised in a barrier environment. After 7 days of adaptive feeding, A375 cells (1 x 10 6 / 100 μl / each) were injected subcutaneously into the right front limb of the mice, and the tumors were visible to the naked eye after about 7 days.

[0100] 7.2 Drug administration and determination results

[0101] When the tumor length was >0.5 cm, tail vein injection was started, and the specific grouping and dosing were as follows:

[0102] The drug was administered once every 3 days (Day 1, Day 4, Day 7), a total of 3 times, and the mice were weighed and the tumor size was measured at the same time. When the tumor length of any mouse was >2 cm, the experiment was stopped at any time, the mice were euthanized with CO2, the tumor was peeled off, photographed, weighed, and the length, width, and thickness were measured with a vernier caliper. The volume was calculated according to the formula, and the results are shown in Figure 12.

[0103] Example 8: Antibody sequence sequencing

[0104] 8.1 Detection of P007 and P008 monoclonal antibody subtypes

[0105] The two P2X1 functional antibodies P007 and P008 selected were detected using the antibody subtype detection kit (SEK003) from Beijing Yiqiao God Science and Technology Co., Ltd., and their IgG subtypes were IgG1 and IgG2a, respectively. The hybridoma cells were expanded and cultured, and part of them were frozen. Then part of the cells were collected for sequencing.

[0106] 8.2 Extraction of P007 and P008 monoclonal antibody heavy chain and light chain variable region genes

[0107] After adding Trizol Reagent (Life, 15596026) to the collected hybridoma cell centrifuge tube, use a pipette to repeatedly blow. Place at room temperature for 5 min, and wait for the nucleic acid and protein to be fully dissociated. Add 0.2 mL of chloroform to each 1 mL of Trizol Reagent reagent homogenate, shake vigorously for 15 s, and then stand at room temperature for 2-3 min. 4°C, 12000g, centrifuge for 10 min. Carefully aspirate the upper aqueous phase (colorless) into a new test tube, and calculate the volume of the aspirated aqueous phase. Then add an equal volume of pre-cooled isopropanol to the aspirated aqueous phase, tightly cap the tube, and gently shake. Stand at room temperature for 10 min, and wait for the RNA to be fully precipitated. 4°C, 12000g centrifuge for 10 min. After discarding the supernatant, add 1 mL of 80% ethanol to each tube, tightly cap the tube, and gently shake the centrifuge tube to remove residual isopropanol and salt. 4°C, 7500g centrifuge for 5 min. Discard the supernatant, open the tube cap, and dry the RNA precipitate. Finally, dissolve the RNA precipitate in an appropriate amount of RNase Free ddH2O. Take 1 μL, and use 1% agarose gel electrophoresis for identification. After that, the extracted RNA is reverse transcribed into cDNA by cDNA Synthesis Kit (Nanjing Novozyme, R312-02), and stored.

[0108] 8.3 Sequencing of monoclonal antibody variable region genes

[0109] The above cDNA genes were sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing of variable region genes.

[0110] 8.4 Analysis of gene sequencing results of monoclonal antibody heavy and light chain variable domains

[0111] According to the sequencing results, the heavy and light chain variable region genes of the anti-P2X1 monoclonal antibody were analyzed by the database on the NCBI website (https: / / www.ncbi.nlm.nih.gov / igblast / ), and the heavy and light chain variable domain genes of the hybridoma cell strain antibody were successfully obtained. The P007 clone heavy chain variable region contains the high variable region 7H-CDR1, 7H-CDR2 and 7H-CDR3, and the light chain variable region sequence contains the high variable region 7L-CDR1, 7L-CDR2 and 7L-CDR3; the P008 clone heavy chain variable region contains the high variable region 8H-CDR1, 8H-CDR2 and 8H-CDR3, and the light chain variable region sequence contains the high variable region 8L-CDR1, 8L-CDR2 and 8L-CDR3. The amino acid sequences of the heavy chain variable regions of the P007 and P008 antibodies are shown in SEQ ID NO. 1 and SEQ ID NO. 3, and the corresponding nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 4; the amino acid sequences of the light chain variable regions are shown in SEQ ID NO. 5 and SEQ ID NO. 7, and the corresponding nucleotide sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 8.

[0112] The IgBLAST sequence alignment of the antibody heavy chain sequence conforms to the characteristics of the mouse IgG variable region gene, and the antibody light sequence also conforms to the characteristics of the mouse IgG variable region gene. According to the kabat rule, it is determined that the P007 and P008 antibodies each contain 3 complementarity determining regions (Complementarity-determining-region, CDR) and 4 frame region sequences (Frame region). The heavy chain complementarity determining region sequence includes the high variable region sequences H-CDR1, H-CDR2 and H-CDR3, and the light chain complementarity determining region sequence includes the high variable region sequences L-CDR1, L-CDR2 and L-CDR3. The two functional P2X1 monoclonal antibody positive clones each include a heavy chain variable region and a light chain variable region, as shown in Table 1.

[0113] Table 1 Amino acid sequences of the heavy and light chain variable complementarity determining regions of the P007 and P008 clones

[0114] Example 9: Preparation and functional verification of semi-humanized antibodies

[0115] The P007 antibody heavy chain variable region SEQ ID NO: 2 is fused with the Fc segment of human IgG, the nucleotide sequence of which is shown as SEQ ID NO: 9 in the sequence table 3, and the amino acid sequence is shown as SEQ ID NO: 10 in the sequence table 3 containing 7H-CDR1, 7H-CDR2 and 7H-CDR3 three complementarity determining regions. The genetically engineered semi-humanized functional P2X1 monoclonal antibody is prepared by taking the sequence of SEQ ID NO: 10 as the heavy chain and the sequence of SEQ ID NO: 6 as the light chain, and is identified as hP007. The same dose of semi-humanized antibody hP007 and its murine antibody P007 are verified to have equivalent functional effects by intracellular free calcium ion assay experiment, and there is no difference, as shown in FIG. 13. The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, and they should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are all known technologies.

Claims

1. A method of producing a monoclonal antibody to a functional P2X1 membrane protein, characterized by, The method comprises the following steps: 1) preparing human P2X1 protein with natural spatial structure and activity; 2) preparing functional P2X1 monoclonal antibody by using mouse hybridoma monoclonal antibody preparation technology; The specific operation method of step 1) is as follows: hP2X1mFc.copGFP / 293 stable transfected cells with human P2X1 protein with high expression and a mouse IgG Fc segment purification tag are amplified, homogenized in a Tris hydrochloride buffer under ice bath, centrifuged, and the supernatant is removed. The wet weight of the membrane fragment precipitate is weighed, and the wet weight of the membrane fragment precipitate is not less than 100 grams. The Tris hydrochloride buffer is added to prepare a membrane suspension. In the membrane suspension, 5% (W / V) of styrene-maleic acid copolymer freeze-dried powder is added and uniformly mixed. After oscillation and centrifugation, the supernatant is collected and filtered through a 0.45 μm filter membrane. The filtrate is passed through a Protein A column. After the column is washed with a PBS buffer, the column is equilibrated with 2 times the column volume of EK enzyme digestion buffer. Finally, an equal column volume of EK enzyme digestion buffer is added, and the EK enzyme is shaken at room temperature for 12 hours. Then, the column is eluted by gravity flow, and the eluate is replaced with a PBS buffer by using a 10 kD ultrafiltration tube and concentrated by 100 times to obtain soluble human P2X1 protein nanoparticles with natural spatial structure and activity, which are still in the membrane lipid bilayer and are coiled by styrene-maleic acid.

2. The method for preparing a monoclonal antibody against a functional P2X1 membrane protein as described in claim 1, characterized in that, The specific operation process of step 2) is as follows: the human P2X1 protein with natural spatial structure and activity prepared in step 1) is used as an antigen to immunize Balb / c mice by using Quick Antibody water-soluble adjuvant. The human P2X1 protein with natural spatial structure and activity is used as a coating antigen, and an enzyme-labeled goat anti-mouse IgG F(ab)2 is used as a secondary antibody. The serum titer of the immunized mice is detected by using an ELISA method. The immunized mice whose serum is diluted by 204800 times and whose OD value is greater than or equal to 1 are used for subsequent hybridoma preparation. The human P2X1 protein with natural spatial structure and activity is used as a coating antigen, and an enzyme-labeled goat anti-mouse IgG F(ab)2 is used as a secondary antibody. Positive clones are screened by using an ELISA method. The clones with an OD value greater than 1 are obtained. The hybridoma cells of several clones with larger OD values are selected by using a half optimal method. The culture solution of the hybridoma cells is diluted by 200 times in sequence, and the OD value is detected by using the same ELISA method. The clones with an OD value greater than 0.5 after dilution by 200 times are used as candidate positive clones to obtain functional P2X1 monoclonal antibody.

3. The use of the functional P2X1 monoclonal antibody prepared by the preparation method according to claim 1, characterized in that, The method comprises the following steps: screening the functional P2X1 monoclonal antibody by P2X1 cell intracellular free calcium ion assay experiment, and requiring that the screened clone restores the abnormal intracellular free calcium concentration of P2X1 overexpressed cells to more than 95% of the normal group cell level, so as to obtain two P2X1 monoclonal antibodies with good functions, and it is proved by the neutrophil chemotaxis experiment that they can restore the neutrophil chemotaxis ability inhibited by LPS to the normal group level; then, the influence of the P2X1 monoclonal antibody on the survival rate of sepsis model mice, the influence on the division and proliferation of cultured tumor cells, the influence on the growth of tumor-bearing mice, and the like are determined, the best clone is selected, the mouse Fc segment is replaced by the human Fc segment, and a semi-humanized chimeric genetic engineering antibody is prepared by using the transgenic cell method.

4. The use of a functional P2X1 monoclonal antibody according to claim 3, characterized in that, The two P2X1 monoclonal antibodies with good functions obtained are marked as P007 and P008, and each comprises three complementarity determining regions of a heavy chain variable region and three complementarity determining regions of a light chain variable region. The three complementarity determining regions of the heavy chain variable region of the P007 clone comprise the following amino acid sequences: 7H-CDR1: GYSFTGYS, 7H-CDR2: VSCYNGAT, and 7H-CDR3: VRGDDYDRAGAY. The three complementarity determining regions of the light chain variable region of the P007 clone comprise the following amino acid sequences: 7L-CDR1: ESVDNSGISF, 7L-CDR2: GAS, 7L-CDR3: QQSKEVPP. The three complementarity determining regions of the heavy chain variable region of the P008 clone comprise the following amino acid sequences: 8H-CDR1: SYNMH, 8H-CDR2: AIYPGNGDTSYNQKFKG, 8H-CDR3: RYRHGDGAHFDY. The three complementarity determining regions of the light chain variable region of the P008 clone comprise the following amino acid sequences: 8L-CDR1: RSSQSLVHSNGNTYLH, 8L-CDR2: KVSNRFS, 8L-CDR3: SQSTHVPWT.

5. The use of a functional P2X1 monoclonal antibody according to claim 4, characterized in that, The amino acid sequence of the heavy chain variable region of the P007 antibody is SEQ ID NO. 1, the nucleotide sequence is SEQ ID NO. 2, the amino acid sequence of the light chain variable region of the P007 antibody is SEQ ID NO. 5, the nucleotide sequence is SEQ ID NO. 6, the amino acid sequence of the heavy chain variable region of the P008 antibody is SEQ ID NO. 3, the nucleotide sequence is SEQ ID NO. 4, the amino acid sequence of the light chain variable region of the P008 antibody is SEQ ID NO. 7, the nucleotide sequence is SEQ ID NO. 8, the amino acid sequence of the heavy chain of the semi-humanized chimeric genetic engineering antibody hP007 is SEQ ID NO. 9, and the nucleotide sequence is SEQ ID NO. 10.

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