Anti-human CD226 antibody and application thereof

Abstract

The invention provides an anti-human CD226 antibody and application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, the CDR1, the CDR2 and the CDR3 of the heavy chain variable region are amino acid sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively; the CDR1, the CDR2 and the CDR3 of the light chain variable region are amino acid sequences as shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively. The anti-human CD226 antibody disclosed by the invention is detected by using a flow cytometer, and the expression condition of CD226 on cells is judged according to the intensity change of a CD226 fluorescence signal, so that the functional state of human immune cells is judged in an auxiliary manner.

Description

TECHNICAL FIELD

[0001] The present application relates to a diagnostic detection antibody, in particular to an anti-human CD226 antibody and application. BACKGROUND

[0002] CD226, also known as DNAM-1, is a cell surface receptor belonging to the immunoglobulin superfamily. CD226 is a type I transmembrane glycoprotein, and its structure can be divided into three main parts: extracellular region, transmembrane region and intracellular region. The extracellular region is the key part of CD226 binding to ligand, which contains two immunoglobulin-like domains: 1. Membrane distal V-type domain, which is the main area of ligand binding; 2. Membrane proximal C2-type-like domain, which mainly plays a supporting and stabilizing role. The transmembrane region is a single alpha helix structure, which anchors the CD226 molecule to the cell membrane, connecting the extracellular region and the intracellular region. The tyrosine phosphorylation site in the intracellular region is responsible for transmitting the signal received by the extracellular to the intracellular, playing a role in activating the signal. CD226 is mainly expressed in natural killer cells (NK cells), T cells (including CD8+ killer T cells and CD4+ helper T cells), monocytes, platelets, etc.

[0003] When immune cells (such as NK cells or T cells) recognize target cells (such as cancer cells or infected cells), just through the main recognition receptor (such as T cell receptor TCR) signal is not enough, other auxiliary signals are also needed to fully activate the immune cells, so that they can effectively kill the target or secrete cytokines. CD226 is one of the key signals to provide such signals. But CD226 must bind to ligand to start the program. Its ligands are PVR (also known as CD155) and Nectin-2 (also known as CD112). In normal tissue cells, the expression of PVR and Nectin-2 is very low, but in cancer cells, virus-infected cells and some stem cells, the expression will be significantly increased, so as to be recognized and removed by immune cells expressing CD226.

[0004] The function of CD226 is not isolated, but in a very delicate regulatory network, competing with two other receptors TIGIT and CD96 for binding to the same ligand (PVR and Nectin-2). In contrast to CD226, TIGIT mainly plays a role in inhibiting immune activation. In a healthy state, TIGIT slightly prevails to prevent the immune system from over-attacking the body's own tissues. In the tumor microenvironment, cancer cells will use this mechanism to highly express ligands such as PVR, which seems to be an activation signal, but at the same time will attract and bind a large amount of TIGIT, resulting in the strong inhibition of immune cells even if they express CD226, thereby achieving immune escape. Therefore, the CD226 / TIGIT axis is an extremely important target in the field of tumor immunotherapy. Generally, the expression level of CD226 of the functionally exhausted T cells will be reduced, therefore, detecting the expression of CD226 in human peripheral blood can be used to assist in judging the occurrence of some diseases, and providing great help for clinical diagnosis and treatment. SUMMARY

[0005] Therefore, the present application aims to overcome the defects in the prior art and proposes an anti-human CD226 antibody and application.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: The present application also provides an anti-human CD226 antibody, which comprises a heavy chain variable region and a light chain variable region. The CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The CDR1, CDR2 and CDR3 of the light chain variable region are respectively the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

[0007] Further, the FR1, FR2, FR3 and FR4 of the heavy chain variable region are respectively the amino acid sequences shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10. The FR1, FR2, FR3 and FR4 of the light chain variable region are respectively the amino acid sequences shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14.

[0008] Further, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16.

[0009] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-human CD226 antibody.

[0010] The present application also provides an expression vector comprising the nucleic acid molecule.

[0011] The present application also provides a cell comprising the nucleic acid molecule or the expression vector.

[0012] The present application also provides use of the anti-human CD226 antibody in preparation of a detection reagent for CD226 molecules.

[0013] The present application also provides use of the anti-human CD226 antibody in preparation of a detection reagent for chronic glomerulonephritis.

[0014] The present application also provides an immunological detection kit comprising the anti-human CD226 antibody.

[0015] Compared with the prior art, the present application has the following advantages: The anti-human CD226 antibody of the present application is detected by a flow cytometer, and the expression of CD226 on cells is judged according to the change in the fluorescence signal strength of CD226, thereby assisting in determining the functional state of human immune cells.

[0016] The anti-human CD226 antibody of the present application can be used to determine the functional state of human immune cells, and provides an anti-human CD226 monoclonal antibody light chain variable region gene and a heavy chain variable region gene, and a hybridoma cell strain capable of producing a CD226 specific antibody. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A purification electropherogram of the anti-human CD226 antibody described in the embodiments of the present application; Figure 2 An activity detection diagram of the anti-human CD226 antibody described in the embodiments of the present application; Figure 3 An affinity detection diagram of the anti-human CD226 antibody described in the embodiments of the present application; Figure 4 An activity detection diagram of the anti-human CD226-APC antibody described in the embodiments of the present application. DETAILED DESCRIPTION

[0018] Unless defined, the technical terms used in the following embodiments have the same meanings as generally understood by those skilled in the art to which the present application belongs. The experimental reagents used in the following embodiments are conventional biochemical reagents unless otherwise specified; and the experimental methods described are conventional methods unless otherwise specified.

[0019] The present application will be described in detail below with reference to examples.

[0020] Example 1 Cloning of human CD226 antigen sequence In the process of plasmid construction, the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP was used as a blank, and the CD226 gene (encoding human CD226 protein) was inserted into the multiple cloning site to construct (GENE ID: NM_001303618.2, https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001303618.2). Meanwhile, the prepared packaging plasmid pMD2.G and psPAX2 were extracted. The above-mentioned plasmids were extracted using a plasmid extraction kit, and the obtained target plasmid was frozen and stored in a -20°C refrigerator.

[0021] Example 2 Preparation of lentivirus carrying human CD226 antigen sequence and immune cells (1) Lentivirus packaging: 293T cells in good condition were uniformly plated in a cell culture dish; 16 hours later, the three plasmids extracted in advance were mixed with the transfection reagent in a certain proportion and reacted at room temperature; the plasmid-transfection reagent mixture after reaction was gently mixed, added to the 293T cells, and the cells were avoided to be blown up; after 48-72 hours of culture, the supernatant was collected; then the supernatant was centrifuged using a high-speed centrifuge to obtain virus precipitate; the concentrated virus was small-volume packaged for subsequent infection experiments and stored in a -80°C refrigerator.

[0022] (2) Lentivirus infection of 3T3 cells: 3T3 cells in good growth condition were plated in a 24-well plate, ensuring that the density reached 50%-70% the next day, and cultured overnight in a 37°C incubator. The virus was thawed in ice water, diluted to the appropriate concentration with fresh culture medium, and then polybrene was added to a final concentration of 2-10 μg / ml, and gently mixed. The original culture medium of the cells was aspirated, and the mixed solution containing the virus and polybrene was added for culture. After 24 hours, fresh culture medium was replaced. After a few days, the GFP fluorescence intensity of 3T3 cells was observed under a filter, and the cells were cultured and expanded after obtaining the ideal brightness. The cells were named CD226-3T3.

[0023] (3) Flow cytometry analysis of lentivirus infection efficiency: CD226-3T3 cells cultured after infection were collected, labeled with corresponding antibodies, and the positive rate data of the cells were detected by flow cytometry; when the positive rate of CD226-3T3 cells reached more than 95%, the antigen could be used to immunize animals.

[0024] Example 3 Animal immunization A sufficient number of CD226-3T3 cells were collected as immunogens, 4-5 week old female BALB / c mice were selected, and the mice were immunized by intraperitoneal injection; booster immunization was performed at 3 weeks and 5 weeks after the initial immunization, respectively; and the serum of the mice was detected for titer.

[0025] Example 4 Fusion and screening of cells (1) 3 days before fusion, the mice were shocked by tail vein injection; on the day of fusion, mice with high immune titer were selected, and the spleen was carefully removed in a biological safety cabinet, and the spleen cells were collected by grinding the spleen; at the same time, SP2 / 0 cells in the logarithmic growth phase and in good growth state were collected, and the SP2 / 0 cells and the spleen cells were mixed in a certain proportion to prepare for fusion; the fusion process was carried out in a 37°C water bath environment, and PEG was used for cell fusion; after fusion, the cells were resuspended with a special medium, and the cells were plated in a flat-bottom 96-well plate and cultured in a cell incubator.

[0026] (2) Screening of hybridoma cells secreting specific CD226-targeted antibodies and subcloning: when the cell clones were observed to be large enough under a microscope, the supernatant of the corresponding well was taken for flow cytometry detection; when a positive well was screened, the well was expanded and retested; if the detection result was still positive, a round of subcloning was performed by limiting dilution method; if the detection result was negative, it was discarded; the clones with positive detection results in the first round of subcloning continued to be subcloned in the second round; the clones with positive detection results in the second round of subcloning were subcloned in the third round; at least three rounds of subcloning were generally performed, and the clones obtained in the third round of subcloning were all positive clones, i.e., stable hybridoma cell lines were obtained.

[0027] Example 5 Preparation and purification of anti-human CD226 antibody (1) Collection of mouse ascites: a certain number of hybridoma cells in good growth state were prepared, and the mice were injected intraperitoneally; when the abdomen of the mouse was significantly distended, the ascites could be collected; the collected ascites was stored at -20°C.

[0028] (2) Purification of anti-human CD226 antibody: the ascites collected in step (1) was purified by Protein A affinity column, PBS was used to balance the column, then the sample was loaded, after the loading was completed, glycine with pH 3.0 was used for elution, and the purified antibody was replaced with G25 column, the solution was replaced with PBS, then it was aliquoted and stored at -20°C, and the purification electrophoretogram is shown in Figure 1 .

[0029] Example 6 Activity determination of anti-human CD226 antibody Normal human peripheral blood detection: add 100 μL of normal human anticoagulant peripheral blood to the flow tube, add different amounts of CD226 antibody, incubate at room temperature for 30 minutes; add 2 ml hemolysin, react at 24°C for 10 minutes in the dark, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add 2 ml of pre-cooled PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add 0.5 μg of APC-labeled mouse secondary antibody, incubate at room temperature for 30 minutes; add 2 ml of pre-cooled PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add matching antibodies CD45-PerCP and CD3-FITC, react for 20 minutes, add 2 ml of pre-cooled PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add 500 μL of PBS buffer, and detect by flow cytometry.

[0030] Flow cytometry detection, the results are shown in Figure 2 The average fluorescence intensity of the self-screening CD226 antibody prepared by the application is 1675 when the amount of antibody added is 0.125 μg / T, at which time the average fluorescence intensity reaches a maximum value, which is the optimal working concentration of the antibody. When the amount of antibody added is too high, the antibody exhibits a significant HOOK effect, and the average fluorescence intensity shows a decreasing trend.

[0031] Example 7 Affinity determination of anti-human CD226 antibody Normal human peripheral blood detection: add 100 μL of normal human anticoagulant peripheral blood to the flow tube, add different amounts of CD226 antibody, incubate at room temperature for 30 minutes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add PBS buffer and resuspend, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add 0.5 μg of APC-labeled secondary antibody, incubate at room temperature for 30 minutes; add 2 mL of hemolysin, react at room temperature for 10 minutes in the dark, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add matching CD45-PerCP and CD3-FITC, incubate at room temperature for 30 minutes; add 1 mL of cold PBS buffer, centrifuge at 1000 rpm for 5 minutes, discard the supernatant; add 500 μL of PBS buffer containing 1% paraformaldehyde, and detect by flow cytometry.

[0032] Flow cytometry detection, the results are shown in Figure 3The data were analyzed by GraphPad, and the affinity curve of the CD226 antibody prepared in the application was a standard inverted "S" curve. When the amount of the antibody was 1.25 μg / ml, the average fluorescence intensity reached the maximum, which was 4796. With the decrease of the amount of the antibody, the average fluorescence intensity decreased, and when the amount of the antibody was 0.039063 μg / ml, the average fluorescence intensity tended to be stable. According to the curve fitting, the affinity of the self-screened CD226 antibody was 0.108 μg / ml.

[0033] Example 8: Activity determination of the APC-labeled anti-human CD226 antibody The anti-human CD226 antibody was reduced using a reducing agent, and was mixed with activated APC uniformly, and was stirred at 25°C for 1 hour. Then, the APC-labeled CD226 antibody was purified by using an S200 increase purification column.

[0034] Example 8: Activity determination of the APC-labeled anti-human CD226 antibody Normal human peripheral blood detection: 100 μL of normal human anticoagulant peripheral blood was added to each tube, different amounts of the antibody CD226-APC (1.0 μg, 0.5 μg, 0.25 μg, 0.13 μg, 0.06 μg, 0.03 μg) were added, and then the antibody CD45-PerCP was added, and the mixture was incubated at room temperature in the dark for 30 minutes. After 2 ml of hemolysin was added and reacted at room temperature in the dark for 10 minutes, the mixture was centrifuged at 1000 rpm / min for 5 minutes, and the supernatant was discarded. Then, 1 ml of cold PBS buffer was added, and the mixture was centrifuged at 1000 rpm / min for 5 minutes, and the supernatant was discarded. Then, 500 μL of PBS buffer was added, and the mixture was detected by flow cytometry.

[0035] The results of flow cytometry detection are shown in Figure 4 The self-screened CD226 antibody prepared in the application and the commercially available antibody were compared on the broken line graph, and it was found that the average fluorescence intensity of the self-screened CD226 antibody was more than twice that of the commercially available antibody. The average fluorescence intensity of the self-screened CD226 antibody on lymphocytes was 6985, and the average fluorescence intensity of the commercially available antibody on lymphocytes was 3318. Therefore, the average fluorescence intensity of the self-screened antibody on lymphocytes was much higher than that of the commercially available antibody, and the self-screened antibody could be used to replace the commercially available antibody.

[0036] The above only describes the preferred embodiments of the application, and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. An anti-human CD226 antibody, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; The CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively the amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; The CDR1, CDR2 and CDR3 of the light chain variable region are respectively the amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO.

6.

2. The anti-human CD226 antibody of claim 1, characterized in that: The FR1, FR2, FR3 and FR4 of the heavy chain variable region are respectively the amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO.

10. The FR1, FR2, FR3 and FR4 of the light chain variable region are respectively the amino acid sequences shown as SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO.

14.

3. The anti-human CD226 antibody of claim 1, wherein: The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

16.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule comprises a nucleotide sequence encoding the anti-human CD226 antibody of any one of claims 1-3.

5. An expression vector, characterized by: The expression vector comprises the nucleic acid molecule of claim 4.

6. A cell, characterized by: The cell comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. Use of the anti-human CD226 antibody of any one of claims 1-3 in the preparation of a detection reagent for CD226 molecules.

8. Use of the anti-human CD226 antibody of any one of claims 1-3 in the preparation of a detection reagent for chronic glomerulonephritis.

9. An immunoassay kit characterized by: The kit comprises the anti-human CD226 antibody of any one of claims 1-3.

Images