Anti-human CD275 antibody and application thereof

Abstract

The present invention provides an anti-human CD275 antibody and application, 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 amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the CDR1, CDR2, and CDR3 of the light chain variable region are amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The anti-human CD275 antibody of the present invention can be detected by flow cytometry, and its expression level on cells can be accurately determined according to the intensity change of the fluorescent signal of CD275.

Description

Technical Field

[0001] The present invention relates to an antibody for diagnosis and detection, in particular to an anti-human CD275 antibody and application thereof. Background Art

[0002] CD275, also known as ICOSL, B7-rp1, B7-h2, is a member of the B7 family with a molecular weight of 40KDa. CD275 can be expressed on a variety of cells, including dendritic cells, macrophages, B cells, T cell subsets, etc. CD275 can also be induced to express in epithelial cells, vascular endothelial cells, fibroblasts and many tumor cells. CD275 plays a key role in many immune processes by binding to its receptor ICOS, such as T cell activation, effector function, regulation of inflammatory factors, autoimmune thyroid disease, etc. Inflammatory response refers to the activation of neutrophils to migrate to the inflammatory focus and release active substances for the purpose of defense when the body is infected or damaged, causing a series of local pathological changes such as increased vascular permeability, plasma infiltration into tissues, and fibrin deposition and thrombin generation. In the process of T cell activation, CD275 and its receptor can induce the binding of the co-stimulatory factor ICOS, promoting the synthesis and secretion of cytokines such as IL-1, IL-5, IL-10, and IL-21 by activated T cells. At the same time, ICOS / CD275 promotes thymus-dependent antigen response by promoting the induction, maintenance and follicular homing of T follicular helper cells (Tfh), and drives antibody affinity maturation in the germinal center (GC), and can also enhance or inhibit Th1 and Th2 inflammatory responses. Therefore, we can determine whether an inflammatory response occurs in the human body by detecting the expression level of CD275.

[0003] ICOS / CD275 interaction promotes the production of type 2 innate lymphoid cell (ILC2s) factors through STAT5 signaling. The expression of CD275 is increased in thyroid follicular cells (TFCs) of patients with autoimmune thyroid disease (GD), proving that ICOS / CD275 signals play a direct role in the proliferation and differentiation of TFCs, and may play an important role in the initiation, maintenance and amplification of local tissue autoimmune reactions. ICOS binds to CD275 to promote the secretion of Th2 cytokines, regulate Th1 / Th2 cell polarization, enhance T cell-dependent B cell function, and promote the formation of germinal centers. Blocking ICOS co-stimulatory signals reduces the secretion of Th2 cytokines such as IL-10, impairs immunoglobulin type switching, and increases apoptosis of memory T cells. If CD275 antibodies are used to test blood in the early stages of autoimmune thyroid disease, further deterioration of the disease can be prevented.

[0004] Although the importance of CD275 in immune regulation has been widely recognized, existing technologies have limitations in the detection and application of CD275. For example, the lack of highly sensitive and specific detection methods limits the early diagnosis and treatment of inflammatory and autoimmune diseases. Summary of the invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes an anti-human CD275 antibody and its application.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] The present invention provides an anti-human CD275 antibody, which comprises a heavy chain variable region and a light chain variable region;

[0008] The CDR1, CDR2 and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0009] The CDR1, CDR2 and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0010] Furthermore, FR1, FR2, FR3, and FR4 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively;

[0011] The FR1, FR2, FR3 and FR4 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0012] Furthermore, 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.

[0013] The present invention also provides a nucleic acid molecule, which comprises a nucleotide sequence encoding the anti-human CD275 antibody.

[0014] The present invention also provides an expression vector, which contains the nucleic acid molecule.

[0015] The present invention also provides a cell, which contains the nucleic acid molecule or the expression vector.

[0016] The present invention also provides an application of the anti-human CD275 antibody in preparing a detection reagent for CD275 molecules.

[0017] The present invention also provides an application of the anti-human CD275 antibody in preparing a detection reagent for auxiliary diagnosis of diseases related to abnormal expression of CD275 molecules.

[0018] The present invention also provides an application of the anti-human CD275 antibody in preparing a detection reagent for inflammatory factor expression or autoimmune thyroid disease.

[0019] The present invention also provides an immunoassay kit, which comprises the anti-human CD275 antibody.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The anti-human CD275 antibody of the present invention can be detected by flow cytometry, and the expression level of CD275 on cells can be accurately determined according to the intensity change of the fluorescent signal of CD275.

[0022] The anti-human CD275 antibody of the present invention has high sensitivity and specificity, can be used to detect the expression level of inflammatory factors in the human body, and as an auxiliary diagnostic tool for autoimmune thyroid disease. In addition, the development of this antibody provides a new way to study the mechanism of action of CD275 in immune regulation, and is expected to bring breakthroughs in the diagnosis and treatment of inflammatory and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the electrophoresis diagram of the purification of the anti-human CD275 antibody described in Example 5 of the present invention;

[0024] Figure 2 This is a graph showing the activity detection of the anti-human CD275 antibody described in Example 6 of the present invention;

[0025] Figure 3 This is a graph showing the affinity detection of the anti-human self-screened CD275 antibody described in Example 7 of the present invention;

[0026] Figure 4 Activity detection diagram of the commercially available antibody described in Example 9 of the present invention: wherein, Figure A shows the commercially available antibody distinguishing granulocytes, monocytes and lymphocytes, Figure B shows CD3, and Figure C shows CD19;

[0027] Figure 5 The activity detection diagram of the self-screening anti-human CD275-PE antibody described in Example 9 of the present invention: wherein, Figure A is a commercially available antibody for distinguishing granulocytes, monocytes and lymphocytes, Figure B is CD3, and Figure C is CD19. DETAILED DESCRIPTION

[0028] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0029] The present invention will be described in detail below with reference to the embodiments.

[0030] Example 1 Cloning of human CD275 antigen sequence

[0031] The lentiviral expression vector used in this project was constructed by inserting the CD275 gene (encoding human CD275 protein) into the multiple cloning site of pCDH-CMV-MCS-EF1-copGFP as an empty vector (GENE ID: NM_015259, https: / / www.ncbi.nlm.nih.gov / nuccore / NM_015259), and preparing packaging vectors pMD2.G and psPAX2 at the same time. The plasmids were extracted using a plasmid mini-extraction kit, and the obtained target plasmids were stored in a -20℃ refrigerator to avoid repeated freezing and thawing.

[0032] Example 2 Lentivirus Coating and Immune Cell Preparation Carrying Human CD275 Antigen Sequence

[0033] 1. Lentivirus coating: Collect 293T cells in the proliferation stage with normal cell morphology and evenly spread them in a cell culture dish; the next day, use the three previously extracted plasmids and transfection reagents to mix in a certain proportion; gently and evenly add the plasmid-transfection reagent mixture after incubation to the 293T cells to avoid blowing the cells; after 48-72 hours of culture, collect the supernatant to obtain the virus; then use a high-speed centrifuge to concentrate the virus; the concentrated virus is directly used for subsequent infection experiments, or packaged in small volumes and stored in a -80℃ refrigerator.

[0034] 2. Infect 3T3 cells with lentivirus: 3T3 cells in the logarithmic growth phase were inoculated into 24-well culture plates and cultured in an incubator overnight. The next day, concentrated lentivirus was added to the well plate containing cells, and polybrene was added to promote infection. The cells were gently mixed and cultured in an incubator. After a few days, the GFP fluorescence intensity of 3T3 cells was observed under a filter. After the ideal brightness was obtained, the cells were cultured and expanded. The cells were named CD275-3T3.

[0035] 3. Flow cytometry analysis of lentiviral infection efficiency: Collect CD275-3T3 cells that have been amplified after infection, and obtain the positive rate data of the cells by using antibody labeling and flow cytometry detection; when the cell positive rate reaches more than 95%, it can be used to immunize animals.

[0036] Example 3 Animal Immunization

[0037] CD275-3T3 cells in the logarithmic growth phase were collected as immunogens and immunized 4-5 week old female BALB / c mice by intraperitoneal injection; booster immunizations were performed at the 3rd and 5th weeks after the initial immunization; and the serum of the mice was then tested for titer.

[0038] Example 4 Cell fusion and screening

[0039] 1. Three days before fusion, mice were immunized by tail vein injection; on the day of fusion, mice with high immune titers were selected, the spleens of mice were carefully removed in a biosafety cabinet, and the spleens were ground to collect spleen cells; SP2 / 0 cells in good growth state in the logarithmic growth phase were collected at the same time, and SP2 / 0 cells were mixed with spleen cells in a certain proportion to prepare for fusion; the fusion process should be carried out in a 37°C water bath environment, and PEG was used to fuse the cells; after fusion, the cells were resuspended in a special culture medium, and the cells were plated in a flat-bottom 96-well plate and cultured in a cell culture incubator.

[0040] 2. Screening of hybridoma cells and subclones that secrete antibodies specifically targeting CD275: When the cell clones are large enough under a microscope, take the supernatant of the corresponding wells for flow cytometry detection; when a positive well is screened, expand the culture of the well and retest. If the test result is still positive, perform a round of subcloning using the limiting dilution method. If the test result is negative, discard it; clones that are still positive in the first round of subcloning will continue to undergo a second round of subcloning; clones that are positive in the second round of subcloning will undergo a third round of subcloning. Generally, at least three rounds of subcloning are performed, and the clones obtained from the three rounds of subcloning are all positive clones, that is, a stable hybridoma cell line is obtained.

[0041] Example 5 Preparation and purification of anti-human CD275 antibodies

[0042] 1. Production of monoclonal antibodies in mice: Collect hybridoma cells in good growth state and inject them into the mouse peritoneum; when the abdomen of the mouse is obviously swollen, start collecting ascites; freeze the collected ascites at -20℃.

[0043] 2. Purification of anti-human CD275 antibody: Purify the ascites collected in the previous step using a Protein A affinity column. After equilibration of the column with PBS, load the sample and elute with glycine at pH 3.0. Use a G25 column to replace the solution with PBS, then aliquot and freeze at -20°C. The purified electrophoresis is shown in the following figure: Figure 1 shown.

[0044] Example 6 Activity determination of anti-human CD275 antibody

[0045] Normal human peripheral blood test: add 100μl normal human anticoagulated peripheral blood to each tube, add different amounts of CD275 antibody, incubate at room temperature in the dark for 30 minutes; add 2ml hemolysin, react at room temperature in the dark for 10 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 2ml cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; then add 0.5μg APC-labeled mouse secondary antibody, incubate at room temperature in the dark for 30 minutes; add 2ml cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add matching antibodies CD3-QB450, CD45-PerCP and CD19-FITC, react for 20 minutes, add 2ml cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 250μl PBS buffer, and detect by flow cytometry. The results are as follows Figure 2 shown.

[0046] The self-screening CD275 antibody prepared by the present invention has an average fluorescence intensity of 4389 when the antibody addition amount is 1 μg / T. As the antibody addition amount decreases, the average fluorescence intensity gradually decreases.

[0047] Example 7 Affinity determination of anti-human CD275 antibodies

[0048] Normal human peripheral blood test: add 100 μL normal human anticoagulated peripheral blood to each tube, add different amounts of CD275 antibody, incubate at room temperature for 30 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add PBS buffer to resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; then add 0.5 μg APC-labeled secondary antibody, incubate at room temperature in the dark for 30 min; add 2 mL hemolysin to react at room temperature in the dark for 10 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant; add matching CD19-FITC, CD45-PerCP and CD3-QB450, incubate at room temperature in the dark for 30 min; add 2 mL cold PBS buffer, centrifuge at 1000 rpm for 5 min, and discard the supernatant; add 300 μL PBS buffer containing 1% paraformaldehyde, and detect by flow cytometry. The results are as follows Figure 3 shown.

[0049] The antibody affinity data was obtained through GraphPad analysis. The principle of GraphPad analysis is: when using different concentrations of antibodies for flow cytometry detection, the antibody concentration corresponding to half of the sum of the highest fluorescence intensity and the lowest fluorescence intensity. At half of the sum of the highest fluorescence intensity (1639) and the lowest fluorescence intensity (1068), the antibody dosage corresponding to the CD275 antibody prepared by the present invention is 0.145 μg / mL. Figure 4-5 It can be seen that the positive rate of the self-screening CD275 antibody on lymphocytes after PE labeling is 3.57, and the positive rate of the commercially available antibody on lymphocytes is 3.77. There is no significant difference between the two results. It can be inferred that the self-screening CD275 and the commercially available CD275 have the same specificity. Therefore, the CD275 self-screening antibody dosage of 0.145μg / mL (obtained from affinity data analysis) can be used for detection.

[0050] Example 8 PE labeling of anti-human CD275 antibody

[0051] The anti-human CD275 antibody was reduced using a reducing agent, mixed evenly with the activated PE, stirred at 25° C., and reacted for 1 hour; then purified using an S200 increase purification column to obtain a PE-labeled CD275 antibody.

[0052] Example 9 Activity determination of anti-human CD275-PE

[0053] Normal human peripheral blood test: add 100μl normal human anticoagulated peripheral blood to each tube, add different amounts of antibody CD275-PE (1.0μg, 0.5μg, 0.25, 0.13μg, 0.06μg, 0.03μg), then add antibodies CD45-PerCP, CD3-QB450, CD19-FITC, incubate at room temperature in the dark for 30 minutes; add 2ml hemolysin, react at room temperature in the dark for 10 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 2ml cold PBS buffer, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 300μl PBS buffer, and detect by flow cytometry. The results are as follows: Figure 4-5 shown.

[0054] The self-screening CD275 antibody prepared by the present invention and the commercially available antibody (purchased from Biolegend) show very similar performances on the scatter plot. The positive rate of the self-screening CD275 antibody on lymphocytes is 3.57, and the positive rate of the commercially available antibody on lymphocytes is 3.77. It can be seen that there is little difference in the activity detection of the self-screening antibody and the commercially available antibody on lymphocytes, and the self-screening antibody can be used to replace the commercially available antibody.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-human CD275 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 the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; The CDR1, CDR2 and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. The anti-human CD275 antibody according to claim 1, characterized in that: The FR1, FR2, FR3, and FR4 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; The FR1, FR2, FR3 and FR4 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

3. The anti-human CD275 antibody according to claim 1, characterized in that: 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.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule comprises a nucleotide sequence encoding the anti-human CD275 antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 4.

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

7. Use of the anti-human CD275 antibody according to any one of claims 1 to 3 in preparing a detection reagent for CD275 molecules.

8. Use of the anti-human CD275 antibody according to any one of claims 1 to 3 in the preparation of a detection reagent for autoimmune thyroid disease.

9. An immunoassay kit, characterized in that: The kit comprises the anti-human CD275 antibody according to any one of claims 1 to 3.

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