Anti-human cd95 antibody and use thereof

Abstract

This invention provides an anti-human CD95 antibody and its applications. The antibody includes 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. The anti-human CD95 antibody of this invention is detected using flow cytometry. The expression status of CD95 in cells is determined based on changes in the intensity of the CD95 fluorescence signal. This antibody has significant application value and can serve as a prognostic marker for certain cancers, used to detect the occurrence and related progression of cancer, and can also be used to detect apoptosis.

Description

Technical Field

[0001] This invention relates to an antibody for diagnostic testing, and more particularly to an anti-human CD95 antibody and its application. Background Technology

[0002] CD95, also known as Fas or APO-1, is an important cell surface receptor belonging to the tumor necrosis factor superfamily. The structure of CD95 consists of three parts: an extracellular portion, a transmembrane domain, and an intracellular portion. The extracellular portion contains 2-6 cysteine-rich repeats, which bind to the ligand FasL (Fas ligand). The transmembrane domain is a single α-helix that anchors the receptor to the cell membrane. The intracellular portion contains a death domain, a key region for inducing apoptosis. CD95 binds to its ligand FasL to form the death-inducing signaling complex (DISC), which contains FADD (Fas-associated protein with death domain) and caspase-8. The death domain binds to the adaptor protein FADD, leading to the activation of caspase-8, which in turn activates the caspase cascade, ultimately resulting in apoptosis. This process is crucial for maintaining tissue homeostasis, regulating the immune system, and clearing abnormal or damaged cells.

[0003] Abnormal expression or dysfunction of CD95 is associated with a variety of diseases, including autoimmune diseases, cancer, and certain viral infections. For example, some tumor cells may evade the immune system's surveillance and clearance by downregulating CD95 expression or function. In systemic lupus erythematosus and rheumatoid arthritis, CD95 may be overexpressed, leading to the erroneous induction of apoptosis in normal cells and triggering immune system dysfunction. Therefore, CD95 and its signaling pathways are important targets in cancer treatment and immunotherapy research. Summary of the Invention

[0004] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes an anti-human CD95 antibody and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0006] The present invention also provides an anti-human CD95 antibody, which includes a heavy chain variable region and a light chain variable region;

[0007] 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.

[0008] 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.

[0009] Furthermore, 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;

[0010] 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.

[0011] 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.

[0012] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned anti-human CD95 antibody.

[0013] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0014] The present invention also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned expression vector.

[0015] The present invention also provides the application of the aforementioned anti-human CD95 antibody in the preparation of a detection reagent for CD95 molecules.

[0016] This invention also provides the application of anti-human CD95 antibody in the preparation of detection reagents for the auxiliary diagnosis of diseases related to abnormal CD95 molecule expression.

[0017] The present invention also provides the application of anti-human CD95 antibody in the preparation of a detection reagent for systemic lupus erythematosus or rheumatoid arthritis.

[0018] The present invention also provides an immunoassay kit comprising the aforementioned anti-human CD95 antibody.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The anti-human CD95 antibody described in this invention is detected using flow cytometry. The expression of CD95 in cells is determined based on changes in the intensity of the CD95 fluorescence signal. This antibody has important application value and can be used as a prognostic marker for certain cancers to detect the occurrence and related progression of cancer. It can also be used to detect apoptosis. Attached Figure Description

[0021] Figure 1 This is an electrophoresis image of the purified anti-human CD95 antibody described in an embodiment of the present invention;

[0022] Figure 2 The following is an activity detection graph of the commercially available CD95 antibody (2 μg) described in this embodiment of the invention: Graph A shows CD45 distinguishing granulocytes, monocytes and lymphocytes, Graph B shows the percentage of CD95-positive cells, and Graph C shows the percentage of CD4-positive cells.

[0023] Figure 3 The following is an activity detection graph of the commercially available CD95 antibody (1 μg) described in the embodiments of the present invention: In the graph A, CD45 distinguishes granulocytes, monocytes and lymphocytes; in the graph B, CD95 positive cells account for the percentage of cells positive; and in the graph C, CD4 positive cells account for the percentage of cells positive.

[0024] Figure 4 The following is an activity detection diagram of the CD95 self-screening antibody (2μg) described in the embodiments of the present invention: Figure A shows the differentiation of granulocytes, monocytes and lymphocytes by CD45, Figure B shows the percentage of CD95 positive cells, and Figure C shows the percentage of CD4 positive cells.

[0025] Figure 5 The following is an activity detection diagram of the CD95 self-screening antibody (1 μg) described in the embodiments of the present invention: Figure A shows the differentiation of granulocytes, monocytes and lymphocytes by CD45, Figure B shows the percentage of CD95 positive cells, and Figure C shows the percentage of CD4 positive cells.

[0026] Figure 6 This is an affinity detection diagram of the CD95 self-screening antibody described in an embodiment of the present invention;

[0027] Figure 7 The following is an activity detection graph of the commercially available CD95-APC antibody (2μg) described in the embodiments of the present invention: In the graph A, CD45 distinguishes granulocytes, monocytes and lymphocytes; in the graph B, CD95 positive cells account for the percentage of cells positive; and in the graph C, the positive rate of lymphocytes.

[0028] Figure 8 The following is an activity detection graph of the commercially available CD95-APC antibody (1 μg) described in the embodiments of the present invention: In the graph A, CD45 distinguishes granulocytes, monocytes and lymphocytes; in the graph B, CD95 positive cells account for the percentage of cells positive; and in the graph C, the positive rate of lymphocytes.

[0029] Figure 9 The following is an activity detection diagram of the CD95-APC self-screening antibody (2μg) described in the embodiments of the present invention: Figure A shows the differentiation of granulocytes, monocytes and lymphocytes by CD45, Figure B shows the percentage of CD95 positive cells, and Figure C shows the positive rate of lymphocytes.

[0030] Figure 10 The following is an activity detection diagram of the CD95-APC self-screening antibody (1 μg) described in the embodiments of the present invention: Figure A shows the differentiation of granulocytes, monocytes and lymphocytes by CD45, Figure B shows the percentage of CD95 positive cells, and Figure C shows the positive rate of lymphocytes. Detailed Implementation

[0031] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0032] The present invention will be described in detail below with reference to the embodiments.

[0033] Example 1: Cloning of the human CD95 antigen sequence

[0034] The lentiviral expression vector used in this invention was constructed by inserting the CD95 gene (encoding the human CD95 protein) into the multiple cloning site of pCDH-CMV-MCS-EF1-copGFP as the empty vector (GENE ID: NM_000043.6, https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000043.6). Packaging vectors pMD2.G and psPAX2 were also prepared. The plasmids were extracted using a plasmid mini-extraction kit, and the obtained target plasmids were stored frozen at -20°C to avoid repeated freeze-thaw cycles.

[0035] Example 2: Preparation of Lentiviral Cells Carrying Human CD95 Antigen Sequence

[0036] (1) Lentiviral coating: Collect 293T cells with normal cell morphology in the proliferation phase and spread them evenly in a cell culture dish; the next day, use three pre-extracted plasmids and transfection reagents to mix in a certain ratio; gently and evenly add the plasmid-transfection reagent mixture after incubation to the 293T cells, avoiding blowing the cells up; after culturing for 48-72 hours, collect the supernatant to obtain the virus; then use a high-speed centrifuge to concentrate the virus; use the concentrated virus directly for subsequent infection experiments, or aliquot it into small volumes and store it in a -80℃ freezer.

[0037] (2) Lentiviral infection of 3T3 cells: 3T3 cells in the logarithmic growth phase were seeded into 24-well culture plates and cultured overnight in an incubator; the next day, concentrated lentivirus was added to the well plates containing cells, and polybrene was added to promote infection. The cells were gently mixed and placed in an incubator for culture; a few days later, the GFP fluorescence intensity of 3T3 cells was observed under a filter. After obtaining the ideal brightness, the cells were cultured and expanded, and the cells were named CD95-3T3.

[0038] (3) Flow cytometry analysis of lentivirus infection efficiency: CD95-3T3 cells expanded after infection were collected, and the positive rate data of the cells were obtained by using antibody labeling and flow cytometry detection; when the cell positive rate reaches more than 95%, it can be used to immunize animals.

[0039] Example 3 Animal Immunization

[0040] Log-growth-phase CD95-3T3 cells were collected as immunogens and immunized 4-5 week old female BALB / c mice via intraperitoneal injection. Booster immunizations were administered at 3 and 5 weeks after the initial immunization. Serum titers of the mice were then measured.

[0041] Example 4: Cell Fusion and Screening

[0042] (1) Three days before fusion, mice were given a shock immunization via tail vein injection. On the day of fusion, mice with high immune titers were selected, and the spleens of the mice were carefully removed in a biosafety cabinet. The spleens were then ground and the spleen cells were collected. At the same time, SP2 / 0 cells in good growth condition during the logarithmic growth phase were collected. The SP2 / 0 cells and spleen cells were mixed in a certain ratio to prepare for fusion. The fusion process was carried out in a 37°C water bath environment. 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-bottomed 96-well plate and placed in a cell culture incubator for culture.

[0043] (2) Screening hybridoma cells and subclones that secrete antibodies specifically targeting CD95: When the cell clones are large enough under a microscope, the supernatant of the corresponding wells is taken for flow cytometry detection; when a positive well is screened, the well is enlarged and cultured and retested. If the test result is still positive, a first round of subcloning is performed using the limiting dilution method. If the test result is negative, it is discarded; clones that are still positive after the first round of subcloning are subjected to a second round of subcloning; clones that are positive after the second round of subcloning are subjected to a third round of subcloning. Generally, at least three rounds of subcloning are performed. All clones obtained from the three rounds of subcloning are positive clones, that is, a stable hybridoma cell line is obtained.

[0044] Example 5: Preparation and purification of anti-human CD95 antibody

[0045] (1) Production of monoclonal antibodies in mice: Collect hybridoma cells in good growth condition and inject them into mice via intraperitoneal injection; collect ascites fluid when the mouse abdomen is significantly distended; freeze the collected ascites fluid at -20℃.

[0046] (2) Purification of anti-human CD95 antibody: The ascites fluid collected in the previous step was purified using a Protein A affinity column. After equilibrating the column with PBS, the sample was loaded and eluted with glycine at pH 3.0. The purified antibody was then replaced with PBS using a G25 column. The solution was then aliquoted and stored at -20°C. The purification electrophoresis results are shown in the figure. Figure 1 As shown.

[0047] Example 6: Activity Assay of Anti-Human CD95 Antibody

[0048] Peripheral blood testing from healthy individuals: Add 100 μl of anticoagulated peripheral blood from healthy individuals to each tube, add different amounts of CD95 antibody, and incubate at room temperature in the dark for 30 minutes; add 2 ml of hemolysin and react at room temperature in the dark for 10 minutes, then centrifuge at 1000 rpm for 5 minutes and discard the supernatant; add 2 ml of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes and discard the supernatant; add 0.5 μg of APC-labeled mouse secondary antibody and incubate at room temperature in the dark for 30 minutes; add 2 ml of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes and discard the supernatant; add the matching antibodies CD45-PerCP and CD4-PE-Cy7, react for 20 minutes, add 2 ml of cold PBS buffer, resuspend, centrifuge at 1000 rpm for 5 minutes and discard the supernatant; add 250 μl of PBS buffer and perform flow cytometry analysis.

[0049] Flow cytometry analysis, results as follows Figures 2-5 As shown, when the antibody addition amount is 2 μg / T, the percentage of CD4 and CD95 positive cells in the self-screened CD95 antibody prepared in this invention is 35.3%, while that in the commercially available CD95 antibody is 38.4%. When the antibody addition amount is 1 μg / T, the percentage of CD4 and CD95 positive cells in the self-screened CD95 antibody is 35.4%, while that in the commercially available CD95 antibody is 38.8%. This indicates that the self-screened antibody and the commercially available antibody show very similar behavior on the scatter plot, although the percentage of positive cells in the self-screened antibody is slightly lower than that in the commercially available antibody. Therefore, the self-screened antibody can be used as a substitute for the commercially available antibody.

[0050] Example 7 Affinity Detection of Anti-Human CD95 Antibody

[0051] Peripheral blood testing from healthy individuals: 100 μL of anticoagulated peripheral blood from healthy individuals was added to each tube, along with varying amounts of CD95 antibody. After incubation at room temperature for 30 minutes, the tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The tube was resuspended in PBS buffer and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 2 mL of hemolysin was added, and the tube was incubated at room temperature in the dark for 10 minutes, followed by centrifugation at 1000 rpm for 5 minutes, and the supernatant was discarded. The tube was then incubated with the corresponding CD4-FITC and CD45-PerCP buffers at room temperature in the dark for 30 minutes. 2 mL of cold PBS buffer was added, and the tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 300 μL of PBS buffer containing 1% paraformaldehyde was added, and the results were analyzed by flow cytometry. The results are shown below. Figure 6 As shown.

[0052] Antibody affinity data were obtained through GraphPad analysis. The principle of GraphPad analysis is to determine the antibody concentration corresponding to half the sum of the highest and lowest fluorescence intensities when using different concentrations of antibody for flow cytometry detection. At half the sum of the highest fluorescence intensity (3080) and the lowest fluorescence intensity (435), the antibody dosage corresponding to the CD95 antibody prepared in this invention is 0.19 μg / mL, while the antibody dosage corresponding to the commercially available CD95 antibody is 0.12 μg / mL. Figure 6 It can be seen that when the antibody concentration is too high, a significant hook effect is observed, and the average fluorescence intensity shows a decreasing trend. When the antibody concentration is reduced to 10 μg / mL, the curve tends to stabilize. The line graph shows that the average fluorescence intensity of the self-screened antibody is significantly higher than that of commercially available antibodies. Therefore, a CD95 self-screening antibody concentration of 0.19 μg / mL (obtained from affinity data analysis) is suitable for detection.

[0053] Example 8: APC labeling of anti-human CD95 antibody

[0054] The anti-human CD95 antibody was reduced using a reducing agent, mixed thoroughly with activated APC, stirred at 25°C, and reacted for 1 hour; then purified using an S200 increase purification column to obtain APC-labeled CD95 antibody.

[0055] Example 9: Activity assay against human CD95-APC

[0056] Peripheral blood testing from healthy individuals: 100 μl of anticoagulated peripheral blood from healthy individuals was added to each tube, along with different amounts of antibody CD95-APC (1.0 μg, 0.5 μg, 0.25 μg, 0.13 μg, 0.06 μg, 0.03 μg), and then antibody CD45-QBV500 and CD4-PerCP-Cy5.5 were added. The mixture was incubated at room temperature in the dark for 30 minutes. 2 ml of hemolysin was added, and the mixture was reacted at room temperature in the dark for 10 minutes. The mixture was then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 2 ml of cold PBS buffer was added, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 300 μl of PBS buffer was added, and the mixture was analyzed by flow cytometry.

[0057] Flow cytometry analysis, results as follows Figures 7-10 As shown in the scatter plot, the self-screened CD95 antibody prepared in this invention performs better than commercially available antibodies. When the antibody dosage is 1.0 μg, the positive rate of the self-screened CD95 antibody on lymphocytes is 37.4%, while that of the commercially available antibody is 32.9%. When the antibody dosage is 0.5 μg, the positive rate of the self-screened CD95 antibody on lymphocytes is 35.1%, while that of the commercially available antibody is 32.0%. Therefore, the self-screened antibody exhibits higher activity on lymphocytes than the commercially available antibody and can be used as a replacement for commercially available antibodies.

[0058] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-human CD95 antibody, characterized in that: The antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

2. The anti-human CD95 antibody according to claim 1, characterized in that The amino acid sequences of FR1, FR2, FR3, and FR4 in the heavy chain variable region are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The amino acid sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, respectively.

3. The anti-human CD95 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 encodes the anti-human CD95 antibody as described in any one of claims 1-3.

5. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule as described in claim 4.

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

7. The use of the anti-human CD95 antibody according to any one of claims 1-3 in the preparation of a detection reagent for CD95 molecules.

8. An immunoassay kit characterized by: The kit contains the anti-human CD95 antibody as described in any one of claims 1-3.

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