Monoclonal antibody targeting all subtypes of CD45 and application thereof

Abstract

The invention discloses a monoclonal antibody targeting all CD45 subtypes and application of the monoclonal antibody, and belongs to the field of monoclonal antibody preparation. The hybridoma cell strain capable of stably secreting all anti-human CD45 subtype antibodies is successfully obtained by stably expressing short and long molecular subtypes of human CD45 extracellular regions in L929 mouse fibroblasts, immunizing BALB / c mice with L929 cells expressing the two molecular subtypes of human CD45, and fusing splenocytes with SP20 myeloma cells after the serum titer reaches the standard. And the monoclonal antibody targeting all subtypes of CD45 is obtained. The monoclonal antibody provided by the invention shows a high-affinity binding characteristic with all human CD45 molecular subtypes, has important application value in the aspect of human CD45 molecular detection, and can be used as a therapeutic antibody in the fields of tumor immunotherapy, autoimmune disease treatment, transplant rejection resistance and the like.

Description

Technical Field

[0001] The present invention relates to the field of monoclonal antibody preparation, and particularly to a monoclonal antibody targeting all subtypes of CD45 and its applications. Background Art

[0002] Human CD45 (also known as leukocyte common antigen, LCA) is a type I transmembrane protein tyrosine phosphatase (PTP) that is widely expressed on the surface of almost all hematopoietic cells. CD45 plays a key role in signal transduction of immune cells, regulates signal transduction of T cell receptor (TCR) and B cell receptor (BCR), and plays an important function in immune response, cell differentiation and activation processes. Therefore, CD45 has become one of the therapeutic targets in immunological research, detection of immune cell markers and specific diseases (such as leukemia and lymphoma).

[0003] CD45 has multiple splice variants, and its extracellular region consists of three variable exons (A, B, C). Different combinations of these exons form different subtypes of CD45, including CD45RA, CD45RB, CD45RC, CD45RO, etc. Among them, CD45RO is the shortest splice variant, mainly expressed in activated T cells, and CD45RABC is the longest splice variant, commonly found in immature hematopoietic cells and some B cells. Due to the different roles of different CD45 subtypes in immune cell differentiation, maturation and disease progression, monoclonal antibodies (mAbs) against CD45 are widely used in basic research, clinical detection and immunotherapy.

[0004] For clinical diagnosis and immunotherapy, monoclonal antibodies that can recognize all CD45 subtypes have important value in flow cytometry (FACS), immunohistochemistry (IHC) and development of therapeutic antibodies. However, most of the anti-CD45 monoclonal antibodies on the market at present are directed against specific subtypes. For example, the UCHL1 antibody against CD45RO is used to detect activated T cells, while the HI100 antibody against CD45RA is used to identify naive T cells. The application scope of these antibodies is limited and it is difficult to cover all CD45 subtypes simultaneously. At the same time, differences in the expression levels of different subtypes may cause signal deviation in detection, affecting the accuracy of quantitative analysis. In addition, some antibodies have problems of insufficient affinity or cross-reactivity, further limiting their reliability in detections such as flow cytometry and immunohistochemistry. Screening with the shortest splice variant CD45RO and the longest splice variant CD45RABC together is expected to obtain CD45 monoclonal antibodies against all subtypes. Summary of the Invention

[0005] The object of the present invention is to provide a monoclonal antibody targeting all subtypes of CD45 and its applications, so as to solve the problems existing in the above-mentioned prior art. The monoclonal antibody provided by the present invention shows high-affinity binding characteristics with all molecular subtypes of human CD45, has important application value in the detection of human CD45 molecules, and can be used as a therapeutic antibody in the fields of tumor immunotherapy, treatment of autoimmune diseases, and anti-transplant rejection, etc.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a monoclonal antibody targeting all subtypes of CD45, and the monoclonal antibody includes monoclonal antibody 1A9, monoclonal antibody 4A10, monoclonal antibody 5B10 or monoclonal antibody 7C7; the amino acid sequence of the monoclonal antibody includes a heavy-chain variable region and a light-chain variable region;

[0008] The heavy-chain variable region of the monoclonal antibody 1A9 includes: heavy-chain CDRH1 with the amino acid sequence shown in SEQ ID NO.10, heavy-chain CDRH2 with the amino acid sequence shown in SEQ ID NO.11, and heavy-chain CDRH3 with the amino acid sequence shown in SEQ ID NO.12; the light-chain variable region includes: light-chain CDRL1 with the amino acid sequence shown in SEQ ID NO.15, light-chain CDRL2 with the amino acid sequence shown in SEQ ID NO.16, and light-chain CDRL3 with the amino acid sequence shown in SEQ ID NO.17;

[0009] The heavy-chain variable region of the monoclonal antibody 4A10 includes: heavy-chain CDRH1 with the amino acid sequence shown in SEQ ID NO.20, heavy-chain CDRH2 with the amino acid sequence shown in SEQ ID NO.21, and heavy-chain CDRH3 with the amino acid sequence shown in SEQ ID NO.22; the light-chain variable region includes: light-chain CDRL1 with the amino acid sequence shown in SEQ ID NO.25, light-chain CDRL2 with the amino acid sequence shown in SEQ ID NO.26, and light-chain CDRL3 with the amino acid sequence shown in SEQ ID NO.27;

[0010] The heavy chain variable region of the monoclonal antibody 5B10 includes: heavy chain CDRH1 with the amino acid sequence shown in SEQ ID NO.30, heavy chain CDRH2 with the amino acid sequence shown in SEQ ID NO.31, and heavy chain CDRH3 with the amino acid sequence shown in SEQ ID NO.32; the light chain variable region includes: light chain CDRL1 with the amino acid sequence shown in SEQ ID NO.15, light chain CDRL2 with the amino acid sequence shown in SEQ ID NO.16, and light chain CDRL3 with the amino acid sequence shown in SEQ ID NO.17;

[0011] The heavy chain variable region of the monoclonal antibody 7C7 includes: heavy chain CDRH1 with the amino acid sequence shown in SEQ ID NO.35, heavy chain CDRH2 with the amino acid sequence shown in SEQ ID NO.36, and heavy chain CDRH3 with the amino acid sequence shown in SEQ ID NO.37; the light chain variable region includes: light chain CDRL1 with the amino acid sequence shown in SEQ ID NO.40, light chain CDRL2 with the amino acid sequence shown in SEQ ID NO.41, and light chain CDRL3 with the amino acid sequence shown in SEQ ID NO.42.

[0012] Optionally, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A9 is as shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14;

[0013] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4A10 is as shown in SEQ ID NO.19, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.24;

[0014] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5B10 is as shown in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14;

[0015] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 7C7 is as shown in SEQ ID NO.34, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.39.

[0016] The present invention also provides a nucleic acid molecule comprising a nucleic acid molecule encoding the monoclonal antibody described above.

[0017] Optionally, the nucleic acid molecule encoding the monoclonal antibody 1A9 includes the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.13 and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO.18;

[0018] The nucleic acid molecule encoding monoclonal antibody 4A10 comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO.23 and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO.28;

[0019] The nucleic acid molecule encoding monoclonal antibody 5B10 comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO.33 and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO.18;

[0020] The nucleic acid molecule encoding monoclonal antibody 7C7 comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO.38 and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO.43.

[0021] The present invention also provides the use of the monoclonal antibody or the nucleic acid molecule in the preparation of a product for in vitro detection of human CD45 molecule, and the product recognizes all CD45 molecular subtypes including CD45RA, CD45RB, CD45RC and CD45RO by binding to the conserved epitope of the human CD45 molecule.

[0022] The present invention also provides the use of the monoclonal antibody or the nucleic acid molecule in the preparation of a drug for tumor immunotherapy.

[0023] The present invention also provides the use of the monoclonal antibody or the nucleic acid molecule in the preparation of a diagnostic reagent for autoimmune diseases.

[0024] Optionally, using the monoclonal antibody as a label, the immune cells related to the autoimmune disease are analyzed and detected.

[0025] The present invention also provides the use of the monoclonal antibody or the nucleic acid molecule in the preparation of a drug for treating autoimmune diseases or anti-organ transplant rejection related to CD45.

[0026] Optionally, the drug comprises the monoclonal antibody or the nucleic acid molecule, an immunosuppressant and a pharmaceutically acceptable carrier.

[0027] The present invention discloses the following technical effects:

[0028] In the present invention, the shortest splice variant CD45RO and the longest splice variant CD45RABC subtype of human are stably expressed in L929 mouse fibroblasts. The above two types of L929 cells overexpressing CD45 are used to immunize BALB / c mice. After the serum titer reaches the standard, spleen cells are taken and fused with SP20 myeloma cells, and a hybridoma cell line that stably secretes antibodies against all subtypes of human CD45 is successfully obtained. The variable region gene sequence of the antibody is cloned by RT-PCR technology, and its binding specificity is confirmed by methods such as flow cytometry and Western Blot.

[0029] The monoclonal antibody provided by the present invention shows high-affinity binding characteristics with all molecular subtypes of human CD45, with excellent Kd values; it has strong specificity and only recognizes human CD45-positive cells; it can effectively recognize linearized human CD45 protein. Based on these characteristics, the monoclonal antibody provided by the present invention has important application value in the detection of human CD45 molecules, and can also be used as a therapeutic antibody in the fields of tumor immunotherapy, treatment of autoimmune diseases, and anti-transplant rejection.

[0030] The monoclonal antibody provided by the present invention not only provides a high-quality tool antibody for CD45-related research, but also lays an important foundation for the development of new immunotherapy drugs. Its unique binding characteristics show good application prospects in both the development of diagnostic reagents and therapeutic applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a specific flow chart for preparing monoclonal antibodies;

[0033] Figure 2 It is the protein quantity, position and size of the 4 antibodies obtained detected by SDS-PAGE method, where "M" represents the molecular marker, and "reduction" means that the antibody is reduced into two double strands by SDS;

[0034] Figure 3 It is the affinity constant situation of the 4 antibodies obtained with two subtypes (RO and RABC) of overexpressed CD45;

[0035] Figure 4 It is the binding situation of the 4 antibodies obtained with hematopoietic cell lines Jurkat / kasumi (CD45+) and nalm6 (CD45-), further confirming the specificity of the antibody;

[0036] Figure 5 The binding of the four obtained antibodies 1A9 (A), 4A10 (B), 5B10 (C) and 7C7 (D) to human peripheral blood cells. Detailed implementation manners

[0037] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0041] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0042] The present invention provides a monoclonal antibody against human CD45, which can specifically bind to all subtypes of CD45, including the shortest CD45RO and the longest CD45RABC, overcoming the problems of subtype limitations and insufficient application scope of existing anti-CD45 antibodies, and having significant advantages in basic research, clinical detection and immunotherapy, etc.

[0043] In the present invention, when immunizing mice at an early stage, a multi-antigen combined immunization strategy is adopted. Mouse fibroblast L929 cells overexpressing the shortest splice variant CD45RO and the longest splice variant CD45RABC of human are used for multiple immunizations to induce the generation of B cells that can broadly recognize CD45. This strategy helps to obtain antibodies with strong specificity and less cross-binding, avoiding the antibody bias caused by the limitation of immunizing antigens in the traditional monoclonal antibody screening process.

[0044] Preferably, after obtaining hybridoma cells, in order to ensure that the screened antibodies can efficiently bind to all CD45 subtypes, the present invention sets a strict screening criterion: three different types of cells are mixed as target cells, namely CD45RO highly expressing cells (representing the shortest splice variant), CD45RABC highly expressing cells (representing the longest splice variant), and CD45 negative cells (used to remove non-specific binding antibodies), and the three cells are mixed at a ratio of 1:1:1. During the screening process, flow cytometry (FACS) or immunofluorescence is used to detect the hybridoma supernatant, and it is required that the antibodies in the supernatant can bind to both CD45RO and CD45RABC cells simultaneously, but have no binding to CD45 negative cells. This strategy ensures that the screened antibodies cover all CD45 subtypes and avoids the problem that existing antibodies only recognize specific subtypes due to screening bias.

[0045] Preferably, in the mixed cell system, the total binding positive rate of the hybridoma supernatant to CD45RO and CD45RABC cells should reach 65%-75% to ensure that it has a high affinity for both splice variants. Antibodies with too low a positive rate (<65%) may have insufficient affinity for a certain splice variant, while antibodies with too high a positive rate (>75%) may have non-specific binding. The screened antibodies are more specific and consistent in subsequent applications.

[0046] Preferably, it is found that when using the obtained monoclonal antibody to label mononuclear cells from healthy humans, the positive rate is consistent with that of commercial antibodies, demonstrating its practical application potential.

[0047] The specific experimental flow chart of the present invention is as Figure 1 shown.

[0048] Example

[0049] 1. Immunize animals

[0050] Using a lentiviral vector to construct an overexpressed human CD45 molecule (including all subtypes of CD45RO and CD45RABC, the plasmid contains a GFP marker gene, and the successfully overexpressed cells show FITC+ in flow cytometry) in the mouse fibroblast cell line L929 as an immunogen, immunize Balb / c mice by intraperitoneal injection, and perform booster immunizations at the 3rd week and 5th week after the primary immunization respectively. On the 8th day after the booster immunization, collect mouse tail blood, let it stand at room temperature for 1 hour, centrifuge at 4°C and 15,000 rpm for 10 minutes, collect the serum, and dilute it with PBS to different concentrations: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800. Collect L929 cells expressing human CD45 molecules, wash them once with PBS, count the cells, and use 5×10 5 cells for each sample. Add 100 μL of sera with different dilutions to the cells respectively. The negative control group is L929 cells that do not express CD45 molecules. Incubate at room temperature for 30 minutes and wash twice with PBS. Add 200 μL of APC-labeled anti-mouse IgG antibody and incubate at room temperature in the dark for 30 minutes; resuspend the cells in 200 μL of PBS buffer, and detect the binding percentage and fluorescence intensity of the antibody in the serum to the cells by flow cytometry. Select mice whose sera can bind CD45RO and CD45RABC but do not bind wild-type L929 for subsequent experiments.

[0051] 2. Cell fusion and hybridoma screening

[0052] Three days before fusion, use L929 cells expressing CD45 molecules as an immunogen and inject them into the selected immunized mice via the tail vein for boost immunization. Take the immunized mice, sacrifice them by cervical dislocation, and soak them in 75% ethanol for disinfection. Take out the spleen cells of the immunized mice, and fuse the mouse spleen cells and the hybridoma cell line SP20 at a ratio of 10:1 under the water bath condition of 37°C. Centrifuge at 800 rpm for 6 minutes to remove the supernatant, and then resuspend the cells with 1640 selection medium containing HAT. Transfer 200 μL of the cell suspension into a 96-well plate. Culture the cells at 37°C and 5% CO2. Continuously culture for 10 - 14 days. When observing that the hybridoma cells grow into round and plump clones (about 1 mm × 1 mm in size), use a stereomicroscope to pick the clones in a biosafety cabinet and transfer them into a 96-well plate for continued culture.

[0053] When the cloned cells grow to an appropriate density, take the cell supernatant as the primary antibody, and mix wild-type L929, L929 cells overexpressing CD45RO and CD45RABC at a ratio of 1:1:1 (2×10 5Using these as target cells, flow cytometry was used to detect their binding to screen for positive clones. The definition of a positive clone was that it only bound to CD45RO-L929 and CD45RABC-L929 and did not bind to wild-type L929, that is, the proportion of the FITC+APC+ cell population should reach 65%-75%.

[0054] The positive clones obtained by screening were transferred to a 24-well plate for continued culture to further screen for positive clones. The positive clone cells screened in the 24-well plate were subcloned and transferred to a 96-well plate for continued culture for 10-14 days, and the supernatant was taken for further flow cytometry detection to further screen for positive clones that could stably secrete the target antibody. After subcloning and screening, all detection wells were positive, and finally 4 different stable hybridoma cell lines were obtained.

[0055] 3. Antibody production and purification

[0056] Ascites preparation: Liquid paraffin was injected into mice intraperitoneally at 500 μL / mouse, and then different hybridoma cells obtained were injected into different mice intraperitoneally at 5×10 5 hybridoma cells per mouse, and mouse ascites was collected 7-10 days later.

[0057] Crude extraction of the antibody from the collected ascites: Take the collected ascites (taking 5 ml as an example), add an equal volume (5 ml) of PBS buffer, and mix well. Then slowly add an equal volume (5 ml) of saturated ammonium sulfate solution drop by drop while stirring to ensure uniform mixing. Place the mixture in a 4°C environment and let it stand overnight. Centrifuge the mixture at 10,000 rpm for 10 minutes and discard the supernatant. Dissolve the precipitate with a small amount of PBS buffer and mix gently. Dialyze with PBS buffer at 4°C for 24 hours, changing the dialysis solution 3 times during this period to remove ammonium sulfate.

[0058] Purification of the crudely extracted antibody: The crudely purified antibody was finely purified using an AKTA protein purification system through a 5 ml Protein G purification pre-packed column according to the purification operation guide provided by GE Company to obtain the purified antibody. The monoclonal antibodies secreted by 4 different hybridoma cell lines were monoclonal antibodies 1A9, 4A10, 5B10, and 7C7 respectively.

[0059] The purified antibody samples were used for subsequent antibody detection and functional experiments.

[0060] 4. Antibody affinity constant detection

[0061] First, the protein concentration of the antibody was quantified by the BCA colorimetric method, and the protein amounts of the purified samples were adjusted to be the same. Then, electrophoresis was performed in an SDS-PAGE electrophoresis system. The resulting gel was stained in Coomassie Brilliant Blue staining solution for 30 min and then decolorized overnight, during which the decolorizing solution was changed 3 - 5 times. Then, the gel was exposed to obtain the protein bands of the antibodies secreted by different hybridoma cells( Figure 2 ).

[0062] The purified CD45 antibody was incubated with 100 μL of 5×10 5 CD45RO and CD45RABC L929 cell lines at 800 nM, 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.57 nM, 0.78 nM, 0.39 nM, 0.19 nM, 0.095 nM, 0.048 nM, and 0.024 nM for 30 min at room temperature, washed three times with PBS, and then incubated with a mouse anti-human IgG secondary antibody at room temperature for 30 min and washed three times with PBS. Then, the fluorescence intensity was detected on a flow cytometry instrument, and the Kd value of the antibody with the CD45RO / CD45RABC L929 cell line was calculated using the data analysis software GraphPad Prism 9.5.0( Figure 3 ).

[0063] 5. Cloning and sequence identification of antibody variable region genes

[0064] First, the total RNA of the selected hybridoma cells was extracted using the SPAReasy cell RNA rapid extraction kit, and then the extracted cellular RNA was reverse transcribed into cDNA using the vazyme HiScript IV All-in-One Ultra RT SuperMix for qPCR kit.

[0065] The following primers were used for PCR amplification of the heavy and light chain variable region genes of the antibodies secreted by different hybridoma cells:[[]]

[0066] Heavy chain framework region upstream primer P1: SAGGTGMAGCTKCASSARTCWGG (SEQ ID NO.1);

[0067] Heavy chain variable region downstream primer P2: TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA (SEQ ID NO.2);

[0068] Light chain leader peptide upstream primer:

[0069] P3: ATGGAGACAGACACACTCCTGCTAT (SEQ ID NO.3);

[0070] P4: ATGGATTTTCAAGTGCAGATTTTCAG (SEQ ID NO.4);

[0071] P5: ATGGAGWCACAKWCTCAGGTCTTTRTA (SEQ ID NO.5);

[0072] P6: ATGKCCCCWRCTCAGYTYCTKGT (SEQ ID NO.6);

[0073] P7: ATGAAGTTGCCTGTTAGGCTGTTG (SEQ ID NO.7);

[0074] Downstream primer P8 for the light chain variable region: GGATACAGTTGGTGCAGCATCAGCCCGTTT (SEQ ID NO.8).

[0075] PCR reaction system:

[0076] First, prepare a 50 μL PCR reaction system, including 2 μL cDNA, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 25 μL mix, and make up to 50 μL with ddH2O. The reaction conditions are: pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of amplification (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute), and finally extension at 72°C for 10 minutes. Separate the PCR products by agarose gel electrophoresis and recover the target VL and VH fragments. Ligate the recovered VL and VH fragments with the pMD19-T (Simple) vector (Takara) respectively. The ligation system includes 70 ng VL or VH PCR product, 1 μL pMD19-T (Simple) vector, 5 μL Solution I ligation reaction solution, and make up to 10 μL with ddH2O, and ligate overnight at 4°C. Transform the ligation products into E. coli DH5α competent cells. After overnight culture at 37°C, send the strains to the company for sequencing until the sequencing results of at least three samples are consistent. Finally, successfully clone the heavy chain and light chain variable region sequences of different CD45 antibodies, and their sequences conform to the characteristics of typical antibody variable region sequences. Then use the RACE kit of vazyme to further obtain the terminal base sequences of the antibody heavy and light chains (VL and VH), and the obtained sequences are the true sequences of the monoclonal antibodies.

[0077] (1) Monoclonal antibody 1A9

[0078] The amino acid sequence of the heavy chain variable region is: QVQLQQSGAEVARPGASVKMSCKASGYTFTSYTVHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADRSSSTLYMQLNSLTSEDSVVYYCARAGDGFYMSWFAYWGQGTLVTVSA (SEQ ID NO.9);

[0079] The CDR sequences of this heavy chain variable region contain the following three segments:

[0080] CDRH1: GYTFTSYTVH (SEQ ID NO.10);

[0081] CDRH2: YINPSSGYTNYNQKFKD (SEQ ID NO.11);

[0082] CDRH3: AGDGFYMSWFAY (SEQ ID NO.12);

[0083] The nucleotide sequence encoding this heavy chain variable region is: CAGGTCCAGCTGCAGCAGTCTGGGGCTGAAGTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACACGGTGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGTAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAGATCCTCCAGCACACTCTACATGCAACTGAACAGCCTGACATCTGAGGACTCTGTAGTCTATTACTGTGCAAGAGCAGGAGATGGTTTCTACATGTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO.13);

[0084] The amino acid sequence of the light chain variable region is: SIVMTQTPKFLLVSAGDRVTMTCKASQSVNNDVAWYQQKPGQSPKLLIYHVSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGG TQLEIKR (SEQ IDNO.14);

[0085] The CDR sequences of the light chain variable region contain the following three segments:

[0086] CDRL1: KASQSVNNDVA (SEQ ID NO.15);

[0087] CDRL2: HVSNRYT (SEQ ID NO.16);

[0088] CDRL3: QQDYSSPWT (SEQ ID NO.17);

[0089] The nucleotide sequence encoding the light chain variable region is: AGTATTGTGATGACCCAGACTCCCAAATTCCTG CTTGTATCAGCAGGAGACAGGGTTACCATGACCTGCAAGGCCAGTCAGAGTGTGAATAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACCATGTATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAGGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCTTGGACGTTCGGTGGAGGCACCCAGCTGGAAATCAAACGG (SEQ ID NO.18).

[0090] (2) Monoclonal antibody 4A10

[0091] The amino acid sequence of the heavy chain variable region is: DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQ APEKGLEWVAYINGGSTTFYYADTVKGRFTISRDNPKNTLYLQMTSLRSEDTAMYYCARTT VPTAWSAYWGQGTLVTVSS (SEQ ID NO.19);

[0092] The CDR sequences of the heavy chain variable region contain the following three segments:

[0093] CDRH1: GFTFSSFGMH (SEQ ID NO.20);

[0094] CDRH2: YINGGSTTFYYADTVKG (SEQ ID NO.21);

[0095] CDRH3: TTVPTAWSAY (SEQ ID NO.22);

[0096] The nucleotide sequence encoding the heavy chain variable region is: GAGGTCCAGTTGCATGAAAGCGGGGGAGGGTT GGTCCAACCAGGCGGGTCCCGAAAGTTATCTTGCGCTGCTTCGGGATTCACTTTCTCCAGTTTTGGTATGCACTGGGTGCGTCAAGCACCAGAGAAGGGCTTAGAGTGGGTTGCGTACATTAACGGAGGCTCGACCACGTTTTATTACGCCGACACTGTGAAGGGGAGATTCACAATAAGTCGTGATAATCCGAAGAACACGCTGTACCTCCAGATGACATCGTTACGTAGTGAAGACACAGCAATGTACTACTGTGCCCGTACGACCGTGCCAACGGCATGGAGCGCGTACTGGGGACAGGGCACGCTTGTTACCGTATCTTCC (SEQ ID NO.23);

[0097] The amino acid sequence of the light chain variable region is: NIMMTQSPSSLAVSAGEKVTMSCKSSQSVFYSSNQKNYLA WYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISNVQTEDLAVYYCHQYLSSW TFGGGTKLEIKR (SEQ IDNO.24);

[0098] The CDR sequences of the light chain variable region include the following 3 segments:

[0099] CDRL1: KSSQSVFYSSNQKNYLA (SEQ ID NO.25);

[0100] CDRL2: WASTRES (SEQ ID NO.26);

[0101] CDRL3: HQYLSSWT (SEQ ID NO.27);

[0102] The nucleotide sequence encoding the light chain variable region is: AATATTATGATGACACAGTCACCTTCTTCGCTAG CAGTCTCGGCAGGGGAGAAAGTCACGATGAGTTGTAAATCGTCACAATCTGTGTTCTATTCGTCGAATCAGAAGAACTACCTAGCTTGGTACCAACAAAAACCAGGTCAATCTCCTAAACTACTCATTTATTGGGCCAGCACCAGGGAGAGTGGCGTGCCTGACAGATTCTCGGGCTCTGGTTCTGGTACCGATTTTACTTTGACAATTAGTAACGTGCAAACAGAGGACCTCGCCGTGTACTATTGTCATCAATATCTGTCTAGCTGGACTTTTGGCGGCGGTACTAAGTTGGAGATTAAGAGG(SEQ ID NO.28).

[0103] (3) Monoclonal antibody 5B10

[0104] The amino acid sequence of the heavy chain variable region is: DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHWVRQ TPEKGLEWVAYISSGSTTFFYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYFCATTTVI TGWFAYWGQGTLVTVSA(SEQ ID NO.29);

[0105] The CDR sequences of the heavy chain variable region contain the following 3 fragments:

[0106] CDRH1: GFTFSNFGMH(SEQ ID NO.30);

[0107] CDRH2: YISSGSTTFFYADTVKG(SEQ ID NO.31);

[0108] CDRH3: TTVITGWFAY(SEQ ID NO.32);

[0109] The nucleotide sequence encoding the heavy chain variable region is: GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTT AGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTTTGGAATGCACTGGGTTCGTCAGACTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTACTACCTTCTTCTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTTCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTTCTGTGCAACAACTACTGTGATTACGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.33);

[0110] The amino acid sequence of the light chain variable region is: SIVMTQTPKFLLVSAGDRVTMTCKASQSVNNDVAWYQQK PGQSPKLLIYHVSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGG TQLEIKR(SEQ IDNO.14);

[0111] The CDR sequences of the light chain variable region contain the following 3 segments:

[0112] CDRL1: KASQSVNNDVA(SEQ ID NO.15);

[0113] CDRL2: HVSNRYT(SEQ ID NO.16);

[0114] CDRL3: QQDYSSPWT(SEQ ID NO.17);

[0115] The nucleotide sequence encoding the light chain variable region is: AATATTATGATGACACAGTCACCTTCTTCGCTAG CAGTCTCGGCAGGGGAGAAAGTCACGATGAGTTGTAAATCGTCACAATCTGTGTTCTATTCGTCGAATCAGAAGAACTACCTAGCTTGGTACCAACAAAAACCAGGTCAATCTCCTAAACTACTCATTTATTGGGCCAGCACCAGGGAGAGTGGCGTGCCTGACAGATTCTCGGGCTCTGGTTCTGGTACCGATTTTACTTTGACAATTAGTAACGTGCAAACAGAGGACCTCGCCGTGTACTATTGTCATCAATATCTGTCTAGCTGGACTTTTGGCGGCGGTACTAAGTTGGAGATTAAGAGG(SEQ ID NO.18).

[0116] (4) Monoclonal antibody 7C7

[0117] The amino acid sequence of the heavy chain variable region is: DVQLVESGGGLVQPGGSRKLSCAASGFTFSDFGMHWVRQ TPEKGLEWVAYISSGSTTFYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYFCARTTV ITGWFAYWGQGTLVTVSA(SEQ ID NO.34);

[0118] The CDR sequences of the heavy chain variable region contain the following 3 fragments:

[0119] CDRH1: GFTFSDFGMH(SEQ ID NO.35);

[0120] CDRH2: YISSGSTTFYYADTVKG(SEQ ID NO.36);

[0121] CDRH3: TTVITGWFAY(SEQ ID NO.37);

[0122] The nucleotide sequence encoding the heavy chain variable region is: GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTT AGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTTTGGAATGCACTGGGTTCGTCAGACTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTACTACCTTCTACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTTCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTTCTGTGCAAGAACTACTGTGATTACGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.38);

[0123] The amino acid sequence of the light chain variable region is: NIMMTQSPSSLAVSAGEKVTLSCKSSQSVFVSSNQKNYLA WYQQKPGQSPRLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCHQYLSSWT FGGGTKLEIKR(SEQ IDNO.39);

[0124] The CDR sequences of the light chain variable region contain the following 3 fragments:

[0125] CDRL1: KSSQSVFVSSNQKNYLA(SEQ ID NO.40);

[0126] CDRL2: WASIRES(SEQ ID NO.41);

[0127] CDRL3: HQYLSSWT(SEQ ID NO.42);

[0128] The nucleotide sequence encoding the variable region of the light chain is: AACATTATGATGACACAGTCGCCATCATCTCTGG CTGTGTCTGCAGGAGAAAAGGTCACTTTGAGCTGTAAGTCCAGTCAAAGTGTTTTCGTCAGTTCAAATCAGAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAGACTGCTAATCTACTGGGCATCCATTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAATTTATTACTGTCATCAATACCTCTCCTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG (SEQ ID NO.43).

[0129] 6. Functional verification of monoclonal antibodies

[0130] To comprehensively evaluate the functional characteristics of the obtained anti-human CD45 monoclonal antibodies (1A9, 4A10, 5B10, and 7C7), the present invention uses a variety of human cells for systematic verification. The human cells used for verification include human acute T lymphocyte leukemia cells Jurkat (CD45+), human acute myeloid leukemia cells Kasumi-1 (CD45+), and human B lymphocytic leukemia cell line Nalm6 (CD45-).

[0131] First, the cell lines Jurkat (CD45+), Kasumi-1 (CD45+), and Nalm6 (CD45-) were selected for specificity detection, and the results are as Figure 4 , showing that all four monoclonal antibodies can specifically bind to CD45-positive cells and have no binding to CD45-negative cells, confirming the specific recognition ability of the antibodies to the human CD45 antigen.

[0132] Furthermore, healthy human peripheral blood mononuclear cells (PBMCs) were used to evaluate the recognition ability of the antibodies to each subtype of native CD45. After separating PBMCs by Ficoll density gradient centrifugation, flow cytometry detection found that all four monoclonal antibodies can effectively bind to the CD45+ cell population in PBMCs, and their binding efficiency is comparable to that of the commercial anti-CD45 antibody (BioLegend anti-human CD45-APC, clone number 2D1) ( Figure 5)。Combined with the reported expression profile of CD45 splice variants (J Clin Pathol. 2021; 74(9): 548 - 552), it is confirmed that these monoclonal antibodies can cover all subtypes of CD45 and have the potential for clinical application.

[0133] The experimental results show that the monoclonal antibodies 1A9, 4A10, 5B10 and 7C7 provided by the present invention not only have high specificity, but also can recognize different CD45 splice variants expressed naturally, and show comparable binding ability with commercial antibodies in flow cytometry detection, laying a foundation for subsequent clinical applications.

[0134] The above - described embodiments are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A monoclonal antibody targeting all subtypes of CD45, characterized in that, The monoclonal antibodies include monoclonal antibody 1A9, monoclonal antibody 4A10, monoclonal antibody 5B10, or monoclonal antibody 7C7; the amino acid sequences of the monoclonal antibodies include a heavy chain variable region and a light chain variable region; The heavy chain variable region of monoclonal antibody 1A9 includes: heavy chain CDRH1 with the amino acid sequence shown in SEQ ID NO.10, heavy chain CDRH2 with the amino acid sequence shown in SEQ ID NO.11, and heavy chain CDRH3 with the amino acid sequence shown in SEQ ID NO.12; the light chain variable region includes: light chain CDRL1 with the amino acid sequence shown in SEQ ID NO.15, light chain CDRL2 with the amino acid sequence shown in SEQ ID NO.16, and light chain CDRL3 with the amino acid sequence shown in SEQ ID NO.17; The heavy chain variable region of monoclonal antibody 4A10 includes: heavy chain CDRH1 with the amino acid sequence shown in SEQ ID NO.20, heavy chain CDRH2 with the amino acid sequence shown in SEQ ID NO.21, and heavy chain CDRH3 with the amino acid sequence shown in SEQ ID NO.22; the light chain variable region includes: light chain CDRL1 with the amino acid sequence shown in SEQ ID NO.25, light chain CDRL2 with the amino acid sequence shown in SEQ ID NO.26, and light chain CDRL3 with the amino acid sequence shown in SEQ ID NO.27; The heavy chain variable region of monoclonal antibody 5B10 includes: heavy chain CDRH1 with the amino acid sequence shown in SEQ ID NO.30, heavy chain CDRH2 with the amino acid sequence shown in SEQ ID NO.31, and heavy chain CDRH3 with the amino acid sequence shown in SEQ ID NO.32; the light chain variable region includes: light chain CDRL1 with the amino acid sequence shown in SEQ ID NO.15, light chain CDRL2 with the amino acid sequence shown in SEQ ID NO.16, and light chain CDRL3 with the amino acid sequence shown in SEQ ID NO.17; The heavy chain variable region of monoclonal antibody 7C7 includes: heavy chain CDRH1 with the amino acid sequence shown in SEQ ID NO.35, heavy chain CDRH2 with the amino acid sequence shown in SEQ ID NO.36, and heavy chain CDRH3 with the amino acid sequence shown in SEQ ID NO.37; the light chain variable region includes: light chain CDRL1 with the amino acid sequence shown in SEQ ID NO.40, light chain CDRL2 with the amino acid sequence shown in SEQ ID NO.41, and light chain CDRL3 with the amino acid sequence shown in SEQ ID NO.

42.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of monoclonal antibody 1A9 is as shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14; The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4A10 is shown as SEQ ID NO.19, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.24; The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5B10 is shown as SEQ ID NO.29, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14; The amino acid sequence of the heavy chain variable region of the monoclonal antibody 7C7 is shown as SEQ ID NO.34, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

39.

3. A nucleic acid molecule, characterized in that, A nucleic acid molecule encoding the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, wherein The nucleic acid molecule encoding the monoclonal antibody 1A9 includes the nucleotide sequence encoding the heavy chain variable region shown as SEQ ID NO.13 and the nucleotide sequence encoding the light chain variable region shown as SEQ ID NO.18; The nucleic acid molecule encoding the monoclonal antibody 4A10 includes the nucleotide sequence encoding the heavy chain variable region shown as SEQ ID NO.23 and the nucleotide sequence encoding the light chain variable region shown as SEQ ID NO.28; The nucleic acid molecule encoding the monoclonal antibody 5B10 includes the nucleotide sequence encoding the heavy chain variable region shown as SEQ ID NO.33 and the nucleotide sequence encoding the light chain variable region shown as SEQ ID NO.18; The nucleic acid molecule encoding the monoclonal antibody 7C7 includes the nucleotide sequence encoding the heavy chain variable region shown as SEQ ID NO.38 and the nucleotide sequence encoding the light chain variable region shown as SEQ ID NO.

43.

5. Use of the monoclonal antibody according to claim 1 or 2 or the nucleic acid molecule according to claim 3 or 4 in the preparation of a product for in vitro detection of human CD45 molecule, characterized in that, The product recognizes all CD45 molecular subtypes including CD45RA, CD45RB, CD45RC, and CD45RO by binding to the conserved epitope of the human CD45 molecule.

6. Use of the monoclonal antibody according to claim 1 or 2 or the nucleic acid molecule according to claim 3 or 4 in the preparation of a drug for tumor immunotherapy.

7. Use of the monoclonal antibody according to claim 1 or 2 or the nucleic acid molecule according to claim 3 or 4 in the preparation of a diagnostic reagent for autoimmune diseases.

8. The application according to claim 7, characterized in that, Using the monoclonal antibody as a label, analyze and detect immune cells related to the autoimmune disease.

9. Use of the monoclonal antibody according to claim 1 or 2 or the nucleic acid molecule according to claim 3 or 4 in the preparation of a drug for treating autoimmune diseases or anti-organ transplant rejection related to CD45.

10. The application according to claim 9, wherein, The drug includes the monoclonal antibody or the nucleic acid molecule, an immunosuppressant, and a pharmaceutically acceptable carrier.

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