Monoclonal antibody targeting all isoforms of cd45 and uses thereof

Abstract

The application discloses a monoclonal antibody targeting all subtypes of CD45 and an application thereof, and belongs to the field of monoclonal antibody preparation. The application stably expresses shorter and longer molecular subtypes of an extracellular region of human CD45 in L929 mouse fibroblasts, immunizes BALB / c mice with L929 cells expressing both molecular subtypes of human CD45, fuses spleen cells with SP20 myeloma cells after the serum titer reaches a standard, successfully obtains a hybridoma cell strain stably secreting an antibody against all subtypes of human CD45, and obtains the monoclonal antibody targeting all subtypes of CD45. The monoclonal antibody provided by the application shows high affinity binding characteristics with all molecular subtypes of human CD45, has important application value in human CD45 molecular detection, and can be used as a therapeutic antibody in the fields of tumor immunotherapy, autoimmune disease treatment and anti-transplant rejection and the like.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody preparation, and in particular to a monoclonal antibody targeting all CD45 subtypes and its applications. Background Technology

[0002] Human CD45 (also known as leukocyte common antigen, LCA) is a type I transmembrane protein tyrosine phosphatase (PTP) widely expressed on the surface of almost all hematopoietic cells. CD45 plays a crucial role in immune cell signal transduction, regulating the signal transduction of T cell receptors (TCR) and B cell receptors (BCR), and exerting important functions in immune responses, cell differentiation, and activation. Therefore, CD45 has become one of the targets for immunological research, immune cell marker detection, and the treatment of specific diseases (such as leukemia and lymphoma).

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

[0004] For clinical diagnostics and immunotherapy, monoclonal antibodies capable of recognizing all CD45 subtypes are of significant value in flow cytometry (FACS), immunohistochemistry (IHC), and the development of therapeutic antibodies. However, most commercially available anti-CD45 monoclonal antibodies target 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 recognize naive T cells. The application scope of these antibodies is limited, making it difficult to simultaneously cover all CD45 subtypes. Furthermore, differences in expression levels among different subtypes can cause signal shifts, affecting the accuracy of quantitative analysis. In addition, some antibodies suffer from insufficient affinity or cross-reactivity, further limiting their reliability in flow cytometry, immunohistochemistry, and other detection methods. However, co-screening using the shortest splice variant CD45RO and the longest splice variant CD45RABC holds promise for obtaining CD45 monoclonal antibodies targeting all subtypes. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody targeting all CD45 subtypes and its applications, thereby addressing the problems existing in the prior art. The monoclonal antibody provided by this invention exhibits high affinity binding to all human CD45 molecular subtypes, making it valuable for the detection of human CD45 molecules. It can also be used as a therapeutic antibody in areas such as tumor immunotherapy, treatment of autoimmune diseases, and anti-transplant rejection.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a monoclonal antibody targeting all CD45 isotypes, wherein 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 shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14;

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

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

[0015] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 7C7 is shown in SEQ ID NO.34, and the amino acid sequence of the light chain variable region is 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 monoclonal antibody 1A9 includes a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.13 and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO.18;

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

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

[0020] The nucleic acid molecule encoding the monoclonal antibody 7C7 includes a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.38 and a nucleotide sequence encoding the 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 described herein in the preparation of a product for the in vitro detection of human CD45 molecules, the product recognizing all CD45 molecule subtypes, including CD45RA, CD45RB, CD45RC and CD45RO, by binding to the conserved epitopes 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 medicaments for tumor immunotherapy.

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

[0024] Optionally, the monoclonal antibody can be used as a marker to analyze and detect immune cells associated with the autoimmune disease.

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

[0026] Optionally, the drug may include the monoclonal antibody or the nucleic acid molecule, immunosuppressant, and pharmaceutically acceptable carrier.

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

[0028] This invention successfully obtained a hybridoma cell line that stably secretes antibodies against all human CD45 subtypes by immunizing BALB / c mice with L929 mouse fibroblasts that stably express the shortest human splicing variant CD45RO and the longest splicing variant CD45RABC. After serum titers reached the target levels, spleen cells were fused with SP20 myeloma cells. The variable region gene sequence of the antibody was cloned using RT-PCR, and its binding specificity was confirmed by flow cytometry and Western blotting.

[0029] The monoclonal antibody provided by this invention exhibits high affinity binding to all molecular subtypes of human CD45, with an excellent Kd value; it also demonstrates high specificity, recognizing only human CD45-positive cells; and it can effectively recognize linearized human CD45 protein. Based on these characteristics, the monoclonal antibody provided by this invention has significant application value in the detection of human CD45 molecules, and can also be used as a therapeutic antibody in areas such as tumor immunotherapy, treatment of autoimmune diseases, and anti-transplant rejection.

[0030] The monoclonal antibody provided by this invention not only offers a high-quality tool antibody for CD45-related research, but also lays an important foundation for the development of novel immunotherapeutic drugs. Its unique binding properties make it promising for both diagnostic reagent development and therapeutic applications. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 A flowchart illustrating the specific process for preparing monoclonal antibodies;

[0033] Figure 2 The protein mass and position of the four antibodies obtained by SDS-PAGE detection are shown. "M" represents a molecular marker, and "reduced" means that the antibody was reduced by SDS to two double strands.

[0034] Figure 3 The affinity constants of the four antibodies obtained with the two CD45 isoforms (RO and RABC) overexpressed are shown.

[0035] Figure 4 The binding of the four antibodies obtained to the hematopoietic cell lines Jurkat / kasumi (CD45+) and nalm6 (CD45-) was used to further confirm the specificity of the antibodies.

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

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] This invention provides a monoclonal antibody against human CD45 that can specifically bind to all CD45 subtypes, including the shortest CD45RO and the longest CD45RABC. This overcomes the subtype limitations and insufficient application scope of existing anti-CD45 antibodies and has significant advantages in basic research, clinical testing and immunotherapy.

[0043] In this invention, a multi-antigen combined immunization strategy is employed during early mouse immunization. Multiple immunizations are performed using mouse fibroblasts (L929 cells) overexpressing both the shortest human splice variant CD45RO and the longest splice variant CD45RABC to induce the production of B cells that broadly recognize CD45. This strategy helps obtain antibodies with high specificity and low cross-binding, avoiding antibody bias caused by limitations in immunogenicity during traditional monoclonal antibody screening.

[0044] Preferably, after obtaining hybridoma cells, to ensure that the screened antibodies can efficiently bind to all CD45 subtypes, this invention sets a strict screening criterion: three different types of cells are used as target cells: 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), mixed in a 1:1:1 ratio. During the screening process, flow cytometry (FACS) or immunofluorescence is used to detect the hybridoma supernatant, requiring that the antibodies in the supernatant can bind to both CD45RO and CD45RABC cells simultaneously, but not to CD45 negative cells. This strategy ensures that the screened antibodies cover all CD45 subtypes, avoiding the problem of existing antibodies only recognizing specific subtypes due to screening bias.

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

[0046] Preferably, the use of the obtained monoclonal antibody to label mononuclear cells derived from healthy individuals showed a positive rate consistent with that of commercially available antibodies, demonstrating its potential for practical application.

[0047] The specific experimental flowchart of this invention is as follows: Figure 1 As shown.

[0048] Example

[0049] 1. Immunized animals

[0050] A mouse fibroblast cell line, L929, overexpressing human CD45 molecules (including all CD45RO and CD45RABC isoforms, with a GFP marker gene in the plasmid, and successfully overexpressing cells showing FITC+ in flow cytometry), was constructed using a lentiviral vector. Balb / c mice were immunized intraperitoneally. Booster immunizations were administered at weeks 3 and 5 after the initial immunization. On day 8 after the booster immunization, tail blood was collected from mice, incubated at room temperature for 1 hour, centrifuged at 15,000 rpm for 10 minutes at 4°C, and serum was collected. The serum was diluted with PBS to different concentrations: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. L929 cells expressing human CD45 molecules were collected, washed once with PBS, and cell counts were performed. Each sample was analyzed using 5 × 10⁻⁶ cells. 5 100 μL of serum at different dilutions was added to each cell type. The negative control group consisted of L929 cells that did not express CD45. Cells were incubated at room temperature for 30 minutes and washed twice with PBS. 200 μL of APC-labeled anti-mouse IgG antibody was added, and the cells were incubated at room temperature in the dark for 30 minutes. The cells were then resuspended in 200 μL of PBS buffer, and flow cytometry was used to detect the percentage of antibody binding to cells and the fluorescence intensity. Mice whose serum bound to CD45RO and CD45RABC but not wild-type L929 cells were selected for subsequent experiments.

[0051] 2. Cell fusion and hybridoma screening

[0052] Three days prior to fusion, L929 cells expressing CD45 were injected via tail vein into selected immunized mice as an immunogen for a shock immunization. Immunized mice were euthanized by cervical dislocation and disinfected by immersion in 75% ethanol. Spleen cells from the immunized mice were extracted and fused with SP20 hybridoma cells at a ratio of 10:1 under 37°C water bath conditions. The cells were centrifuged at 800 rpm for 6 min to remove the supernatant, then resuspended in 1640 selective medium containing HAT, and transferred to 96-well plates with 200 μL of medium. Cells were cultured at 37°C and 5% CO2 for 10-14 days. When hybridoma cells grew to a round, plump clone (approximately 1 mm × 1 mm), clones were picked using a stereomicroscope in a biosafety cabinet and transferred to 96-well plates for further culture.

[0053] When the cloned cells reached a suitable density, the cell supernatant was used as the primary antibody, and mixed with wild-type L929 cells and L929 cells overexpressing CD45RO and CD45RABC in a 1:1:1 ratio (2 × 10⁶ cells per cell type). 5Using FITC+APC+ cells as target cells, flow cytometry was used to detect their binding to screen for positive clones. A positive clone was defined as one that only binds to CD45RO-L929 and CD45RABC-L929 and does not bind to wild-type L929, i.e., the proportion of FITC+APC+ cells should reach 65%-75%.

[0054] Positive clones obtained from the screening were transferred to 24-well plates for further culture and screening. Subclones of the positive clones selected from the 24-well plates were then transferred to 96-well plates and cultured for 10-14 days. The supernatant was collected for flow cytometry analysis to further screen for positive clones that stably secrete the target antibody. After subcloning and screening, all wells were positive, ultimately yielding four different stable hybridoma cell lines.

[0055] 3. Antibody production and purification

[0056] Ascites preparation: Liquid paraffin was injected intraperitoneally into mice at a rate of 500 μL per mouse, followed by administration of 5 × 10 μL per mouse. 5 Different hybridoma cells were obtained and injected into different mice via the peritoneum. Ascites fluid was collected from the mice 7-10 days later.

[0057] For crude antibody extraction from the collected ascites fluid: Take 5 ml of the collected ascites fluid and add an equal volume (5 ml) of PBS buffer, mixing thoroughly. Then, slowly add an equal volume (5 ml) of saturated ammonium sulfate solution while stirring to ensure thorough mixing. Place the mixture at 4°C 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 the mixture with PBS buffer at 4°C for 24 hours, changing the dialysate three times during this period to remove ammonium sulfate.

[0058] Purification of crude antibody: The crudely purified antibody was finely purified using the AKTA protein purification system according to the purification guidelines provided by GE, through a 5ml Protein G purification pre-packed column, to obtain the purified antibody. The monoclonal antibodies secreted by four 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 antibody protein concentration was quantified using the BCA colorimetric method. The purified sample protein amounts were then adjusted to be uniform. Electrophoresis was then performed in an SDS-PAGE system. The resulting gel was stained with Coomassie Brilliant Blue staining solution for 30 min, followed by overnight destaining, with the destaining solution changed 3-5 times during this period. The gel was then exposed to light to obtain protein bands of antibodies secreted by different hybridoma cells. Figure 2 ).

[0062] The purified CD45 antibody was dissolved in 100 μL of 5×10⁻⁶ solution at concentrations of 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, respectively. 5 CD45RO and CD45RABC L929 cell lines were incubated at room temperature for 30 min, washed three times with PBS, and then incubated with mouse anti-human IgG secondary antibody at room temperature for 30 min, followed by three washes with PBS. Fluorescence intensity was then detected using flow cytometry, and the Kd value of the antibody against the CD45RO / CD45RABC L929 cell line was calculated using GraphPadPrism 9.5.0. Figure 3 ).

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

[0064] First, total RNA was extracted from the selected hybridoma cells using the SPAReasy Cell RNA Rapid Extraction Kit. 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 to amplify the light and heavy chain variable regions of antibodies secreted by different hybridoma cells by PCR:

[0066] Upstream primer P1 for the heavy chain backbone region: SAGGTGMAGCTKCASSARTCWGG (SEQ ID NO.1);

[0067] Downstream primer P2 for the heavy chain variable region: TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA (SEQ ID NO.2);

[0068] upstream primer of light chain leader peptide:

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

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

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

[0072] P6:ATGKCCCWRCTCAGYTYCTKGT(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, a 50 μL PCR reaction mixture was prepared, consisting of 2 μL cDNA, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 25 μL mix, and ddH2O to a final volume of 50 μL. The reaction conditions were: 95°C pre-denaturation 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 a final extension at 72°C for 10 minutes. The PCR products were separated by agarose gel electrophoresis, and the target VL and VH fragments were recovered. The recovered VL and VH fragments were ligated into the pMD19-T (Simple) vector (Takara), respectively. The ligation mixture consisted of 70 ng of VL or VH PCR product, 1 μL of pMD19-T (Simple) vector, 5 μL Solution I ligation solution, and ddH2O to a final volume of 10 μL. Ligation was performed overnight at 4°C. The ligation product was transformed into E. coli DH5α competent cells and cultured overnight at 37°C. The strains were then sent to a company for sequencing until the sequencing results of at least three samples were consistent. Ultimately, the heavy and light chain variable region sequences of different CD45 antibodies were successfully cloned, and their sequences conformed to the typical antibody variable region sequence characteristics. Then, using the Vazyme RACE kit, the terminal base sequences of the antibody light and heavy chains (VL and VH) were further obtained; the resulting 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: QVQLQQSGAEVARPGASVKMSCKASGYTFTSYTVHWVK QRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADRSSSTLYMQLNSLTSEDSVVYYCARAGDGFYMSWFAYWGQGTLVTVSA (SEQ ID NO.9);

[0079] The heavy chain variable region CDR sequence contains 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 the heavy chain variable region is: CAGGTCCAGCTGCAGCAGTCTGGGGCTGAAGT GGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACACGGTGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGTAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACA AGGCCACATTGACTGCAGACAGATCCCTCCAGCACACTCTACATGCAACTGAACAGCCTGACATCTGAGGACTCTGTAGTCTATTACTGTGCAAGAGCAGGATGGTTTCTACATGTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.13);

[0084] The amino acid sequence of the light chain variable region is: SIVMTQTPKFLLVSAGDRVTMTCKASQSVNNDVAWYQQK PGQSPKLLIYHVSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGG TQLEIKR (SEQ ID NO.14);

[0085] The light chain variable region CDR sequence contains 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 CTTGTATCAGCAGGAGACAGGGTTACCATGACCTGCAAGGCCAGTCAGAGTGTGAATAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACCATGTATCCAATCGCTACACTGGAGTCCCTGAT CGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAGGACCTGGCAGTTTTATTTCTGTCAGCAGGATTATAGCTCTCCTTGGACGTTCGGTGGAGGCACCCAGCTGGAAATCAAACGG(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 heavy chain variable region CDR sequence contains 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 GGTCCAACCAGGCGGGTCCCGAAAGTTATCTTGCGCTGCTTCGGGATTCACTTTCTCCAGTTTTGGTATGCACTGGGTGCGTCAAGCACCAGAGAAGGGCTTAGAGTGGGTTGCGTACATTAACGGAGGCTCGACCACGTTTTATTACGCCGACACTGTGAAGG GGAGATTCACAATAAGTCGTGATAATCCGAAGAACACGCTGTACCTCCAGATGACATCGTTACGTAGTGAAGACACAGCAATGTACTACTGTGCCCGTACGACCGTGCCAACGGCATGGAGCGCGTACTGGGGACAGGGCACGCTTGTTACCGTATCTTCC(SEQ ID NO.23);

[0097] The amino acid sequence of the light chain variable region is: NIMMTQSPSSLAVSAGEKVTMSCKSSQSVFYSSNQKNYLA WYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISNVQTEDLAVYYCHQYLSSW TFGGGTKLEIKR (SEQ ID NO. 24);

[0098] The light chain variable region CDR sequence contains the following three 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: AATATTATGATGACACAGTCACCTTCTTCGCCTAG CAGTCTCGGCAGGGGAGAAAGTCACGATGAGTTGTAAATCGTCACAATCTGTGTTCTATTCGTCGAATCAGAAGAACTACCTAGCTTGGTACCAACAAAAACCAGGTCAATCTCCTAAACTACTCATTTATTGGGCCAGCACCAGGGAGAGTGG CGTGCCTGACAGATTCTCGGGCTCTGGTTCTGGTACCGATTTTACTTTGACAATTAGTAACGTGCAAACAGAGGACCTCGCCGTGTACTATTGTCATCAATATCTGTCTAGCTGGACTTTTGGCGGCGGTACTAAGTTGGAGATTAAGAGG(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 heavy chain variable region CDR sequence contains the following three segments:

[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 AGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTTTGGAATGCACTGGGTTCGTCAGACTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTACTACCTTCTTCTATGCAGACACAGTGAAGG GCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTTCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTTCTGTGCAACAACTACTGTGATTACGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.33);

[0110] The amino acid sequence of the light chain variable region is: SIVMTQTPKFLLVSAGDRVTMTCKASQSVNNDVAWYQQK PGQSPKLLIYHVSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPWTFGGG TQLEIKR (SEQ ID NO.14);

[0111] The light chain variable region CDR sequence contains the following three 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: AATATTATGATGACACAGTCACCTTCTTCGCCTAG CAGTCTCGGCAGGGGAGAAAGTCACGATGAGTTGTAAATCGTCACAATCTGTGTTCTATTCGTCGAATCAGAAGAACTACCTAGCTTGGTACCAACAAAAACCAGGTCAATCTCCTAAACTACTCATTTATTGGGCCAGCACCAGGGAGAGTGG CGTGCCTGACAGATTCTCGGGCTCTGGTTCTGGTACCGATTTTACTTTGACAATTAGTAACGTGCAAACAGAGGACCTCGCCGTGTACTATTGTCATCAATATCTGTCTAGCTGGACTTTTGGCGGCGGTACTAAGTTGGAGATTAAGAGG(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 heavy chain variable region CDR sequence contains the following three segments:

[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 AGGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTTTGGAATGCACTGGGTTCGTCAGACTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTACTACCTTCTACTATGCAGACACAGTGAAGG GCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTTCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTTCTGTGCAAGAACTACTGTGATTACGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.38);

[0123] The amino acid sequence of the light chain variable region is: NIMMTQSPSSLAVSAGEKVTLSCKSSQSVFVSSNQKNYLA WYQQKPGQSPRLLIYWASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCHQYLSSWT FGGGTKLEIKR (SEQ ID NO. 39);

[0124] The light chain variable region CDR sequence contains the following three segments:

[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 light chain variable region is: AACATTATGATGACACAGTCGCCATCATCTCTGG CTGTGTCTGCAGGAGAAAAGGTCACTTTGAGCTGTAAGTCCAGTCAAAGTGTTTTCGTCAGTTCAAATCAGAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAGACTGCTAATCTACTGGGCATCCATTAGGGAATCTGG TGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAATTTATTACTGTCATCAATACCTCTCCTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG(SEQ ID NO.43).

[0129] 6. Functional validation of monoclonal antibodies

[0130] To comprehensively evaluate the functional properties of the obtained anti-human CD45 monoclonal antibodies (1A9, 4A10, 5B10, and 7C7), this invention employed various human cell lines for systematic validation. The human cell lines used for validation included human acute T-lymphoblastic leukemia cells Jurkat (CD45+), human acute myeloid leukemia cells Kasumi-1 (CD45+), and the human B-lymphoblastic leukemia cell line Nalm6 (CD45-).

[0131] First, specificity assays were performed using Jurkat (CD45+), Kasumi-1 (CD45+), and Nalm6 (CD45-) cell lines. The results are as follows: Figure 4 The results showed that all four monoclonal antibodies could specifically bind to CD45-positive cells, but not to CD45-negative cells, confirming the antibodies' specific recognition ability of human CD45 antigen.

[0132] The ability of antibodies to recognize different subtypes of natural CD45 was further evaluated using peripheral blood mononuclear cells (PBMCs) from healthy individuals. After separating PBMCs using Ficoll density gradient centrifugation, flow cytometry analysis revealed that all four monoclonal antibodies effectively bound to the CD45+ cell population in PBMCs, with binding efficiency comparable to that of the commercially available anti-CD45 antibody (BioLegend anti-human CD45-APC, clone number 2D1). Figure 5Combined with the CD45 splice variant expression profiles reported in the literature (J Clin Pathol. 2021; 74(9): 548-552), it was confirmed that these monoclonal antibodies can cover all CD45 subtypes and have clinical application potential.

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

[0134] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody targeting all isoforms of CD45, characterized in that, The monoclonal antibody is monoclonal antibody 1A9; the amino acid sequence of the monoclonal antibody includes a heavy chain variable region and a light chain variable region; 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.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

14.

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

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecule encoding monoclonal antibody 1A9 includes a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.13 and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO.

18.

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

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