Anti-human cd45ra antibodies, encoding nucleic acid molecules, expression vectors and uses thereof

Abstract

The application provides an anti-human CD45RA antibody, a coding nucleic acid molecule, an expression vector and application thereof. The anti-human CD45RA antibody or an antigen binding fragment thereof provided by the application has high binding affinity to CD45RA, and can be used for detection of CD45RA and treatment of tumors with high expression of CD45RA, such as acute myeloid leukemia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to antibodies targeting CD45RA, their antigen-binding fragments, encoding nucleic acid molecules, expression vectors, and their applications in CD45RA antigen detection and treatment of acute myeloid leukemia. Background Technology

[0002] CD45RA is a subtype of the common leukocyte antigen (CD45), belonging to the high molecular weight (approximately 205-220 kDa) isoform. The CD45 family includes multiple isoforms, with CD45RA being a major form, particularly highly expressed on immature and naive T cells and some B cells. CD45RA protein, through its phosphatase activity, participates in regulating the phosphorylation state of T cell receptor (TCR) and B cell receptor (BCR) signal transduction processes, thereby affecting the activation, proliferation, and differentiation of immune cells.

[0003] CD45RA plays a crucial regulatory role in the development of immune cells. In the thymus, CD45RA participates in the development and selection of T cells, helping immature T cells to mature into mature T cells with specific antigen specificity through positive and negative selection. CD45RA also plays a similar role in B cell development, promoting the migration and maturation of B cells from the bone marrow to peripheral lymphoid organs.

[0004] During the immune response, CD45RA affects the activation threshold and response intensity of immune cells by regulating the phosphorylation status of the TCR and BCR signaling pathways. Naïve T cells and some B cells highly express CD45RA, which allows them to maintain a low activation threshold upon encountering antigen stimulation and rapidly initiate an immune response. However, as the immune response progresses, the expression of CD45RA is gradually downregulated, replaced by other isoforms of CD45, such as CD45RO, marking the transition of immune cells from a naïve state to a memory or effector cell state.

[0005] Due to its specific expression pattern on immune cells, CD45RA has become an important biomarker for the diagnosis and prognostic assessment of many immune-related diseases. For example, in autoimmune diseases, changes in the ratio of CD45RA+ T cells to B cells can reflect disease activity and treatment efficacy. Furthermore, in tumor immunotherapy, the infiltration of CD45RA+ T cells is also considered an important indicator for assessing the immune status of the tumor microenvironment.

[0006] Acute myeloid leukemia (AML) is a malignant clonal disease originating from myeloid hematopoietic stem / progenitor cells in the bone marrow, accounting for approximately 60-70% of adult acute leukemia cases. Despite a high initial remission rate, the overall relapse rate exceeds 50%. Immune escape and self-renewal of leukemia stem cells (LSCs) are key reasons for AML relapse and drug resistance. Although immune checkpoint inhibitors, such as anti-PD-1 / CTLA-4 inhibitors, are highly effective in solid tumors, their efficacy in AML is limited, suggesting the existence of novel immune escape mechanisms in leukemia stem cells. Therefore, elucidating the immune escape mechanisms of LSCs and developing new targeted intervention strategies has become an urgent need in AML treatment.

[0007] Normal HSCs (hematopoietic stem cells) are CD45RA negative, while AML cells (including leukemia stem cells) are CD45RA positive. CD45RA is highly expressed in acute myeloid leukemia cells, while normal hematopoietic stem cells (HSCs) do not express or express low levels of this subtype. Therefore, CD45RA is a very promising therapeutic target for AML, especially suitable for strategies that target and eliminate AML cells while protecting normal HSCs. Summary of the Invention

[0008] In one aspect, the present invention provides an anti-human CD45RA antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain CDR1 sequence is shown in SEQ ID NO: 13, the heavy chain CDR2 sequence is shown in SEQ ID NO: 15, and the heavy chain CDR3 sequence is shown in SEQ ID NO: 17; and the light chain CDR1 sequence is shown in SEQ ID NO: 5, the light chain CDR2 sequence is shown in SEQ ID NO: 7, and the light chain CDR3 sequence is shown in SEQ ID NO: 9.

[0009] In some embodiments, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 11, and the light chain variable region sequence of the antibody is shown in SEQ ID NO: 3.

[0010] In some implementations, the antigen-binding fragment is in the form of Fab, F(ab')2, or scFv.

[0011] On the other hand, the present invention provides a nucleic acid molecule encoding the anti-human CD45RA antibody or its antigen-binding fragment as described in the claims.

[0012] On the other hand, the present invention provides an expression vector containing the aforementioned nucleic acid molecules.

[0013] On the other hand, the present invention provides a host cell comprising the above-mentioned nucleic acid molecule or expression vector, or expressing the above-mentioned anti-human CD45RA antibody or its antigen-binding fragment.

[0014] On the other hand, the present invention provides an immunoassay kit comprising the anti-human CD45RA antibody or its antigen-binding fragment as described above.

[0015] On the other hand, the present invention provides a pharmaceutical composition comprising: 1) The above-mentioned anti-human CD45RA antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, or host cell; and 2) Pharmaceutically acceptable excipients.

[0016] On the other hand, the present invention provides the use of the above-mentioned anti-human CD45RA antibody or its antigen-binding fragment, nucleic acid molecule, expression vector or host cell in the preparation of tumor therapeutic drugs.

[0017] In some implementations, the tumor is acute myeloid leukemia. Attached Figure Description

[0018] Figure 1 The HPLC detection results of the anti-human CD45RA antibody of the present invention are shown, and the purity of the antibody is close to 100%.

[0019] Figure 2 The results are from flow cytometry. When the amount of CD45RA purchased antibody added was 0.125 μg, CD45-PerCP-Cy5.5 antibody was used for gating, with lymphocytes at the bottom.

[0020] Figure 3 The results are from flow cytometry analysis. When 0.125 μg of the purchased CD45RA antibody was added, the... Figure 2 The histogram of lymphocyte entry points shows CD45RA-negative cells on the left and CD45RA-positive cells on the right.

[0021] Figure 4 The results are from flow cytometry. In this invention, when the amount of CD45RA antibody added was 0.125 μg, CD45-PerCP-Cy5.5 antibody was used for gating, with lymphocytes at the bottom.

[0022] Figure 5 The results are from flow cytometry analysis. When the amount of CD45RA antibody added in this invention is 0.125 μg, from... Figure 4 The histogram of lymphocyte entry points shows CD45RA-negative cells on the left and CD45RA-positive cells on the right.

[0023] Figure 6The results are from flow cytometry analysis. The flow cytometry curves show the antibody levels added: 1.0 μg, 0.5 μg, 0.25 μg, 0.125 μg, 0.0625 μg, and 0.03125 μg. "Self-developed CD45RA" refers to the antibody of this invention, while the purchased control CD45RA is the HI100 antibody.

[0024] Figure 7 The results are from flow cytometry. In this invention, when 50 μg of CD45RA antibody was added, CD45-PerCP antibody was used for gating, with lymphocytes at the bottom.

[0025] Figure 8 The results are from flow cytometry analysis. When the amount of CD45RA antibody added in this invention is 50 μg, from... Figure 7 The histogram of lymphocyte entry shows CD45RA-negative cells on the left and CD45RA-positive cells on the right. As can be seen from the figure, after blocking with 50 μg of the antibody of this invention, the positive rate was 0.17%, with almost no clustering.

[0026] Figure 9 The results are from flow cytometry. In this invention, when 10 μg of CD45RA antibody was added, CD45-PerCP antibody was used for gating, with lymphocytes at the bottom.

[0027] Figure 10 The results are from flow cytometry analysis. When the amount of CD45RA antibody added in this invention is 10 μg, from... Figure 9 The histogram of lymphocyte entry points shows CD45RA-negative cells on the left and CD45RA-positive cells on the right. As can be seen from the figure, after blocking with 10 μg of the antibody of this invention, the positive rate was 22.8%, and the clustering was not obvious.

[0028] Figure 11 The results are from flow cytometry. In this invention, when the amount of CD45RA antibody added was 0.4 μg, CD45-PerCP antibody was used for gating, with lymphocytes at the bottom.

[0029] Figure 12 The results are from flow cytometry analysis. When the amount of CD45RA antibody added in this invention is 0.4 μg, from... Figure 11 The histogram of lymphocytes entering the lymphocyte population shows CD45RA-negative cells on the left and CD45RA-positive cells on the right. As can be seen from the figure, after blocking with 0.4 μg of the antibody of this invention, the positive rate was 31.8%, and cell clustering was observed.

[0030] Figure 13The results are from flow cytometry. In this invention, when the amount of CD45RA antibody added was 0.02 μg, CD45-PerCP antibody was used for gating, with lymphocytes at the bottom.

[0031] Figure 14 The results are from flow cytometry analysis. When the amount of CD45RA antibody added in this invention is 0.02 μg, from... Figure 13 The histogram of lymphocyte entry points shows CD45RA-negative cells on the left and CD45RA-positive cells on the right. As can be seen from the figure, after blocking with 0.02 μg of the antibody of this invention, the positive rate was 35.4%, and the clustering was obvious.

[0032] Figure 15 The results are from flow cytometry. In this invention, when the amount of CD45RA antibody added was 0 μg, CD45-PerCP antibody was used for gating, with lymphocytes at the bottom.

[0033] Figure 16 The results are from flow cytometry analysis. When the amount of CD45RA antibody added in this invention is 0 μg, from... Figure 15 The histogram of lymphocyte entry points shows CD45RA-negative cells on the left and CD45RA-positive cells on the right. As can be seen from the figure, after blocking with 0 μg of the antibody of this invention, the positive rate was 43.7%, and the clustering was very obvious. Detailed Implementation

[0034] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0035] "Antibody" refers to an immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). This foreign substance is correspondingly called an antigen. The basic structure of a classic antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. Based on the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The interaction between the heavy chain variable region (VH) and the light chain variable region (VL) forms the antigen-binding site (Fv). In this context, "antibody" can refer to a complete antibody or its antigen-binding fragment, and can be a human antibody, mouse antibody, humanized antibody, chimeric antibody, etc. Antibodies can be of any type, such as IgG, IgE, IgM, IgD, IgA, or IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, etc.

[0036] An antibody's "antigen-binding fragment" refers to a polypeptide that includes a portion of the source antibody's sequence (especially the CDR sequence) and possesses the specificity for binding to the source antibody. This antigen-binding fragment typically includes both the light chain variable region and the heavy chain variable region of the source antibody, thus enabling antigen binding. Antigen-binding fragments come in various forms, such as Fab, F(ab')2, and single-chain antibodies (scFv). scFv is composed of the antibody's heavy chain variable region and light chain variable region linked together by a short peptide chain. Through proper folding, the variable regions from the heavy and light chains interact non-covalently to form the Fv segment, thus allowing scFv to retain its antigen-binding activity relatively well.

[0037] Methods for preparing antibodies are known in the art, including but not limited to isolating cells expressing specific antibodies after immunizing animals with antigens (e.g., preparing antibodies via hybridoma technology), antibody screening techniques using phage antibody libraries, and transforming cells using genetic engineering techniques to express antibody molecules. For example, in one specific embodiment, antibody molecules are prepared by introducing a recombinant expression vector encoding the heavy and light chains of an antibody into 293T cells.

[0038] An "expression vector," also known as a "recombinant expression vector," refers to a nucleic acid molecule containing various expression elements for expressing a target protein (such as the light and / or heavy chains of an antibody molecule) in a host cell. For expression vectors used to express the target protein in eukaryotic cells (mammalian cells, insect cells, plant cells, etc.), these expression elements typically include promoters, enhancers, and polyadenylation signal sequences. To facilitate amplification in *E. coli*, expression vectors often also include *E. coli* replicon sequences. Expression vectors may also include antibiotic resistance genes or selection marker genes for screening (such as the ampicillin resistance gene (AmpR), thymidine kinase gene (TK), etc.) and multiple cloning sites (MCS) for inserting the target protein coding sequence. Expression vectors can be plasmid vectors or viral vectors.

[0039] The expression vectors provided herein are suitable for expressing the antibody molecules or fragments thereof in a variety of host cells, particularly in mammalian cells (e.g., mouse, rat, pig, sheep, monkey, chimpanzee, or human cells). In some embodiments, the mammalian cells may be selected from CHO cells, HEK293 cells, or BHK cells. Additionally, the host cells used herein also include hybridoma cells capable of expressing the antibody molecules of the present invention.

[0040] When referring to pharmaceutical compositions, the term "pharmaceuticalally acceptable excipient" refers to solid or liquid diluents, fillers, antioxidants, stabilizers, and other substances that can be safely administered to humans and / or animals without excessive adverse side effects, and which are suitable for maintaining the activity of the drug or active agent contained therein.

[0041] It is known in the art that antibody molecules can be modified in various ways, such as PEGylation, glycosylation, forming antibody conjugates (e.g., ADCs), adding purification tags, or fusing with other proteins, for example, to form bispecific antibodies. These antibody derivatives obtained by modifying the antibody molecules provided herein should also be covered within the scope of this invention.

[0042] "Treatment" refers to relieving, reducing, improving, or inhibiting (e.g., preventing the development) a disease that a subject has exhibited or has previously experienced. For a specific disease, "treatment" can include "curing" the disease, but in most cases it does not require the complete elimination of all symptoms. For example, if administration of a relevant drug results in a reduction or elimination of at least one symptom in the subject, it can be considered treatment.

[0043] The anti-human CD45RA antibody of the present invention specifically targets CD45RA and has a binding affinity superior to the commercially available antibody HI100.

[0044] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0045] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0046] Example 1: CD45RA plasmid lentivirus coating and preparation of immune cells 1) Lentiviral Packaging: A three-plasmid packaging system was used, consisting of one packaging plasmid (psPAX2), one envelope plasmid (pMD2G), and one target gene plasmid. The nucleotide sequence of the human CD45RA antigen (SEQ ID NO: 19) was ligated into the target gene plasmid (pCDH-CMV-MCS-EF1-copGFP). The packaging plasmid, envelope plasmid, and target gene plasmid were extracted separately and mixed at a ratio of 3:1:4. The total mass of the plasmids was then mixed with PEI at a ratio of 1:3 to form a PEI-DNA mixture. When the confluence of 293T cells (gifted by the Institute of Hematology, Chinese Academy of Medical Sciences) was between 90% and 95%, the DNA-PEI mixture was added to the mixture. After 48 hours, the viral supernatant was collected, and 10 mL of pre-warmed 293T culture medium was added to the cell culture dish. The collected viral supernatant was stored at 4°C. The viral supernatant collected at 72 hours was then mixed with the viral supernatant collected at 48 hours. Centrifuge at 25,000 rpm for 120 min to concentrate the virus, aliquot and store at -80℃.

[0047] 2) Preparation of CD45RA-3T3 cells for immunization: 3T3 cells in logarithmic growth phase were digested, counted, and the cell density was adjusted to 2×10⁻⁶. 5 Cells were seeded at a density of 100 cells / mL into 6-well plates, with 2 mL of DMEM medium (containing 10% fetal bovine serum) added to each well. After 24 hours of culture, when cell confluence was approximately 30%-50%, the 6-well plates were removed from the incubator, the old medium was aspirated and discarded, and 2 mL of fresh, complete DMEM (containing 10% fetal bovine serum) containing virus (50 μL) and Polybrene (Solepro, catalog number H8761, concentration 10 mg / mL, dilution 1:2000) was added. The plate was returned to the incubator and gently agitated to distribute the liquid evenly. Cell infection efficiency was observed to be greater than 80% under a fluorescence microscope 72 hours post-infection. Subculture was then performed, and the cells were suitable for use in immunizing animals.

[0048] Nucleotide sequence of human CD45RA antigen (SEQ ID NO: 19): Example 2 Animal Immunization Expanding the culture of CD45RA-3T3 transfected cells prepared in Example 1, the cells were administered via intraperitoneal injection at a dose of 1×10⁻⁶. 6 Cell dosage per mouse: A total of 3 mice were immunized, with 4-week-old female BALB / c mice selected for immunization. Immunization was performed once a week for a total of 3 times. Tail blood was collected from the mice, and the titer was detected by ELISA. Horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (manufacturer: Solarbio, catalog number: SE131) was diluted 1:5000 and then detected. The mice with the highest titer were selected for subsequent cell fusion.

[0049] Example 3: Cell Fusion and Screening 1) Cell preparation: Select mice with the highest titer and inject them intraperitoneally with the transfected cell line (CD45RA-3T3) at a dose of 5 × 10⁻⁶. 6 Cells per mouse were used for shock immunization; after 3 days, cell fusion was performed, and the spleen of the mouse was removed under sterile conditions, the spleen was ground, and the spleen cells were collected by sieve and washed once with PBS; at the same time, myeloma cells in the logarithmic growth phase were collected and the myeloma cells were mixed with the collected spleen cells at a ratio of 1:5, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0050] 2) Induction of fusion: Add 1 mL of PEG solution as an inducer, shake gently, and incubate at 37°C for 1 min to allow mouse spleen cells and mouse myeloma cells to fuse.

[0051] 3) Screening hybrid cells: Inoculate the hybrid cells into a selective medium (such as HAT medium), discard unfused cells and homonuclear fusion cells, and retain heteronuclear hybrid cells.

[0052] 4) Cloning culture and identification: Using the secondary antibody Anti-Mouse IgG (H+L) F(ab')2 Fragment (Alexa Fluor® 647, CST 4410S), positive clone wells were screened by flow cytometry. Three rounds of subcloning were performed using the limiting dilution method. The antibody activity in the cell supernatant was identified by flow cytometry, and the clones with the best activity were selected for expansion culture for later use.

[0053] Example 4 Preparation of anti-human CD45RA antibody 1) Preparation of CD45RA mouse ascites: Hybridoma cells in good growth condition (from Example 3) were collected and injected intraperitoneally into mice at a concentration of 1×10⁻⁶. 6 Cells per mouse; when the mouse's abdomen is noticeably distended and the skin is taut and shiny, ascites can be collected in batches; the collected ascites is then stored at -20°C.

[0054] 2) Purification of anti-human CD45RA antibody: The harvested ascites fluid was centrifuged at 10,000 rpm for 30 min, filtered through a 0.45 μm filter membrane, and then purified using a Protein A column. The column was first equilibrated with PBS, then loaded with the sample, and finally eluted with glycine at pH 3.5. The solution was then replaced with G25 molecular sieves, and finally replaced with PBS buffer. 100 μL of the solution was used for HPLC purity identification. Figure 1 Simultaneously, hybridoma cells used for ascites preparation were sent to a third party for cell sequencing, yielding the antibody nucleotide and amino acid sequences as follows.

[0055] Antibody variable region nucleotide sequence CD45RA light chain variable region nucleotide sequence (SEQ ID NO: 1) GACATTTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGAAAGTCTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATC CAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA; CD45RA heavy chain variable region nucleotide sequence (SEQ ID NO: 2) CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAGGTGGTAATACTAATTACAATGAGACTTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCGACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATCGTCGTTTGGTTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA; Amino acid sequence of antibody variable region (CDRs defined by IMGT) CD45RA light chain variable region (SEQ ID NO: 3) DIVLTQSPASLAVSLGQRATISCRASESLDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK FR1: DIVLTQSPASLAVSLGQRATISCRAS (SEQ ID NO: 4) CDR1: ESLDNYGISF (SEQ ID NO: 5) FR2: MNWFQQKPGQPPKLLIY (SEQ ID NO: 6) CDR2: AAS (SEQ ID NO: 7) FR3: NQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFC (SEQ ID NO: 8) CDR3: QQSKEVPWT (SEQ ID NO: 9) FR4: FGGGTKLEIK (SEQ ID NO: 10) CD45RA heavy chain variable region QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGGGNTNYNETFKGKATFTADTSSNTAYMRLSSLTSEDSAVYYCARSSFGYYFDYWGQGTTLTVSS (SEQ ID NO: 11) FR1: QVQLQQSGAELMKPGASVKISCKAT (SEQ ID NO: 12) CDR1: GYTFSSYW (SEQ ID NO: 13) FR2: IEWVKQRPGHGLEWIGE (SEQ ID NO: 14) CDR2: ILPGGGNT (SEQ ID NO: 15) FR3:NYNETFKGKATFTADTSSNTAYMRLSSLTSEDSAVYYC (SEQ ID NO: 16) CDR3: ARSSFGYYFDY (SEQ ID NO: 17) FR4: WGQGTTLTVSS (SEQ ID NO: 18) Example 5: Activity assay of anti-human CD45RA antibody Add 1.0 μg, 0.5 μg, 0.25 μg, 0.125 μg, 0.0625 μg, and 0.03125 μg of the anti-human CD45RA antibody prepared in Example 4 or a commercially available anti-human CD45RA antibody (manufacturer: Thermo, catalog number 14-0458-82, clone number HI100) to each flow cytometry tube; add 100 µL of peripheral blood sample from a healthy individual to each tube, vortex thoroughly, and incubate at room temperature in the dark for 20 minutes; add 2 mL of fixative-free hemolysin to each tube, vortex thoroughly, and incubate at room temperature in the dark for 15 minutes; add 1 mL of PBS to each tube, vortex thoroughly, centrifuge at 300 g for 5 minutes, and discard the supernatant; dilute Tonbo's Goat anti-Mouse IgG (H&L)-FITC secondary antibody (catalog number 35-8055-U500) 1:700, add 50 μL of the diluted secondary antibody to each tube, vortex thoroughly, and incubate at room temperature in the dark for 20 minutes; add 1 mL of PBS to each tube. Add PBS, vortex thoroughly, centrifuge at 300 g for 5 minutes, and discard the supernatant; add the matching reagent CD45-PerCP-Cy5.5 to each tube, vortex thoroughly, and incubate at room temperature in the dark for 20 minutes; add 1 mL PBS to each tube, vortex thoroughly, centrifuge at 300 g for 5 minutes, and discard the supernatant; add 250 µL PBS to each tube, vortex thoroughly, and perform flow cytometry analysis. Results are as follows: Figure 6 As shown ( Figure 2-5 The flow cytometry results are shown when the antibody concentration is 0.125 μg. The anti-human CD45RA antibody prepared in Example 4 maintained stable antigen-binding ability over a wide concentration range of approximately 0.03–1.0 μg / tube. Even when diluted to lower concentrations, the signal did not show significant attenuation, demonstrating better antibody affinity and titer compared to the commercially available antibody HI100.

[0056] Example 6: Blocking experiment of anti-human CD45RA antibody (antigen epitope competition inhibition experiment) Add 50 μg, 10 μg, 2 μg, 0.4 μg, 0.08 μg, 0.02 μg, and 0 μg of the anti-human CD45RA antibody prepared in Example 4 to flow cytometry tubes, respectively. Add 100 µL of peripheral blood sample from healthy individuals to each tube and vortex thoroughly. Incubate at room temperature in the dark for 20 minutes. Add 2 mL of fixative-free hemolysin to each tube and vortex thoroughly. Incubate at room temperature in the dark for 15 minutes. Add 1 mL of PBS to each tube, vortex thoroughly, centrifuge at 300 g for 5 minutes, and discard the supernatant. Add 20 μL each of commercially available CD45RA-FITC (manufacturer: Thermo, catalog number 11-0458-42, clone number HI100) and CD45-PerCP to each tube and vortex thoroughly. Incubate at room temperature in the dark for 20 minutes. Add 1 mL of PBS to each tube, vortex thoroughly, centrifuge at 300 g for 5 minutes, and discard the supernatant. Add 250 µL of PBS to each tube, vortex thoroughly, and then perform flow cytometry analysis.

[0057] Some representative results are as follows: Figures 7-16 As shown. Figure 7 The results of gating with CD45-PerCP antibody are shown when the amount of CD45RA antibody added in this invention is 50 μg, with lymphocytes at the bottom. Figure 8 This invention demonstrates that when the amount of CD45RA antibody added is 50 μg, from... Figure 7 The histogram results of lymphocyte entry show that the left side represents CD45RA negative cells and the right side represents CD45RA positive cells. As can be seen from the figure, after blocking with 50 μg of the antibody of this invention, the positive rate was 0.17%, with almost no clustering. Figure 9 The results of gating with CD45-PerCP antibody are shown when the amount of CD45RA antibody added in this invention is 10 μg, with lymphocytes at the bottom. Figure 10 This invention demonstrates that when the amount of CD45RA antibody added is 10 μg, from... Figure 9 The histogram results of lymphocyte entry show that the left side represents CD45RA negative cells and the right side represents CD45RA positive cells. As can be seen from the figure, after blocking with 10 μg of the antibody of this invention, the positive rate is 22.8%, and the clustering is not obvious. Figure 11The results of gating with CD45-PerCP antibody are shown when the amount of CD45RA antibody added in this invention is 0.4 μg, with lymphocytes at the bottom. Figure 12 The results show that when the amount of CD45RA antibody added in this invention is 0.4 μg, from... Figure 11 The histogram results of lymphocyte entry show that the left side represents CD45RA negative cells and the right side represents CD45RA positive cells. As can be seen from the figure, after blocking with 0.4 μg of the antibody of this invention, the positive rate is 31.8%, and clustering is visible. Figure 13 The results of gating with CD45-PerCP antibody are shown when the amount of CD45RA antibody added in this invention is 0.02 μg, with lymphocytes at the bottom. Figure 14 The results show that when the amount of CD45RA antibody added in this invention is 0.02 μg, from... Figure 13 The histogram results of lymphocyte entry show that the left side represents CD45RA negative cells and the right side represents CD45RA positive cells. As can be seen from the figure, after blocking with 0.02 μg of the antibody of this invention, the positive rate is 35.4%, and the clustering is obvious. Figure 15 The results of gating with CD45-PerCP antibody are shown when the amount of CD45RA antibody added in this invention is 0 μg, with lymphocytes at the bottom. Figure 16 This invention demonstrates that when the amount of CD45RA antibody added is 0 μg, from Figure 15 The histogram results of lymphocyte entry show that the left side represents CD45RA negative cells and the right side represents CD45RA positive cells. As can be seen from the figure, after blocking with 0 μg of the antibody of this invention, the positive rate is 43.7%, and the clustering is very obvious.

[0058] In summary, as the concentration of the antibody of this invention increases, the fluorescence intensity of HI100-FITC decreases in a dose-dependent manner, indicating that the antibody of this invention can competitively bind to commercially available HI100 antibodies. The anti-human CD45RA antibody prepared in this invention recognizes the same or spatially overlapping antigenic epitopes as commercially available HI100 antibodies, and the two exhibit direct competition, confirming the antigen specificity of the antibody of this invention.

[0059] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An anti-human CD45RA antibody or its antigen-binding fragment, characterized in that, The antibody comprises a heavy chain and a light chain, wherein the heavy chain CDR1 sequence is shown in SEQ ID NO: 13, the heavy chain CDR2 sequence is shown in SEQ ID NO: 15, and the heavy chain CDR3 sequence is shown in SEQ ID NO: 17; the light chain CDR1 sequence is shown in SEQ ID NO: 5, the light chain CDR2 sequence is shown in SEQ ID NO: 7, and the light chain CDR3 sequence is shown in SEQ ID NO:

9.

2. The anti-human CD45RA antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 11, and the light chain variable region sequence of the antibody is shown in SEQ ID NO:

3.

3. The anti-human CD45RA antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antigen-binding fragment is in the form of Fab, F(ab')2, or scFv.

4. A nucleic acid molecule, characterized in that, Encodes the anti-human CD45RA antibody or its antigen-binding fragment as described in any one of claims 1-3.

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

6. A host cell, characterized in that, It includes the nucleic acid molecule of claim 4 or the expression vector of claim 5, or expresses the anti-human CD45RA antibody or its antigen-binding fragment of any one of claims 1-3.

7. An immunoassay kit, characterized in that, Includes the anti-human CD45RA antibody or its antigen-binding fragment as described in any one of claims 1-3.

8. A pharmaceutical composition, characterized in that, include 1) The anti-human CD45RA antibody or its antigen-binding fragment according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6; and 2) Pharmaceutically acceptable excipients.

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