Anti-CD45RO monoclonal antibody and application thereof in preparation of drugs for treating immune diseases

Abstract

The invention relates to an anti-CD45RO monoclonal antibody and application thereof in preparation of drugs for treating autoimmune diseases, the anti-CD45RO monoclonal antibody comprises an antibody heavy chain variable region and / or an antibody light chain variable region: a) the antibody heavy chain variable region comprises three complementary determining regions: CDR1, CDR2 and CDR3; and b) an antibody light chain variable region, wherein the antibody light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3. The invention also comprises application of the anti-CD45RO monoclonal antibody in preparation of drugs for treating immune diseases. The monoclonal antibody aiming at the CD45RO can specifically recognize and combine CD45RO molecules on the surfaces of the memory T cells, so that the activation and function of the memory T cells are blocked, and the inflammatory response of immune diseases is reduced by reducing the activity of the memory T cells, so that the symptoms of patients are improved. The compound is expected to be used for preparing medicines for treating immune diseases such as nephrotic syndrome.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibodies, specifically to anti-CD45RO monoclonal antibodies and their application in the preparation of drugs for treating immune diseases such as nephrotic syndrome (NS) and systemic lupus erythematosus (SLE). Background Technology

[0002] Nephrotic syndrome, systemic lupus erythematosus, and other autoimmune diseases are caused by abnormalities in B and T cell immunity. Studies have found that the total number of B cells increases significantly during the onset or relapse of nephrotic syndrome, with CD27 cells being particularly prominent. + Memory B cells increased more than 10-fold. The disease was cured after 24 months of treatment with a novel rituximab regimen for nephrotic syndrome; CD27 levels increased after rituximab treatment. + Memory B cells are cleared and then gradually rise to return to normal levels; however, each relapse of nephrotic syndrome and systemic lupus erythematosus is preceded by an increase in memory B cells. Therefore, CD27 + Memory B cells are the pathogenic cells in nephrotic syndrome and systemic lupus erythematosus. CD27 + Memory B cells, through specific antigen recognition, target glomerular podocytes or other organ target cells, leading to damage to podocytes and other organ cells, resulting in massive proteinuria and multi-organ damage. Rituximab-induced sustained B-cell clearance for approximately 6 months allows for the continued or permanent discontinuation of steroids; sustained B-cell clearance for more than 2 years allows for the discontinuation of rituximab, ultimately achieving relapse-free nephrotic syndrome and systemic lupus erythematosus remission maintained solely by mycophenolate mofetil and hydroxychloroquine without steroids. The mechanism is related to the prolonged natural decay of T cells during sustained B-cell clearance. Our study found that during treatment, CD45RO... + Memory T cells gradually and passively decline, while other studies have found a significant increase in CD45RO+ memory T cells during systemic lupus erythematosus activity. Our research on nephrotic syndrome has also revealed a series of findings related to CD45RO+. + Signs associated with memory T cells. Basic research has identified CD45RO in nephrotic syndrome. + Memory T cells drive excessive activation of extrafollicular B cells in lymph nodes, while simultaneously inhibiting germinal center responses, leading to CD27... + Abnormal proliferation of memory B cells; while in systemic lupus erythematosus, CD45R + Memory T cells simultaneously enhance both extrafollicular and germinal center responses, leading to the proliferation of memory B cells (including CD27) from both extrafollicular and germinal center sources. + Subgroups) increased together; therefore, CD45RO +Memory T cells are key regulators driving the pathological activities of nephrotic syndrome and systemic lupus erythematosus. Blocking memory T cells can significantly improve the prognosis of these diseases and even achieve a cure, avoiding the long-term use of hormones and their related side effects.

[0003] Currently, treatment strategies targeting memory T cells mainly rely on non-specific immunosuppressants or broad-spectrum antibodies, which generally suffer from insufficient specificity and significant side effects. Memory T cells refer to CD3+ cells. + In T lymphocytes, some T cells, including CD4, express the 45R antigen on their cell membrane surface as 45RO. + / CD8 + 45RO + Memory T cells. CD45RO + The regulation of memory T cell production is closely related to CD45R antigen expression. The extracellular portion of the CD45R antigen consists of three parts: A, B, and C. Cells expressing all three (CD45ABC) are B cells; those expressing only CD45RA or CD45RB are naive T cells; and those expressing none of A, B, or C are CD45RO. + These are memory T cells. CD45RO, as a characteristic surface molecule of memory T cells, has been shown to effectively inhibit the activation process of memory T cells through specific blocking, demonstrating significant therapeutic potential in the treatment of autoimmune diseases (such as nephrotic syndrome and systemic lupus erythematosus) and transplant rejection. However, current technologies lack highly efficient and specific monoclonal antibodies (especially humanized antibodies) targeting human CD45RO, as well as antibody-drug conjugates (ADCs) developed based on it.

[0004] Therefore, this invention aims to provide a humanized anti-CD45RO monoclonal antibody and further synthesize ADC drugs. The development of this antibody will not only effectively fill this technological gap, but is also expected to significantly block the activation of memory T cells by specifically targeting the CD45RO molecule (which can be further applied to ADC construction). By reducing the activity of memory T cells, it will reduce the inflammatory response in autoimmune diseases, thereby improving patient symptoms; thus providing a novel, safer, and more effective precision treatment strategy for autoimmune diseases such as nephrotic syndrome and systemic lupus erythematosus. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art, provide an anti-CD45RO monoclonal antibody, and further humanize it for use in blocking human memory T cells, so as to fill the gap in the prior art of not having a target for blocking CD45RO memory T cells.

[0006] Another technical problem that this invention aims to solve is to synthesize ADC antibodies using CD45RO monoclonal antibodies to increase the blocking effect.

[0007] Another technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an application of anti-CD45RO monoclonal antibody in the preparation of drugs for treating immune diseases.

[0008] In a first aspect of the invention, a heavy chain variable region of an antibody is provided, said heavy chain variable region having three complementarity-determining regions (CDRs) selected from the group consisting of:

[0009] (a) CDR1, CDR2 and CDR3 shown in SEQ ID No: 2, 3 and 4;

[0010] Or (b) CDR1, CDR2 and CDR3 as shown in SEQ ID No: 10, 11 and 12.

[0011] In another preferred embodiment, the heavy chain variable region includes four skeleton regions FR, which are separated by the aforementioned CDR1, CDR2 and CDR3.

[0012] In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No:1.

[0013] In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No:9.

[0014] In a second aspect of the invention, a light chain variable region of an antibody is provided, said light chain variable region having three complementarity-determining regions (L-CDRs) with sequences as shown in SEQ ID No: 6, 7, and 8;

[0015] Alternatively, the light chain variable region may have three complementary determinant regions (L-CDRs) as shown in SEQ ID No: 14, 15, and 16.

[0016] In another preferred embodiment, the light chain variable region includes four skeleton regions FR, which are separated by the aforementioned CDR1, CDR2 and CDR3.

[0017] In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No:5.

[0018] In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No:13.

[0019] In a third aspect of the invention, an antibody is provided, the antibody having:

[0020] (1) the heavy chain variable region as described in the first aspect of the invention; and / or

[0021] (2) The light chain variable region as described in the second aspect of the present invention.

[0022] In another preferred embodiment, the antibody has a heavy chain variable region as shown in SEQ ID No:9; and / or as shown in SEQ ID No:9.

[0023] The light chain variable region shown in ID No:13.

[0024] In another preferred embodiment, the antibody is a humanized antibody, a chimeric antibody, or a murine antibody.

[0025] In another preferred embodiment, the antibody is a high-affinity binding CD45RO.

[0026] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0027] In another preferred embodiment, the antibody is a monoclonal antibody.

[0028] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0029] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0030] In a fourth aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0031] (i) a heavy chain variable region as described in the first aspect of the invention, a light chain variable region as described in the second aspect of the invention, or an antibody as described in the third aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0032] In another preferred embodiment, the tag sequence includes a 6His tag.

[0033] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0034] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0035] In a fifth aspect of the invention, an antibody preparation is provided, the antibody preparation comprising:

[0036] (a) the antibody as described in the third aspect of the present invention; and

[0037] (b) Carrier or excipient.

[0038] In another preferred embodiment, the preparation is a pharmaceutical composition.

[0039] In another preferred embodiment, the excipient or carrier is a pharmaceutically acceptable carrier or excipient.

[0040] In another preferred embodiment, the carrier comprises: a buffer, sterile water, and optionally a surfactant.

[0041] In another preferred embodiment, the buffer is selected from the group consisting of PBS buffer systems, citrate buffer systems, histidine buffer systems, or combinations thereof.

[0042] In another preferred embodiment, the pH range of the formulation is 5.0-7.5, more preferably 5.5-7.

[0043] In another preferred embodiment, the formulation is an injectable formulation.

[0044] In a sixth aspect of the invention, a kit is provided containing the antibody described in the third aspect of the invention, and a container holding the antibody.

[0045] In a seventh aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising:

[0046] (a) An antibody portion, said antibody portion being selected from the group consisting of: heavy chain variable regions as described in the first aspect of the invention, light chain variable regions as described in the second aspect of the invention, or antibodies as described in the third aspect of the invention, or combinations thereof; and

[0047] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0048] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0049] In an eighth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: the heavy chain variable region and heavy chain as described in the first aspect of the invention, the light chain variable region and light chain as described in the second aspect of the invention, or an antibody as described in the third aspect of the invention, a recombinant protein as described in the fourth aspect of the invention, an antibody-drug conjugate as described in the seventh aspect of the invention, or a combination thereof, wherein the active ingredient is used for

[0050] (a) Preparation of detection reagents or kits;

[0051] (b) To prepare medicines or formulations for the prevention and / or treatment of CD45RO-related diseases.

[0052] The CD45RO-related diseases are selected from the following group: nephrotic syndrome, systemic lupus erythematosus, or other combinations.

[0053] In another preferred embodiment, the drug or formulation is used to prepare a drug or formulation for the prevention and / or treatment of diseases associated with CD45RO (positive expression).

[0054] In another preferred embodiment, the antibody is in the form of a drug conjugate (ADC).

[0055] In another preferred embodiment, the detection reagent or kit is used to diagnose CD45RO-related diseases.

[0056] In another preferred embodiment, the detection reagent or kit is used to detect CD45RO protein in a sample.

[0057] In another preferred embodiment, the detection reagent is a detection strip.

[0058] In a ninth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0059] (i) an active ingredient selected from the group consisting of: heavy chain variable regions as described in the first aspect of the invention, light chain variable regions as described in the second aspect of the invention, antibodies as described in the third aspect of the invention, recombinant proteins as described in the fourth aspect of the invention, antibody-drug conjugates as described in the seventh aspect of the invention, or combinations thereof; and

[0060] (ii) Pharmaceutically acceptable carriers.

[0061] In another preferred embodiment, the second active ingredient is selected from the group consisting of cytotoxic drugs, toxins, cytokines, enzymes, antibodies, or combinations thereof.

[0062] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0063] In another preferred embodiment, the pharmaceutical composition is an injection.

[0064] In another preferred embodiment, the pharmaceutical composition is used to treat autoimmune diseases.

[0065] In a tenth aspect of the invention, a polynucleotide is provided, said polynucleotide encoding a polypeptide selected from the group consisting of:

[0066] (1) The heavy chain variable region as described in the first aspect of the present invention, the light chain variable region as described in the second aspect of the present invention, or the antibody as described in the third aspect of the present invention; or the recombinant protein as described in the fourth aspect of the present invention.

[0067] In an eleventh aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the tenth aspect of the invention.

[0068] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0069] In a twelfth aspect of the invention, a genetically engineered host cell is provided, said host cell containing a vector as described in an eleventh aspect of the invention or a genome in which polynucleotides as described in a tenth aspect of the invention are integrated.

[0070] In a thirteenth aspect of the invention, a method for in vitro detection of CD45RO protein in samples (including diagnostic or non-diagnostic samples) is provided, the method comprising the steps of:

[0071] (1) In vitro, the sample is contacted with the antibody as described in the third aspect of the present invention;

[0072] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD45RO protein in the sample.

[0073] In a fourteenth aspect of the invention, a kit is provided, the kit comprising:

[0074] (1) A first container containing an antibody as described in the third aspect of the present invention; and / or

[0075] (2) A second container containing a secondary antibody against the antibody as described in the fifth aspect of the present invention;

[0076] In a fifteenth aspect of the invention, a method for treating CD45RO-related diseases, such as nephrotic syndrome, systemic lupus erythematosus, or other autoimmune diseases or cancer, is provided, the method comprising: administering to a desired subject an antibody as described in a third aspect of the invention, an antibody-drug conjugate of the antibody, or CAR-T cells expressing the antibody, or a combination thereof.

[0077] In another preferred embodiment, the object includes humans and non-human mammals.

[0078] In another preferred embodiment, the object is a person.

[0079] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0080] This invention achieves the following beneficial effects: It discloses a monoclonal antibody targeting CD45RO. The antibody binds to the CD45RO molecule on the surface of memory T cells, specifically recognizing CD45RO and thereby specifically blocking the activation and function of memory T cells. By reducing the activity of memory T cells, it lowers the inflammatory response in immune diseases, thus improving patient symptoms. It holds promise for use in the preparation of drugs for treating immune diseases such as nephrotic syndrome. Attached Figure Description

[0081] Figure 1 These are SDS-PAGE and Western blot images of the antigen-human CD45RO of this invention: In the figures, A. Lane M: protein marker, Lane R: reduced protein conditions, Lane NR: unreduced protein conditions. B. Lane M: protein marker, Lane P: mouse anti-His monoclonal antibody (GenScript, Cat. No. A00186) as a positive control, Lane R: reduced protein conditions. B. Lane NR: unreduced protein conditions.

[0082] Figure 2 This is a three-dimensional structural diagram of the anti-CD45RO monoclonal antibody of the present invention;

[0083] Figure 3 This is a three-dimensional diagram of the binding between the anti-CD45RO monoclonal antibody and the antigen of this invention;

[0084] Figure 4 This is a diagram showing the binding effect of the CD45RO monoclonal antibody to CD45RO on the surface of human T cells detected by FACS in this invention. Detailed Implementation

[0085] Through extensive and in-depth research and screening, the inventors obtained several murine antibodies with excellent properties, including high affinity for human CD45RO. Based on these high-affinity murine antibodies, chimeric antibodies and humanization modifications were further developed. The antibodies of this invention can effectively bind to human CD45RO and can be used as monoclonal antibody drugs for targeted therapy. This invention was completed based on these findings.

[0086] The present invention will be further described below with reference to specific embodiments.

[0087] Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available products.

[0088] Example 1: Production of anti-human CD45RO mouse monoclonal antibody

[0089] 1.1 The following reagents, cells, and antibodies are used in antibody production:

[0090]

[0091] All the reagents, cells, and antibodies mentioned above are commercially available products and are from Genescript Biotechnology Co., Ltd.

[0092] 1.2 Preparation method of monoclonal antibodies

[0093] Phase 1: Antigen Preparation

[0094] 1. Cloning scheme design

[0095] EcoRI-Kozak sequence-gp67-PTPRC(CD45RO)-His-stop codon-HindIII

[0096] Protein Length=391 MW=43566.70 Predicted pI=5.69

[0097] Antigen amino acid sequence: as shown in SEQ ID No: 21:

[0098] QSPPTPSPTDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNA SKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK

[0099] 2. The amino acid sequence synthesized in step 1 and the corresponding base sequence are cloned into plasmid pcDNA3.4.

[0100] The plasmid was transfected into HD293F cells, and after culturing in culture medium, the supernatant was collected for SDS-PAGE verification (see experimental results). Figure 1 ), Figure 1 This is an SDS-PAGE and Western blot image of the human CD45RO (PTPRC) antigen of this invention; A. Lane M: Protein Marker, Lane R: Protein reduction conditions, Lane NR: Protein non-reduction conditions. B. Lane M: Protein Marker, Lane P: Mouse anti-His monoclonal antibody (GenScript, Cat. No. A00186) as a positive control, Lane R: Protein reduction conditions, Lane NR: Protein non-reduction conditions. Experimental results show that after HD293F cells express the human CD45RO antigen and secrete it into the culture medium, SDS-PAGE of the culture medium containing human CD45RO shows clear bands. Western blot detection using an anti-HIS tag monoclonal antibody as the primary antibody shows two bands at the same position, proving that both bands are human CD45RO bands, and the antigen expression is successful.

[0101] Phase Two: Animal Immunization

[0102] CD45RO protein was used as an immunogen, and three BALB / c mice were immunized using standard methods. The specific animal immunization procedure is shown in Table 1.

[0103] Table 1. Animal Immunization Procedure

[0104]

[0105] 1. Blood samples were collected for ELISA testing starting 7 days after the second immunization. Protein and His-related proteins were detected indirectly using plate-coated ELISA.

[0106] 2. After the third immunization, serum samples are tested. If the serum titer meets the fusion requirements for protein detection using ELISA (OD value > 1.0 at a 1:8000 dilution), the process continues. If the immunogenicity is low and does not meet the requirements, an improvement plan is implemented.

[0107] Phase 3: Cell Fusion and Screening

[0108] The experimental animals used for fusion are determined based on the results of immune testing.

[0109] 1. Cell fusion and plating: Two mice with the best titers were selected for fusion. The fusion efficiency can reach approximately 2000 B cells to produce one hybridoma cell. Each fusion was plated into 15 96-well plates.

[0110] 2. Initial screening: Protein was used to coat the cells, and indirect ELISA was used to screen the supernatant of fused cells.

[0111] 3. Confirmatory screening: The positive supernatant obtained from the initial screening is selected and subjected to a second ELISA test with proteins. His-irrelevant proteins are coated for reverse screening to identify positive cell lines specifically targeting the target protein.

[0112] Phase 4: Subcloning, Preservation and Cryopreservation

[0113] 1. After the above fusion, the supernatant of the maternal clone was subjected to Western blotting. Five maternal clones were selected based on specificity, and a first round of subcloning was performed using the limiting dilution method.

[0114] 2. After one round of subcloning, the supernatant is further tested. Based on the results, the limiting dilution method is used to determine the subcloning line. Each round of subcloning is tested by ELISA to select cell lines that specifically target the target protein.

[0115] 3. For each selected maternal clone, two daughter clones will be retained for expansion culture, cell cryopreservation, and subtype identification.

[0116] Table 2.13G3-1, 15B10-1, and 19E6-2 cell lines produced subclonal supernatant ELISA

[0117]

[0118] Phase 5: Antibody Production

[0119] 1. Five cell lines were cultured in roller bottles for small-scale antibody production.

[0120] 2. Affinity purification was performed using a protein A / G column, with an expected yield of 2-5 mg of antibody per cell line.

[0121] 3. The purified antibody will be tested by ELISA to determine the antibody titer. Table 3 shows the titration results of ELISA after purification of the 13G3-1 cell line.

[0122] Table 3.13 shows the purified antibodies produced by the G3-1 cell line for ELISA.

[0123]

[0124] ELISA coating antigens: A is CD45RO protein; B is His-tagged protein;

[0125] Coating concentration: 1ug / ml, 100ul / well

[0126] Coating buffer: Phosphate buffer at pH 7.4

[0127] Secondary antibody: peroxidase-labeled goat anti-mouse IgG, Fcγ-specific fragment

[0128] The initial concentration is 1 ug / ml, and the dilution factor is based on the actual concentration.

[0129] The OD value of the blank hole is the average of the two duplicate holes.

[0130] The titer is the highest dilution concentration when the OD value of the test wells / blank wells is >= 2.1.

[0131] Example 2: Sequence of a monoclonal antibody

[0132] 1. RNA extraction

[0133] RNA was extracted from approximately 1–2 × 10⁵ hybridoma cells (Vazyme, RNA Isolation Kit, catalog number: RC112-01). The concentration was then detected using Nanodrop.

[0134] 2. cDNA Acquisition

[0135] mRNA reverse transcription was performed using 5' RACE (rapid amplification of the 5' end of cDNA). A universal sequence was introduced at the 3' end of the cDNA (5' end of the transcript) using template-conversion oligonucleotides (TSO). VH and VL antibody fragments were amplified using constant region (CH1) primers and TSO adapter-specific primers according to the GenScript NGS standard operating procedure. After obtaining the target bands, the PCR products were purified using an agarose gel DNA recovery kit.

[0136] 3. NGS Library Construction and Hybridization

[0137] NGS libraries were constructed using sample-specific barcode primers. The concentration of recovered products was determined, and all samples were mixed in equal volumes to create two libraries: a heavy chain (VH) library and a light chain (VL) library.

[0138] 4. Miseq sequencing and data analysis

[0139] Sequencing was performed using the Miseq platform to ensure that each sample had 10 million reads of VH or VL sequence data. Based on the barcode primer sequence splitting results, the most effective antibody sequences with the highest sequencing depth were selected. Quality control (QC) was performed on the NGS data, and sequence information was summarized.

[0140] In the early stages of this invention, mouse hybridoma cells that produced antibodies binding to CD45RO were screened, namely strains 13G3-1, 15B10-1, and 19E6-2. Each clone was further cloned, and the V region sequences of the heavy and light chains were analyzed.

[0141] Results: The obtained heavy and light chain PCR bands were bright and of the correct size, and the sequencing reaction peaks were normal and uniform. NCBIBlast results showed normal antibody V region sequence characteristics, i.e., a complete Framework region, CDR region, no stop codon, and rearrangement of the VDJ / VJ gene. The V regions of all antibodies were successfully obtained. For the 13G3-1 and 15B10-1 sequences, the VH chain FR1, FR2, FR3, FR4 and CDR1, CDR2 regions were completely identical, with only one base difference in the CDR3 region. For the VL chain, the FR1, FR2, FR3 and CDR1, CDR3 regions were completely identical, with only one base difference in the CDR2 and FR4 regions. Specific sequences are shown in Table 4 below.

[0142] Table 4

[0143]

[0144] Anti-CD45RO monoclonal antibody 13G3-1, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is (142aa) SEQ ID No: 1, as shown below:

[0145] 1 Met Tyr Leu Gly Leu Asn Cys Val Phe Ile Val Phe Leu Leu Lys

[0146] 16 Gly Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu

[0147] 31 Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly

[0148] 46 Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ser Pro

[0149] 61 Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Asn

[0150] 76 Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr

[0151] 91 Ile Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn

[0152] 106 Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Ser Leu

[0153] 121 Ile Thr Ala Val Asp Tyr Tyr Ala Val Asp Tyr Trp Gly Gln Gly

[0154] 136 Thr Ser Val Thr Val Ser Ser

[0155] The amino acid sequence of the light chain variable region is (133 aa) SEQ ID No: 5, as follows:

[0156] 1 Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp Val

[0157] 16 Ser Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser

[0158] 31 Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser

[0159] 46 Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr

[0160] 61 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

[0161] 76 Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

[0162] 91 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln

[0163] 106 Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr

[0164] 121 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[0165] Furthermore, the heavy chain DNA sequence of the monoclonal antibody is (426bp), SEQ ID No: 17, as follows:

[0166] ATGTACTTGGGACTGAACTGTGTATTCATAGTTTTTCTCTTAAAAGGTGT

[0167] CCAGAGTGAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTG

[0168] GAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAAC

[0169] TACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGT

[0170] TGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGT

[0171] CTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTC

[0172] TACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTG

[0173] TACCAGTCTTATCACTGCGGTAGATTACTATGCTGTGGACTACTGGGGTC

[0174] AAGGAACCTCAGTCACCGTCTCCTCA。

[0175] Furthermore, the light chain DNA sequence of the monoclonal antibody is (399bp) SEQ ID No: 18, as shown below:

[0176] ATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGGTATCTGG

[0177] TACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGA

[0178] CAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTA

[0179] AACAGTGGAAATCAAAAGAATTACTTGACCTGGTACCAGCAGAAACCAGG

[0180] GCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGG

[0181] TCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACC

[0182] ATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGA

[0183] TTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0184] Another preferred embodiment is the heavy chain variable region amino acid sequence of the anti-CD45RO monoclonal antibody 19E6-2, which is (139aa) SEQ ID No: 9, as shown below:

[0185] 1 Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala

[0186] 16 Gly Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

[0187] 31 Met Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Arg Ala Thr Gly

[0188] 46 Tyr Thr Phe Ser Ser Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro

[0189] 61 Gly His Gly Leu Glu Trp Ile Gly Glu Ile Phe Pro Gly Ser Gly

[0190] 76 Thr Thr Asn Tyr Ser Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr

[0191] 91 Ala Asp Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu

[0192] 106 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Phe Ser Tyr

[0193] 121 Arg Asn Asp Trp Phe Phe Asp Val Trp Gly Ala Gly Thr Thr Val

[0194] 136 Thr Val Ser Ser

[0195] Another preferred embodiment has the following amino acid sequence in the light chain variable region: (127 aa) SEQ ID No: 13, as shown below:

[0196] 1 Met Glu Phe Gln Thr Gln Val Phe Val Phe Val Leu Leu Trp Leu

[0197] 16 Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe

[0198] 31 Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala

[0199] 46 Ser Gln Asn Val Arg Thr Thr Val Ala Trp Tyr Gln Gln Lys Pro

[0200] 61 Gly Gln Ser Pro Lys Ala Leu Ile Tyr Leu Ala Ser Asn Arg His

[0201] 76 Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

[0202] 91 Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp

[0203] 106 Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu Thr Phe Gly Ala

[0204] 121 Gly Thr Lys Leu Glu Leu Lys

[0205] Another preferred embodiment is that the heavy chain DNA sequence of the monoclonal antibody is (bp), SEQ ID No: 19, as shown below:

[0206] ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGT

[0207] CCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTG

[0208] GGGCCTCAGTGAAGATTTCCTGCAGGGCTACTGGCTACACATTCAGTAGC

[0209] TACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGAT

[0210] TGGAGAGATTTTTCCTGGAAGTGGTACTACTAACTACAGTGAGAAGTTCA

[0211] AGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACGGCCTACATG

[0212] CAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAG

[0213] ATTCTCCTATAGGAACGACTGGTTCTTCGATGTCTGGGGCGCAGGGACCA

[0214] CGGTCACCGTCTCCTCA

[0215] Another preferred embodiment is that the light chain DNA sequence of the monoclonal antibody is (381 bp) SEQ ID No: 20, as shown below:

[0216] ATGGAGTTTCAGACCAGGTCTTTGTATTCGTGTTGCTCTGGTTGTCTGG

[0217] TGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACAT

[0218] CAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTTCGT

[0219] ACTACTGTAGCCTGGTATCAACAGAAACCAGGGCAGTCTCCTAAAGCACT

[0220] GATTTACTTGGCATCCAACCGGCACACTGGAGTCCCTGATCGCTTCACAG

[0221] GCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAATCT

[0222] GAAGACCTGGCAGATTATTTCTGTCTGCAACATTGGAATTATCCGCTCAC

[0223] GTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0224] Example 3: FACS detection of the binding of CD45RO monoclonal antibody to CD45RO protein on the surface of human T lymphocytes

[0225] To determine whether the murine monoclonal antibody disclosed herein binds to the CD45RO protein on the surface of human T lymphocytes, an in vitro test was performed. A commercially available antibody was used as a positive control.

[0226] Collect fresh human blood and transfer peripheral blood to flow cytometry tubes (100 μL per tube, 1 × 10^6 cells recommended). Add the following antibodies for this experiment: commercial antibody (control), 13G3-1, 15B10-1 (similar), and 19E6-2, to a final concentration of 5 μg / mL. Incubate at room temperature (20-25°C) in the dark for 30 minutes. Add CD3 and CD4 antibodies and incubate in the dark for 15 minutes. Add 1.5 mL of hemolysin and hemolyze for 5 minutes. Centrifuge (1700 rpm, 5 minutes, 4°C), discard the supernatant, and vortex. Add 2 mL of wash buffer and centrifuge (400 × g, 5 minutes, 4°C), discard the supernatant. Repeat washing twice to remove unbound primary antibodies. Add 100 μL of diluted APC anti-mouse IgG1 Antibody (1:100 dilution) and gently mix to resuspend the cells. Incubate at room temperature in the dark for 30 minutes (APC fluorescence is easily quenched, avoid prolonged exposure). Add 2 mL of washing buffer, centrifuge (400 × g, 5 min, 4°C), and discard the supernatant. Repeat washing twice to thoroughly remove unbound secondary antibody (reduce background noise). Add 450 μL of washing buffer (or PBS), gently mix to resuspend cells evenly. Perform assays.

[0227] The results showed that CD45RO expression was high on CD3+CD4+ T cells, approximately 80-95%. All three clonal antibodies could bind well to CD45RO on the surface of T cells, and the staining effect was comparable to that of the control antibody.

Claims

1. An anti-CD45RO monoclonal antibody, characterized in that, The monoclonal antibody contains the following antibody heavy chain variable region. and / or antibody light chain variable regions: a) The antibody heavy chain variable region comprises the following three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3, wherein: CDR1 is the amino acid sequence shown in SEQ ID NO:

2. CDR2 is the amino acid sequence shown in SEQ ID NO:

3. CDR3 is the amino acid sequence shown in SEQ ID NO:4; Alternatively, CDR1 may be an amino acid sequence as shown in SEQ ID NO:

10. CDR2 is the amino acid sequence shown in SEQ ID NO:

11. CDR3 is the amino acid sequence shown in SEQ ID NO:12; b) The variable region of the antibody light chain comprises the following three complementarity-determining regions (L-CDRs): L-CDR1, L-CDR2, and L-CDR3, wherein: L-CDR1 has the amino acid sequence shown in SEQ ID NO:

6. L-CDR2 has the amino acid sequence shown in SEQ ID NO:

7. L-CDR3 has the amino acid sequence shown in SEQ ID NO:

8. Alternatively, L-CDR1 may have the amino acid sequence shown in SEQ ID NO:

14. L-CDR2 has the amino acid sequence shown in SEQ ID NO:

15. L-CDR3 is the amino acid sequence shown in SEQ ID NO:

16.

2. An antibody heavy chain, characterized in that, The antibody heavy chain has the antibody heavy chain variable region as described in claim 1 or has the amino acid sequence shown in SEQ ID No:1 or SEQ ID No:

9.

3. An antibody light chain, characterized in that, The antibody light chain has the antibody light chain variable region as described in claim 1 or has the amino acid sequence shown in SEQ ID No:5 or SEQ ID No:

13.

4. A murine antibody, chimeric antibody, or humanized antibody, characterized in that, The antibody has the following characteristics: 1) The heavy chain variable region as described in claim 1 and / or the light chain variable region as described in claim 1; Alternatively, the antibody may have: the antibody heavy chain as described in claim 2; and / or the antibody light chain as described in claim 3.

5. A DNA molecule encoding the anti-CD45RO monoclonal antibody as described in claims 1-4, characterized in that: The nucleic acid sequence encoding the antibody heavy chain is shown in SEQ ID NO: 17 or SEQ ID NO: 19; the nucleic acid sequence encoding the antibody light chain is shown in SEQ ID NO: 18 or SEQ ID NO:

20.

6. The use of an anti-CD45RO monoclonal antibody as described in any one of claims 1-4 in the preparation of a medicament for treating immune diseases.

7. The use of the anti-CD45RO monoclonal antibody according to claim 6 in the preparation of drugs for treating immune diseases, characterized in that, The immune disease mentioned is nephrotic syndrome or systemic lupus erythematosus.

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