An anti-human cd45 antibody or antigen-binding fragment thereof and methods of making the same

Abstract

The application belongs to the technical field of biology and provides an anti-human CD45 antibody or antigen binding fragment thereof, wherein the light chain complementarity determining region has an amino acid sequence as shown in SEQ ID NO:1-6 or has at least one amino acid difference compared with the amino acid sequence; the heavy chain complementarity determining region has an amino acid sequence as shown in SEQ ID NO:7-12 or has at least one amino acid difference compared with the amino acid sequence. The anti-human CD45 antibody or antigen binding fragment can specifically recognize a human CD45 antigen, and the affinity and antibody specificity are close to those of positive antibody CD45 [2D1]. The preparation method of the anti-human CD45 antibody provided by the application does not need to be prepared by animal hybridoma, so that the steps are simple, the antibody gene is not easy to be lost, and the antibody expression yield is much higher than that of the conventional hybridoma cell for preparing the antibody.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-human CD45 antibody or its antigen-binding fragment and its preparation method. Background Technology

[0002] Traditionally, antibodies for scientific research and diagnostic testing are prepared using animal hybridomas. However, due to the instability of hybridomas, antibody genes are easily lost during preparation. Furthermore, traditional hybridoma preparation techniques do not identify the gene and protein sequences of the target antibody, hindering subsequent production, preparation, and research.

[0003] CD45 is a key molecule in cell membrane signal transduction, playing a crucial role in lymphocyte development, maturation, functional regulation, and signal transduction. Some reports indicate that, in addition to mature erythrocytes and platelets, CD45 is expressed in bone marrow progenitor cells, mature cells in peripheral blood and lymph nodes, over 90% of myeloid leukemia cells, and most lymphoid leukemia and lymphoma cells. However, it is not expressed in non-hematopoietic tissues, a characteristic that makes it an ideal antigen for studying targeted therapies for malignant hematologic disorders.

[0004] CD45 plays a crucial role in the differential diagnosis and immunophenotyping of leukemia. It can be used to identify minimal residual disease in leukemia. Currently, anti-CD45 monoclonal antibodies are mainly used for immunophenotyping in hematological diseases. Because CD45 is widely and highly expressed in almost all leukemia specimens, and exhibits no internalization effect after binding to antibodies, and importantly, is not expressed in non-hematopoietic tissues, it has become the preferred antigen for targeted radioimmunotherapy in hematopoietic stem cell patients.

[0005] Chinese patent application 202080086243.5 discloses an anti-CD45 antibody that specifically binds to human CD45, its antigen-binding fragment, and an antibody-drug conjugate (ADC). These antibodies and ADCs can be used in treatment methods, including methods that deplete a patient's CD45+ cells.

[0006] US Patent Application US202318153181 discloses a humanized antibody molecule that binds to CD45, and a construct, such as an antibody molecule containing a binding domain specifically targeting CD45, and a construct, such as an antibody molecule containing a binding domain specifically targeting CD45.

[0007] There is currently a need in this field for more anti-human CD45 monoclonal antibodies. Summary of the Invention

[0008] To achieve the above-mentioned technical objectives, the present invention proposes the following technical solution:

[0009] In a first aspect, the present invention provides an anti-human CD45 antibody or an antigen-binding fragment thereof, wherein the anti-human CD45 antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region;

[0010] The variable region of the light chain has:

[0011] (1) The amino acid sequences of LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:3; or

[0012] (2) The amino acid sequences of LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; or

[0013] (3) LCDR1 having at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:1, LCDR2 having at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:2, and LCDR3 having at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:3; or

[0014] (4) LCDR1, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:4; LCDR2, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:5; and LCDR3, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:6.

[0015] The heavy chain variable region has:

[0016] 1) HCDR1 with amino acid sequences as shown in SEQ ID NO:7, HCDR2 with amino acid sequences as shown in SEQ ID NO:8, and HCDR3 with amino acid sequences as shown in SEQ ID NO:9; or

[0017] 2) HCDR1 with amino acid sequences as shown in SEQ ID NO:10, HCDR2 with amino acid sequences as shown in SEQ ID NO:11, and HCDR3 with amino acid sequences as shown in SEQ ID NO:12; or

[0018] 3) HCDR1, which differs from the amino acid sequence shown in SEQ ID NO:7 by at least one amino acid; HCDR2, which differs from the amino acid sequence shown in SEQ ID NO:8 by at least one amino acid; and HCDR3, which differs from the amino acid sequence shown in SEQ ID NO:9 by at least one amino acid; or

[0019] 4) HCDR1, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:10; HCDR2, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:11; and HCDR3, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:12.

[0020] The amino acid differences mentioned above are achieved through the addition, substitution, deletion, replacement, or modification of amino acids.

[0021] In some embodiments, the light chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:13, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:13; or

[0022] The light chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:14, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:14.

[0023] In some embodiments, the heavy chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:15, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:15; or

[0024] The heavy chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:16, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:16.

[0025] In some embodiments, the light chain of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:17, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:17; or

[0026] The light chain of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:18, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:18.

[0027] In some embodiments, the heavy chain of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:19, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19; or

[0028] The heavy chain of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:20, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:20.

[0029] SEQ ID NO: 1: SASSSVSYMH; SEQ ID NO: 2: GTSNLAS; SEQ ID NO: 3: HQWNGYPWT; SEQ ID NO: 4: RSSSKSLLHSSGITYLY; SEQ ID NO: 5: RMSNLAS; SEQ ID NO: 6: MQHLEYPFT;

[0030] SEQ ID NO:7: HYTIH; SEQ ID NO:8: YINPNSGFNNYNQKFKD; SEQ ID NO:9: YKGSHRYDYVLDY; SEQ ID NO:10: SYSIQ; SEQ ID NO:11: YINPSSGYIKYNQHFRD. SEQ ID NO: 12: NGSGSFDY.

[0031] SEQ ID NO:13:

[0032] QILLTQSPAIMSTFLGEEITLTCSASSSVSYMHWFQQKSGTSPKLLIYGTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWNGYPWTFGGGTKVEIKRA。

[0033] SEQ ID NO:14:

[0034] DIVMTQAAPSVPVTPGESLSISCRSSKSLLHSSGITYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLELKRA。

[0035] SEQ ID NO:15:

[0036] QVQLQQSGAELARPGASVKMSCKASGYTFTHYTIHWIKQRPGQGLEWIGYINPNSGFNNYNQKFKDKATVTADKSSNTAYLQLTSLTSEDSAVYYCARYKGSHRYDYVLDYWGQGTSVTVSS。

[0037] SEQ ID NO:16:

[0038] QVQLQQLGAELARPGASVKMSCKASGYTFTSYSIQWVKQRPGQGLEWIGYINPSSGYIKYNQHFR DRATLTADRSSSTAYMQLSSLTSEDSAVYYCARNGSGSFDYWGQGTTLTVSS。

[0039] SEQ ID NO:17:

[0040] QILLTQSPAIMSTFLGEEITLTCSASSSVSYMHWFQQKSGTSPKLLIYGTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWNGYPWTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0041] The nucleotide sequence encoding SEQ ID NO:17 is shown in SEQ ID NO:21:

[0042] CAGATCCTGCTGACACAGAGCCCTGCCATCATGAGCACCTTCCTGGGCGAGGAGATCACCCTGACCTGCAGCGCTAGCAGCAGCGTGAGCTACATGCACTGGTTTCAGCAGAAGAGCGGCACAAGCCCTAAGCTGCTGATCTACGGCACAAGCAACCTGGCTAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCTTCTACAGCCTGACCATCAGCAGCGTGGAGGCCGAGGACGCCGCCGACTACTACTGCCATCAGTGGAACGGCTACCCTTGGACCTTCGGCGGAGGCACCAAGGTGGAGATCAAGAGAGCCGACGCCGCCCCTACCGTGAGCATCTTCCCTCCTAGCAGCGAGCAGCTGACAAGCGGCGGCGCTAGCGTGGTGTGCTTCCTGAACAACTTCTACCCTAAGGACATCAACGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACAGCTGGACCGACCAAGACAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGAGACACAACAGCTACACCTGCGAGGCCACCCACAAGACAAGCACAAGCCCTATCGTGAAGAGCTTCAACAGAAACGAGTGC。

[0043] SEQ ID NO:18:

[0044] DIVMTQAAPSVPVTPGESLSISCRSSKSLLHSSGITYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0045] The nucleotide sequence encoding SEQ ID NO:18 is shown in SEQ ID NO:22:

[0046] GATATCGTGATGACCCAGGCTGCTCCATCTGTGCCTGTGACACCTGGCGAGTCCCTGTCTATCTCCTGCAGATCCTCCAAGTCTCTGCTGCACTCCTCTGGCATCACCTACCTGTACTGGTTCCTGCAGAGACCCGGCCAAAGCCCTCAGCTCCTGATCTACCGGATGTCCAACCTGGCCTCTGGCGTCCCCGATAGATTCTCCGGCAGCGGCTCCGGCACCGCCTTTACCCTGCGGATCTCTCGGGTGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGCATCTGGAATATCCCTTCACTTTCGGCGGTGGAACCAAGCTGGAACTGAAGAGAGCCGACGCCGCTCCTACAGTGTCCATCTTCCCTCCTAGCTCCGAGCAGCTGACCTCTGGAGGCGCCTCCGTGGTGTGCTTTCTGAACAACTTCTACCCTAAGGACATCAACGTGAAGTGGAAGATCGACGGCTCTGAACGGCAGAACGGCGTGCTGAATTCTTGGACCGACCAGGACTCCAAAGATTCTACCTACTCCATGTCTAGCACCCTGACACTGACCAAGGACGAGTACGAGAGACACAACTCCTACACCTGCGAGGCTACCCACAAGACCTCCACCAGTCCTATCGTCAAATCCTTCAACCGCAACGAGTGT.

[0047] SEQ ID NO:19:

[0048] QVQLQQSGAELARPGASVKMSCKASGYTFTHYTIHWIKQRPGQGLEWIGYINPNSGFNNYNQKFKDKATVTADKSSNTAYLQLTSLTSEDSAVYYCARYKGSHRYDYVLDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0049] The nucleotide sequence encoding SEQ ID NO:19 is shown in SEQ ID NO:23:

[0050]

[0051] SEQ ID NO:20:

[0052] QVQLQQLGAELARPGASVKMSCKASGYTFTSYSIQWVKQRPGQGLEWIGYINPSSGYIKYNQHFRDRATLTADRSSSTAYMQLSSLTSEDSAVYYCARNGSGSFDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK.

[0053] The nucleotide sequence encoding SEQ ID NO:20 is shown in SEQ ID NO:24:

[0054]

[0055] Secondly, the present invention provides an isolated polynucleotide encoding any of the aforementioned anti-human CD45 antibodies or their antigen-binding fragments.

[0056] Thirdly, the present invention provides a recombinant expression vector comprising the isolated polynucleotides.

[0057] In some implementations, the recombinant expression vector includes a eukaryotic expression vector and a prokaryotic expression vector.

[0058] Fourthly, the present invention provides a host cell comprising any of the aforementioned recombinant expression vectors.

[0059] Fifthly, the present invention provides a method for preparing any of the aforementioned anti-human CD45 antibodies or their antigen-binding fragments, the preparation method comprising the following steps:

[0060] (I) The isolated polynucleotide fragment or recombinant expression vector is introduced into host cells for culture, and after culturing the cells, the anti-human CD45 antibody or its antigen-binding fragment is purified and collected.

[0061] (II) Culturing any of the aforementioned host cells, and purifying and collecting anti-human CD45 antibodies or their antigen-binding fragments after culturing the cells.

[0062] In some implementations, the host cell includes a eukaryotic cell or a prokaryotic cell.

[0063] Sixthly, a method for detecting CD45 protein for non-disease diagnostic purposes, said detection method comprising:

[0064] I) Incubate the sample to be tested with any of the aforementioned anti-human CD45 antibodies or their antigen-binding fragments, or a fluorescently labeled antibody containing the aforementioned anti-human CD45 antibody or its antigen-binding fragments;

[0065] Ⅱ) Detect whether there are antibody-antigen conjugates in the sample, or detect the presence of fluorescence by flow cytometry.

[0066] This invention provides two anti-human CD45 antibodies and a genetic engineering-based preparation method. The two anti-human CD45 antibodies provided by this invention can specifically recognize the human CD45 antigen, and their affinity and antibody specificity are close to those of the positive antibody CD45[2D1]. The preparation method of the anti-human CD45 antibodies provided by this invention does not require preparation via animal hybridoma, thus simplifying the process and preventing the easy loss of antibody genes, thereby enabling large-scale preparation and providing more convenient conditions for the detection of CD56 protein. Attached Figure Description

[0067] Figure 1The SDS-PAGE analysis results of three anti-human CD45 antibodies (Biolegend CD45[2D1], CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01]) are shown.

[0068] Figures 2-7 The results of flow cytometry detection of three labeled antibodies (Biolegend CD45[2D1], CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01]) are shown.

[0069] Figures 8-13 Affinity test results for three anti-human CD45 antibodies (Biolegend CD45[2D1], CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01]) are shown.

[0070] Figure 14 The results of antibody-specific detection for CD45 [ZXClone-HCD45-04] are shown.

[0071] Figure 15 The results of antibody-specific detection for CD45 [ZXClone-HCD45-01] are shown.

[0072] Figure 16 The antibody-specificity detection results of the commercially available antibody CD45[2D1]-PE are shown. Detailed Implementation

[0073] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0074] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0075] As used herein, the term "antibody" refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies. The term "antibody" also refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) linked together by disulfide bonds (SS). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (VH). The light chain constant region consists of one domain: CL. Light chains are divided into two categories: kappa-type light chains and lambda-type light chains (for example, in this invention, the light chain constant region Cκ / λ indicates that the light chain constant region is a kappa-type light chain or a lambda-type light chain). The VH and VL regions can be further subdivided into hypervariable regions (also called complementarity-determining regions (CDRs)), interspersed with more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0076] As used herein, the term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody that typically include at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When the antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of an antibody may include conserved or non-conserved amino acid substitutions (referred to as "conserved variants" or "functionally conserved variants" of the antibody) that do not significantly alter its biological activity. The term "binding compound" refers to both the antibody and its binding fragment.

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0078] Example 1: Preparation and Detection of Antibodies

[0079] 1.1 Antibody Preparation

[0080] The inventors prepared two anti-human CD45 mouse antibodies, numbered ZXClone-HCD45-04 and ZXClone-HCD45-01, respectively.

[0081] The amino acid sequence of the light chain of CD45[ZXClone-HCD45-04] is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:13, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:15; in the light chain complementarity-determining region (LCDR), LCDR1 has the amino acid sequence shown in SEQ ID NO:1, LCDR2 has the amino acid sequence shown in SEQ ID NO:2, and LCDR3 has the amino acid sequence shown in SEQ ID NO:3; in the heavy chain complementarity-determining region (HCDR), HCDR1 has the amino acid sequence shown in SEQ ID NO:7, HCDR2 has the amino acid sequence shown in SEQ ID NO:8, and HCDR3 has the amino acid sequence shown in SEQ ID NO:9.

[0082] CD45[ZXClone-HCD45-01] has an amino acid sequence as shown in SEQ ID NO:18 for the light chain and an amino acid sequence as shown in SEQ ID NO:20 for the heavy chain; the variable region of the light chain has an amino acid sequence as shown in SEQ ID NO:14 for the light chain and an amino acid sequence as shown in SEQ ID NO:16 for the heavy chain; in the complementarity-determining region (LCDR), LCDR1 has an amino acid sequence as shown in SEQ ID NO:4, LCDR2 has an amino acid sequence as shown in SEQ ID NO:5 for the light chain and LCDR3 has an amino acid sequence as shown in SEQ ID NO:6 for the heavy chain; in the complementarity-determining region (HCDR), HCDR1 has an amino acid sequence as shown in SEQ ID NO:10 for the heavy chain, HCDR2 has an amino acid sequence as shown in SEQ ID NO:11 for the heavy chain and HCDR3 has an amino acid sequence as shown in SEQ ID NO:12 for the heavy chain.

[0083] Based on the amino acid sequences of the two antibodies mentioned above, the inventors designed nucleotide sequences (SEQ ID NO: 21-24) for expressing the corresponding antibodies. After cloning the nucleotide sequences into a stable CHO cell line, the antibodies were stably expressed. The construct vector was then introduced into CHO cells, and the antibodies were purified by protein A affinity chromatography after cell culture to obtain the two antibodies mentioned above.

[0084] 1.2 Antibody Detection

[0085] Take 10 μg of commercially available antibody CD45[2D1] (Biolegend, CAT#: 368534). Mix 10 μg of the prepared CD45[ZXClone-HCD45-04] and 10 μg of CD45[ZXClone-HCD45-01] with the loading buffer (antibody to loading buffer volume ratio 4:1), incubate at 100℃ for 30 min, and centrifuge at 10000 rpm for 1 min at room temperature to obtain the sample to be tested. Take out the pre-cast gel and install it in the electrophoresis tank, inject the electrophoresis buffer, remove the comb, and load the sample. Among them, 10 μL of protein marker is loaded and 10 μg of sample to be tested is loaded; electrophoresis settings: voltage 80V, electrophoresis time 90 min, stop when the bromophenol blue indicator band reaches the bottom; take out the gel and stain it with Coomassie Brilliant Blue for 30 min, then place it in destaining solution for 1 h, repeat 5 times, and then take pictures.

[0086] The results showed that the anti-human CD45 antibody prepared in this embodiment had intact heavy and light chains, clear bands, and a purity of over 95%. (See attached figures.) Figure 1 .

[0087] Example 2 Anti-human CD45 antibody labeling

[0088] CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01] were labeled with FITC (fluorescein isothiocyanate) or PE.

[0089] 2.1 FITC Marking

[0090] (1) Take 0.2 mg of CD45[ZXClone-HCD45-04] and 0.2 mg of CD45[ZXClone-HCD45-01] into ultrafiltration tubes respectively, wash the antibodies three times with 1 mM PBS. Centrifuge the ultrafiltration tubes at 4000g for 2 min inverted position, collect the concentrated antibody, and dilute it to 10 mg / mL with 1 mM PBS.

[0091] (2) Take the small molecule dye FITC (add dye at a ratio of n (molar mass) antibody: n (molar mass) FITC = 1:15) and add it to the monoclonal antibody reaction solution and mix well.

[0092] (3) React at room temperature of 25℃ for 45 minutes in the dark on a shaker.

[0093] (4) Transfer the reaction solution to an ultrafiltration centrifuge tube, wash the antibody 5 times with 1mM PBS. Centrifuge the ultrafiltration tube inverted at 4000g for 2min to collect the concentrated antibody, and dilute the antibody with 1mM PBS to a final concentration of 0.5mg / mL.

[0094] The labeled antibodies were designated as CD45[ZXClone-HCD45-04]-FITC and CD45[ZXClone-HCD45-01]-FITC.

[0095] 2.2 PE Marking

[0096] According to the method described in the patent "Method and Kit for Labeling Fluorescent Proteins and / or Coupled Proteins with Monoclonal Antibodies (CN202010972671.X)," CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01] were labeled respectively, and the labeled antibodies were denoted as CD45[ZXClone-HCD45-04]-PE and CD45[ZXClone-HCD45-01]-PE.

[0097] 2.3 Detection of the effect of fluorescently labeled antibodies

[0098] 1) Add 100 μL of cell quality control material (Beckman Coulter, CAT#: 6607077) to a 1.5 mL centrifuge tube.

[0099] 2) Add 1 μL of the fluorescently labeled antibodies CD45[ZXClone-HCD45-04]-FITC, CD45[ZXClone-HCD45-04]-PE, CD45[ZXClone-HCD45-01]-FITC, and CD45[ZXClone-HCD45-01]-PE to the sample respectively; add 5 μL of the control fluorescent antibodies CD45[2D1]-FITC (Biolegend, CAT#: 368507) and CD45[2D1]-PE (Biolegend, CAT#: 368509) to the sample respectively.

[0100] 3) All four samples were reacted in the dark for 15 minutes.

[0101] 4) Add 1 mL of hemolysin and continue the reaction for 15 minutes. The hemolysin used is the one from Zhengxi flow cytometer. Hemolysin: Zhejiang Medical Device Registration No. 20200133.

[0102] 5) Centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample to be tested in 500 μL PBS.

[0103] 6) Flow cytometry (Beckman DxFLEX flow cytometer) is used to detect the proportion of positive cells in the sample, the clustering of the chromatogram, and other indicators, thereby determining whether CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01] can be labeled with the fluorescent dyes FITC and PE, and whether the labeled fluorescent antibody is correctly used to detect the target antigen.

[0104] The results showed that both FITC and PE successfully labeled CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01], with proportions similar to the control CD45[2D1] of the commercial Biolegend. Figures 2-7 ).

[0105] Example 3 Anti-human CD45 antibody titer

[0106] First, commercially available CD45[2D1] (Biolegend, CAT#: 368534), antibody CD45[ZXClone-HCD45-04], and antibody CD45[ZXClone-HCD45-01] were diluted to 1 mg / mL with known concentrations. The antibodies were then further diluted with PBS at ratios of 1:100, 1:1000, 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:60000, and 1:96000. The CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01] antibodies served as the experimental group, while the commercially available CD45[2D1] antibody served as the control group.

[0107] The experimental group consisted of two subgroups, and the control group consisted of one subgroup. Each subgroup received 100 μL of healthy human blood or a manufacturer's reference sample. 1 mL of hemolysin was added to each of the three experimental groups. Hemolysin (Zhejiang Medical Device Registration No. 20200133), continue the reaction for 15 min. Centrifuge at 4000 rpm for 2 min, and discard the supernatant.

[0108] The experimental groups were treated with 100 μL of proportionally diluted CD45 [ZXClone-HCD45-04] and CD45 [ZXClone-HCD45-01] antibodies, respectively. The control groups were treated with 100 μL of proportionally diluted commercial anti-human CD45 [2D1] antibody. The samples were vortexed and incubated at room temperature in the dark for 15 min. 900 μL of PBS was added to resuspend the samples, and the mixture was centrifuged at 4000 rpm for 2 min. The supernatant was discarded. 100 μL of PBS was added, and the mixture was vortexed. 1 μL of commercial goat anti-mouse IgG-PE flow cytometry antibody (Biolgend, CAT#: 405307) was added to each group, and the mixture was vortexed and incubated at room temperature in the dark for 15 min. 900 μL of PBS was added to resuspend the samples, and the mixture was centrifuged at 4000 rpm for 2 min. The supernatant was discarded. 1000 μL of PBS was added again to resuspend the samples, and the mixture was centrifuged at 4000 rpm for 2 min. The supernatant was discarded. Resuspend the sample in 500 μL of PBS and perform flow cytometry analysis.

[0109] The results showed that the affinities of the three antibodies were as follows: CD45[2D1] antibody, with an affinity (kd) of 173.6 (1 mg / mL); CD45[ZXClone-HCD45-04] antibody, with an affinity (kd) of 189.8 (1 mg / mL); and CD45[ZXClone-HCD45-01] antibody, with an affinity (kd) of 177.1 (1 mg / mL). Figures 8-13 ).

[0110] The results showed that the CD45[ZXClone-HCD45-04] antibody had similar affinity to the positive control CD45[2D1] antibody; the affinity of the CD45[ZXClone-HCD45-01] antibody was basically the same as that of the positive control CD45[2D1] antibody.

[0111] Example 4: Anti-human CD45 antibody specificity

[0112] The antibody used in this embodiment is:

[0113] CD45 [ZXClone-HCD45-04] prepared in Example 1;

[0114] CD45 [ZXClone-HCD45-01] prepared in Example 1;

[0115] Mouse isotype control antibody (MOPC-21 clone number) (Biolegend, CAT#: 400102);

[0116] CD45[ZXClone-HCD45-04]-PE prepared in Example 2;

[0117] CD45[ZXClone-HCD45-01]-PE prepared in Example 2;

[0118] Anti-human CD45[2D1](Biolegend, CAT#: 368534);

[0119] CD3(SK7)-APC (Biolegend, CAT#: 340440).

[0120] Antibody-specific detection methods:

[0121] Lymphocytes were obtained by taking 100 μL of cell quality control material (Beckman Coulter, CAT#: 6607077). 1 mL of hemolysin was added, and the reaction was continued for 15 minutes. The hemolysin used was the one from Zhengxi flow cytometry. Hemolysin: Zhejiang Medical Device Registration No. 20200133. Centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample in 100 μL of PBS. Prepare three identical samples simultaneously.

[0122] Experimental group: Add 1 μL of anti-human CD45 [ZXClone-HCD45-04] and CD45 [ZXClone-HCD45-01] to two of the above samples, respectively. After reacting in the dark for 15 minutes, resuspend the samples in 500 μL of PBS, centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the samples in 100 μL of PBS. Add Biolegend CD45 [2D1]-PE to the two blocked samples to detect the specificity of the pure antibody. Add Biolegend CD45 [2D1]-PE to the sample blocked with pure anti-CD45 [ZXClone-HCD45-04]. Add 5 μL of Biolegend CD45 [2D1]-PE to the other sample blocked with pure anti-CD45 [ZXClone-HCD45-01].

[0123] Control group: 1 μL of pure mouse isotype control antibody (MOPC-21 clone) was added to one of the above samples. After reacting in the dark for 15 minutes, the sample was resuspended in 500 μL of PBS, centrifuged at 4000 rpm for 2 minutes, the supernatant was discarded, and then 100 μL of PBS was added to resuspend the sample. 5 μL of Biolegend CD45[2D1]-PE was added to each resuspended sample.

[0124] After reacting all samples in the dark for 15 minutes, resuspend the samples in 500 μL of PBS, centrifuge at 4000 rpm for 2 min, discard the supernatant, and then resuspend the samples in 500 μL of PBS.

[0125] Flow cytometry (Beckman DxFLEX flow cytometer) was used to detect indicators such as the proportion of positive cells in the sample and the clustering of cell patterns, in order to determine whether CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01] can specifically recognize the CD45 target antigen.

[0126] The results showed no signal from the CD45[2D1]-PE fluorescent antibody in the experimental group. In the control group, the isotype control antibody failed to block the CD45[2D1]-PE, but a fluorescent signal from CD45[2D1]-PE was observed. This indicates that the two CD45 antibodies prepared from CHO cells, CD45[ZXClone-HCD45-04] and CD45[ZXClone-HCD45-01], can specifically recognize and block the CD45 target antigen (…). Figures 14-16 ).

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-human CD45 antibody or its antigen-binding fragment, characterized in that, The anti-human CD45 antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region; The light chain variable region includes LCDR1, LCDR2 and LCDR3, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3; The CDR sequence of the anti-human CD45 antibody or its antigen-binding fragment is selected from: (1) The amino acid sequence is as shown in SEQ ID NO: 1 for LCDR1, as shown in SEQ ID NO: 2 for LCDR2, and as shown in SEQ ID NO: 3 for LCDR3; and the amino acid sequence is as shown in SEQ ID NO: 7 for HCDR1, as shown in SEQ ID NO: 8 for HCDR2, and as shown in SEQ ID NO: 9 for HCDR3; or (2) The amino acid sequence is LCDR1 as shown in SEQ ID NO: 4, the amino acid sequence is LCDR2 as shown in SEQ ID NO: 5, the amino acid sequence is LCDR3 as shown in SEQ ID NO: 6; and the amino acid sequence is HCDR1 as shown in SEQ ID NO: 10, the amino acid sequence is HCDR2 as shown in SEQ ID NO: 11, and the amino acid sequence is HCDR3 as shown in SEQ ID NO:

12.

2. The anti-human CD45 antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO: 13; or The light chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

14.

3. The anti-human CD45 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain variable region of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

16.

4. The anti-human CD45 antibody or its antigen-binding fragment according to claim 3, characterized in that, The light chain of the anti-human CD45 antibody or its antigen-binding fragment has the amino acid sequence shown in SEQ ID NO: 17; or The light chain of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

18.

5. The anti-human CD45 antibody or its antigen-binding fragment according to claim 4, characterized in that, The heavy chain of the anti-human CD45 antibody or its antigen-binding fragment has the amino acid sequence shown in SEQ ID NO: 19; or The heavy chain of the anti-human CD45 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

20.

6. An isolated polynucleotide, characterized in that, It encodes the anti-human CD45 antibody or its antigen-binding fragment as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, It contains the isolated polynucleotide as described in claim 6.

8. A host cell, characterized in that, It comprises the recombinant expression vector as described in claim 7.

9. A method for preparing an anti-human CD45 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The preparation method includes at least one of the following steps: (I) The isolated polynucleotide of claim 6 or the recombinant expression vector of claim 7 is introduced into a host cell, and the cell is cultured and then purified and collected for anti-human CD45 antibody or its antigen-binding fragment; (II) Cultivate the host cells as described in claim 8, and purify and collect the anti-human CD45 antibody or its antigen-binding fragment after culturing the cells.

10. A method for detecting CD45 protein for non-disease diagnostic purposes, characterized in that, The detection method includes: I) Incubate the sample to be tested with any of the anti-human CD45 antibodies or their antigen-binding fragments as described in claims 1-5, or with a fluorescently labeled antibody containing any of the anti-human CD45 antibodies or their antigen-binding fragments as described in claims 1-5; Ⅱ) Detect whether there are antibody-antigen conjugates in the sample, or detect the presence of fluorescence by flow cytometry.

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