Monoclonal antibodies against CD4 protein and their uses

Abstract

The present invention discloses a monoclonal antibody against CD4 protein and uses thereof. The present invention provides an anti-CD4 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region CDR1 as shown in SEQ ID NO:1, a heavy chain variable region CDR2 as shown in SEQ ID NO:2, a heavy chain variable region CDR3 as shown in SEQ ID NO:3, a light chain variable region CDR1 as shown in SEQ ID NO:9, a light chain variable region CDR2 as shown in SEQ ID NO:10, and a light chain variable region CDR3 as shown in SEQ ID NO:11.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody targeting CD4 protein and a use thereof. Background Art

[0002] CD4 protein, also known as CD4 molecule or CD4 receptor, is a major surface glycoprotein expressed on the surface of T helper cells. + T cells are key players in the human immune system, able to recognize and respond to foreign antigens presented by antigen presenting cells (APCs). + T cells can differentiate into a variety of effector T cell subsets, such as Th1, Th2, Th17 and Treg, to perform different immune functions. CD4 antibodies play an important role in cell sorting and can also be used for flow cytometry analysis and detection of cell surface markers. CD4 antibodies can be used in immunohistochemistry, immunoblotting, immunocytochemistry and immunoprecipitation techniques to detect CD4 protein, and have multiple functions in basic research and clinical applications.

[0003] The large-scale culture technology of hybridoma cells is a relatively simple technology for preparing monoclonal antibodies in large quantities. After a small amount of hybridoma cells are amplified, they are injected into the abdominal cavity of mice. Within 5-10 days, the recombinant expression technology of monoclonal antibodies is a key technology in the field of biopharmaceuticals, which makes the large-scale preparation of monoclonal antibodies possible. Through large-scale production, antibodies with high consistency and specificity are prepared for antibody drug discovery and screening. The recombinant expression technology room is achieved by cloning the gene sequence encoding the antibody into a suitable expression vector and expressing it in the host cell. Commonly used expression hosts are human embryonic kidney cells (HEK293) and Chinese hamster ovary cells (CHO). HEK293 is mainly expressed by transient transfection, with a short cycle and low cost. In addition, the host is a human cell with a low risk of exogenous factors. Integrating the antibody gene into the CHO cell genome can achieve stable expression of the antibody.

[0004] Ascites production of antibodies is a traditional method for preparing monoclonal antibodies, which involves injecting hybridoma cells into the peritoneal cavity of mice, allowing the cells to grow in the peritoneal cavity and produce ascites, thereby obtaining a large amount of monoclonal antibodies. This method has ethical issues of animal experiments and the risk of contaminating animal viruses, and the yield is unstable and the consistency between batches is poor. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The present invention provides an anti-CD4 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region CDR1 as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 as shown in SEQ ID NO: 2, a heavy chain variable region CDR3 as shown in SEQ ID NO: 3, a light chain variable region CDR1 as shown in SEQ ID NO: 9, a light chain variable region CDR2 as shown in SEQ ID NO: 10, and a light chain variable region CDR3 as shown in SEQ ID NO: 11.

[0007] The present invention uses the terms "CDR" and "complementarity determining region" interchangeably and refers to a portion of the variable chain of an antibody that is involved in binding to an antigen. Therefore, a CDR is a portion of an "antigen binding fragment" or an "antigen binding fragment". In some embodiments, a monoclonal antibody comprises three heavy chain variable region CDRs and three light chain variable region CDRs that together form an antigen binding fragment. "CDR" may refer to a CDR defined by any method known in the art (including a combination of methods).

[0008] The term "CD4" used in the present invention refers to the full-length CD4 protein from humans (UNIPORT ID: P01730). CD4 specifically refers to the CD4 membrane glycoprotein of T lymphocytes. The CD4 antigen acts as a co-receptor together with the T cell receptor on T lymphocytes to recognize antigens displayed by antigen presenting cells in the context of class II MHC molecules.

[0009] The term "T cell" or "T lymphocyte" used herein is recognized in the art and is intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 -T cells, αβT cells expressing α and β chains of T cell receptors (TCRs), and γδT cells expressing TCRγ and δ chains, or any other subset of T cells.Other illustrative T cell groups suitable for specific embodiments include memory T cells, suitably early memory T cells.The term "T cell" includes natural T cells (e.g., separated from an organism, such as a mammal, e.g., a human, e.g., a subject), T cells grown in vitro, and genetically engineered T cells within its scope.The term T cell also includes T cells comprising T cell receptors (e.g., natural TCRs, or recombinant TCRs) and T cells (e.g., CAR-T cells) comprising artificial T cell receptors.

[0010] Further, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity with the amino acid sequences described in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity with the amino acid sequences described in SEQ ID NOs: 12-15, respectively;

[0011] The present invention uses the term "sequence identity" to mean that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over a comparison window. The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U, or I) appears in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce a sequence identity percentage.

[0012] Furthermore, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 12-15, respectively;

[0013] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16;

[0014] Furthermore, the light chain amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 17;

[0015] In some embodiments, the light chain is the sum of a light chain variable region and a light chain constant region.

[0016] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 18;

[0017] In some embodiments, the heavy chain is the sum of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region includes a CH1 region and CH2 and CH3 regions.

[0018] Furthermore, the monoclonal antibody or antigen-binding fragment thereof is humanized.

[0019] The present invention uses the term "monoclonal antibody" intended for use in the art, and refers to an antibody obtained from a substantially homologous antibody population that binds to the same antigenic determinant (epitope). "Substantially homologous" means that the individual antibodies are identical except for possible naturally occurring mutations that may be present in small amounts. This is in contrast to polyclonal antibodies, which typically include different antibodies against a variety of different antigenic determinants (epitopes). The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. "Monoclonal antibodies" and their antigen-binding fragments are made in many ways, including, but not limited to, by hybridomas, phage selection, recombinant expression, and transgenic animals.

[0020] The present invention provides a polynucleotide, wherein the nucleic acid molecule encodes the monoclonal antibody or antigen-binding fragment thereof as described above;

[0021] Furthermore, the light chain and the heavy chain of the monoclonal antibody or antigen-binding fragment thereof are connected by a T2A linker;

[0022] Further, the amino acid sequence encoded in the polynucleotide is a light chain + T2A linker + heavy chain connected in sequence;

[0023] Further, the T2A linker is modified by GSG linker;

[0024] Furthermore, the polynucleotide also includes a nucleotide sequence encoding a signal peptide molecule;

[0025] Further, the signal peptide molecule is at the 5' end of the monoclonal antibody or its antigen-binding fragment;

[0026] Furthermore, the amino acid sequence of the signal peptide molecule is shown in SEQ ID NO: 19;

[0027] Furthermore, the amino acid sequence of the T2A linker after transformation from the GSG linker is shown in SEQ ID NO: 20.

[0028] The present invention provides a method for producing the above-mentioned monoclonal antibody or antigen-binding fragment thereof, the method comprising the following steps:

[0029] The polynucleotide described above is provided, transformed into a host cell, the transformed host cell is cultured under appropriate conditions, and the monoclonal antibody or its antigen-binding fragment in the host cell culture fluid is separated and purified.

[0030] The present invention provides use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof in the preparation of a drug for treating or preventing a disease or condition caused by CD4 protein.

[0031] The present invention provides use of the above-mentioned monoclonal antibody or its antigen-binding fragment in preparing a product for detecting CD4 protein.

[0032] The present invention provides use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof in preparing a cell sorting product.

[0033] The present invention provides a vector, which comprises the polynucleotide described above.

[0034] Furthermore, the vector includes plasmid, lentivirus, adenovirus, adeno-associated virus and other vectors.

[0035] The present invention uses the term "vector" to refer to a nucleic acid molecule that can transport another nucleic acid molecule connected to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) are able to replicate autonomously in the host cells into which they are introduced. Other vectors (e.g., non-episodic mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and are thereby replicated together with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA techniques are usually in the form of plasmids.

[0036] The present invention provides a cell, the cell comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, or the aforementioned vector;

[0037] Furthermore, the cell includes any cell that can replicate, preserve, and express the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, or the aforementioned vector.

[0038] Furthermore, the cells are from humans or non-human mammals;

[0039] Furthermore, the cells are from humans.

[0040] In some embodiments, the cell is a HEK293 cell line.

[0041] The present invention uses the terms "cell", "cell line", "host cell" interchangeably and covers plant and animal cells, and includes invertebrates, non-mammalian vertebrates and mammalian cells. All such names include cell populations and progeny. Exemplary non-mammalian vertebrate cells include, for example, avian cells, reptile cells and amphibian cells. Exemplary invertebrate cells include, but are not limited to, insect cells, such as caterpillar (Spodoptera frugiperda) cells, mosquito (Aedes aegypti) cells, fruit fly (Drosophila melanogaster) cells, Schneider cells and silkworm cells. Cells can be differentiated, partially differentiated or undifferentiated, such as stem cells, including embryonic stem cells and pluripotent stem cells. Additional tissue samples derived from organs or organ systems can be used according to the present invention. Exemplary mammalian cells include, for example, cells derived from humans, non-human primates, cats, dogs, sheep, goats, cows, horses, pigs, rabbits, rodents including mice, hamsters, rats and guinea pigs, and any derivatives and progeny thereof.

[0042] The present invention provides a conjugate, which comprises the monoclonal antibody or antigen-binding fragment thereof as described above, and a substance conjugated to the antibody;

[0043] Furthermore, the substance includes fluorescent dyes, chemiluminescent compounds, radioactive isotopes, biotin or antibody-conjugated enzymes.

[0044] In some embodiments, the substance comprises an antibody-conjugated enzyme (an enzyme capable of generating a detectable signal), specifically including horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase. In some embodiments, the substance comprises a chromophore, such as a fluorescent or luminescent dye; a group whose electron density is detected by electron microscopy or by its electrical properties, such as conductivity, current analysis, voltammetry, impedance.

[0045] In some embodiments, exemplary fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6) and rhodamine 110; cyanine dyes, such as Cy3, Cy5 and Cy7 dyes; Alexa dyes, such as Alexa-fluor-555; coumarins, diethylaminocoumarins, umbelliferone; benzylamine dyes, such as Hoechst33258; phenanthridinium dyes, such as Texas red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein chlorotriazinyl, R110, Eosin, JOE, R6G, tetramethylrhodamine, lissamine, ROX and naphthofluorescein.

[0046] Advantages of the present invention:

[0047] According to the sequence of the antibody, an expression plasmid of a humanized anti-CD4 antibody was constructed; HEK293 cells were transiently transfected to express the recombinant anti-human CD4 monoclonal antibody; the recombinant anti-human CD4 monoclonal antibody with high purity was obtained by purification; the binding activity of the anti-CD4 antibody to CD4 in PBMC was compared, and the effects of the mouse antibody and the humanized antibody were comparable. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is the electrophoresis diagram of cell feed solution;

[0049] Figure 2 It is the affinity capture and polishing electrophoresis diagram;

[0050] Figure 3 It is a flowchart of the antibody preparation process;

[0051] Figure 4 This is the result of antibody purity test;

[0052] Figure 5 This is the sensitivity chart of antibody detection by indirect ELISA method. DETAILED DESCRIPTION

[0053] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As will be apparent to those skilled in the art after reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be easily separated or combined with features of any of the other several aspects without departing from the scope or essence of this disclosure.

[0054] It should also be understood that the terms used herein are only used for the purpose of describing specific aspects and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the specification and related art, and should not be interpreted as idealized or overly formal unless explicitly defined herein.

[0055] Example 1 Expression plasmid design, construction and plasmid mass production

[0056] 1. Construct CD4 antibody expression plasmid and extract the plasmid for transient cell transfection

[0057] 1.1 The sequence information of the expressed antibodies is shown in Table 1

[0058] Table 1

[0059]

[0060] 1.2 Vector construction and plasmid extraction

[0061] 1.2.1 Using pCDNA3.4 as the expression vector, Mouse Ig Kappa signal peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 19), modified T2A sequence: RRKRGSGEGRGSLLTCGDVEENPGPMEWIWIFLLILSGTRGVQS (SEQ ID NO: 20) to construct a CD4 antibody expression plasmid named pTL-004Ab, whose reading frame is SEQ ID NO: 17 and SEQ ID NO: 18 connected by the T2A sequence. The vector construction was entrusted to Jiangsu Saisuofei Biotechnology Co., Ltd.

[0062] Example 2 HEK293 cell culture and antibody transient transfection expression

[0063] 1. Using HEK293 as host cells for recombinant expression of antibodies

[0064] 1.1 Main Materials

[0065] HEK293 cells (human embryonic kidney cells), stored by our company; HEK293 serum-free medium (item number: AS-11), Tongli Haiyuan; HEK-293 expression feed medium (item number: AS-18), Tongli Haiyuan; transfection reagent TA-293 (item number: K20001), Zhuhai Kairui; recombinant protein expression enhancer KE-293 (item number: K30001), Zhuhai Kairui; glutamine (item number: 21051024), Gibco; expression plasmid: plasmid extracted in step 1 (pTL-004Ab). Cell shaker, Corning; cell counter, Contstar; cell culture shaker, EppendorfS41i; large-capacity centrifuge, Hunan Xiangyi GL-21M.

[0066] 1.2 Methods

[0067] 1.2.1 Cell culture

[0068] Resuscitate HEK293 cells and culture them at a density of 3-4 × 10 6 / mL, and then subculture. According to the cell density, add fresh 293 serum-free medium and dilute the cell density to 0.3-0.6×10 6 / mL, culture in a cell culture shaker (37°C, CO2 concentration 5%). Requirement: The volume of the culture medium should not exceed 1 / 5 of the volume of the culture flask.

[0069] 1.2.2 Transient Transformation and Feeding

[0070] 1) On the day of transfection, detect cell density and viability and adjust the cell density to 3×10 6 / mL;

[0071] 2) Take 1 mg of plasmid (plasmid number: pJD27, 1013.6 μg / mL, 987 μL) and add it to a 50 mL centrifuge tube (tube A) pre-prepared with 20 mL of 293 serum-free medium, and gently pipette to mix; take 5 mL of TA-293 and add it to a 50 mL centrifuge tube (tube B) pre-prepared with 20 mL of 293 serum-free medium, and gently pipette to mix;

[0072] 3) Mix the liquids in tube A and tube B, mix gently, and leave at room temperature for 10 minutes to prepare the transfection reagent-plasmid complex;

[0073] 4) Use a pipette to slowly add the transfection reagent-plasmid complex to the cells at the pre-adjusted density, shaking the cell shaker flask while adding to evenly disperse the complex. After completion, move the shaker flask into a shaker and start culturing. The culture conditions are: 5% CO2, 37°C, 120rpm;

[0074] 5) After culturing for 24 h (Day 1), samples were taken to detect cell density and viability (retained samples), and 50 mL of feed medium and 6 mL of KE-293 were added in sequence, and the culture continued;

[0075] 6) Day 3, test cell density and viability (keep sample), add 50 mL feed medium, and continue culturing;

[0076] 7) On Day 4, detect cell density and viability (keep samples); on Day 5 or when the cell viability is lower than 70%, terminate the expression, transfer the feed solution into a clean centrifuge bottle, centrifuge at 8000 rpm for 20 min at room temperature, and harvest the cell supernatant for purification.

[0077] 1.3 Results

[0078] The results of cell status monitoring during the expression process are shown in Table 2.

[0079] Table 2

[0080]

[0081] The results of the cell solution electrophoresis (Reduce SDS-PAGE) are as follows Figure 1 shown.

[0082] Example 3 Affinity chromatography and purified antibodies

[0083] 1. Capture of recombinant anti-CD4 antibodies in cell feed using affinity chromatography media

[0084] 1.1 Main Materials

[0085] Affinity chromatography filler: GE, Mab Select suRe filler (5 mL); Chromatography system, GE, AKTA Purifier100;

[0086] 1.2 Methods

[0087] 1.2.1 Column loading: Clean 5 mL pre-loaded column for later use, take 5 mL GE mab select suRe filler according to the correct glue-water ratio, load the lower gasket and lower plug into the pre-loaded column, pour the filler from the top to allow it to settle naturally, and after all the filler is loaded, load the upper gasket and upper plug;

[0088] 1.2.2 Set the flow rate to 5 mL / min;

[0089] 1.2.3 Balance: 20 mM Tris, 100 mM NaCl, pH 7.4, 6 CV;

[0090] 1.2.4 Sample loading: Expression delivery 1L, sample loading volume 300ml;

[0091] 1.2.5 flow through: 2508.716mAU;

[0092] 1.2.6 Re-equilibration: 20 mM Tris, 100 mM NaCl, pH 7.4, 7 CV;

[0093] 1.2.7Wash 1: 44 mM sodium citrate, 1 M NaCl, pH 5.5, 15 CV, 644.529 mAU-30 ml;

[0094] 1.2.8Wash 2: 44 mM sodium citrate, pH 5.0, 6 CV;

[0095] 1.2.9 Elution: 0.1 M glycine, 25 mM arginine, 0.15 M NaCl, 12% trehalose, pH 3.2, 12 CV, 2200.691 mAU-16 ml;

[0096] 1.2.10 Neutralization: Add 1M Tris (pH 9.0) to the eluted sample and adjust the pH to 7.68, labeled as JD27-A;

[0097] 2. Purify the antibody with high purity by G25 liquid exchange and ion exchange chromatography

[0098] 2.1 Main Materials

[0099] Bestdex G-25 filler, Bogeron; Q Bestarose FF filler, Bogeron; SP Bestarose FF filler, Bogeron; chromatography system, GE, AKTA Purifier 100.

[0100] 2.1.1 G25 fluid change

[0101] 1) Column treatment: 5 mL / min, water injection, 10 CV;

[0102] 2) CIP: 8 mL / min, CIP solution flushing 10 CV;

[0103] 3) Equilibration: 8 mL / min, flushing with desalting chromatography equilibration solution (PBS, pH 7.4) until the baseline is stable;

[0104] 4) Sample loading: 5 mL / min, JD27-A (16 mL);

[0105] 5) Balance: 8 mL / min, collect protein peak, 100 mAu-988 mAu-150 mAu, volume 23 mL, labeled as JD27-AG;

[0106] 6) Storage: 8 mL / min, water flushing, 10 CV; 5 mL / min, 20% ethanol flushing, 10 CV.

[0107] 2.1.2 SP purification

[0108] 1) Column loading: Clean 5 mL of the chromatography column pre-loaded with SP filler for later use. The flow rate during the purification process is controlled at 5 ml / min;

[0109] 2) Equilibration: 20 mM NaAC-HAC, pH 6.0, 10 CV;

[0110] 3) Sample treatment: JD27-AG (23 ml), diluted with water to a conductivity of 2.097 mS / cm, adjusted to pH 5.5, final volume 229 ml;

[0111] 4) Solution B: 20mM NaAC-HAC, 1M NaCl, pH 5.5;

[0112] 5) Elution: 10% B elution, 681.694 mAU-16 ml, marked as JD27-AGS;

[0113] 6) Neutralization: add 1M Tris pH 9.0 to adjust to pH 8.05;

[0114] 2.1.3Q purification

[0115] 1) Column loading: Clean 5 mL of a chromatography column pre-loaded with Q filler for later use. The flow rate during the purification process is controlled at 5 ml / min.

[0116] 2) Equilibration: 20 mM Tris, 20 mM NaCl, pH 8.0, 10 CV;

[0117] 3) Sample treatment: JD27-AGS, 16 ml, diluted with water to a conductivity of 1.536 mS / cm, adjusted to pH 8.02, final volume 29 ml;

[0118] 4) Solution B: 20 ​​mM Tris, 1 M NaCl, pH 8.0;

[0119] 5) Elution: 20% B elution, 186.869mAU-18ml;

[0120] 6) Column treatment: flush column with liquid B, 6CV;

[0121] 2.2 Results

[0122] SDS-PAGE electrophoresis analysis of the components of Ni column imidazole gradient elution showed that the purity of the 20% B elution product was >90% after one-step affinity purification. After G25 solution replacement, the protein concentration was 1.63 mg / mL by BCA quantitative detection. The purified electrophoresis is shown in Figure 2 shown.

[0123] The specific process flow chart of Examples 1-3 is as follows Figure 3 shown.

[0124] Example 4 Antibody Property Identification

[0125] Take the purified and amino acid sequenced antibodies and test the purity and titer of the antibodies

[0126] 1. Main materials

[0127] Anti-human CD4 monoclonal antibody, Company A; phosphate buffer, Shanghai Yuanpei; GoatAnti-Human IgG Fc / HRP (ab97225), Abcam; TMB substrate colorimetric kit (CW0050S), Kangwei Century; multi-function microplate reader (INFINITEEPLEX), Tecan; high performance liquid chromatography (InfinityII 126), Agilent; flow cytometer, Millipore.

[0128] 2. Experimental methods

[0129] 2.1 HPLC detection of antibody purity

[0130] Conventional ELISA test: coated with recombinant human CD4 protein, 0.5μg / well; blocked with 5% skim milk powder; recombinant anti-human CD4 antibody

[0131] 2.2ELISA detection of antibody titer

[0132] Conventional ELISA detection: coated with recombinant human CD4 protein, 0.5μg / well; blocked with 5% skim milk powder; recombinant anti-human CD4 antibody (1mg / mL), diluted 500, 2000, 8000, 32000, 128000 times, commercial anti-human CD4 monoclonal antibody (Company A) as positive control, antibody diluent as negative control, incubated at 37℃ for 1h; secondary antibody is goat anti-human IgGFc / HRP, after 10000-fold dilution, 100μL per well, incubated at 37℃ for 50min; TMB color development, read with enzyme reader, and analyze the results.

[0133] 3. Results

[0134] The results of HPLC test for antibody purity are as follows Figure 4 The indirect ELISA method was used to detect the sensitivity of antibodies at a ratio of 1:100 mg / L; 2:10 mg / L; 3:1 mg / L; 4:100 μg / L; 5:10 μg / L; 6:1 μg / L; 7:100 ng / L; 8:0 ng / L. The results are shown in the figure. Figure 5As shown, the antibody was able to detect 100 ng / L of CD4 protein.

[0135] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An anti-CD4 monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region CDR1 as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 as shown in SEQ ID NO: 2, a heavy chain variable region CDR3 as shown in SEQ ID NO: 3, a light chain variable region CDR1 as shown in SEQ ID NO: 9, a light chain variable region CDR2 as shown in SEQ ID NO: 10, and a light chain variable region CDR3 as shown in SEQ ID NO:

11.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100% sequence identity with the amino acid sequences described in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100% sequence identity with the amino acid sequences described in SEQ ID NOs: 12-15, respectively.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or its antigen-binding fragment are shown in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or its antigen-binding fragment are shown in SEQ ID NOs: 12-15, respectively.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The light chain amino acid sequence of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO:

17.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain amino acid sequence of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO:

18.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof is humanized.

8. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7. 9 . The polynucleotide according to claim 8 , wherein the light chain and the heavy chain of the monoclonal antibody or the antigen-binding fragment thereof are connected by a T2A linker.

10. The polynucleotide according to claim 8, wherein the amino acid sequence encoded in the polynucleotide is a light chain + T2A linker + heavy chain connected in sequence. The polynucleotide according to claim 8 , further comprising a nucleotide sequence encoding a signal peptide molecule.

12. The polynucleotide according to claim 11, wherein the signal peptide molecule is at the 5' end of the monoclonal antibody or the antigen-binding fragment thereof.

13. The polynucleotide according to claim 11, wherein the amino acid sequence of the signal peptide molecule is shown in SEQ ID NO:

19. 14 . The polynucleotide according to claim 9 , wherein the amino acid sequence of the T2A linker is shown in SEQ ID NO:

20.

15. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: Providing the polynucleotide according to any one of claims 8 to 14, transforming it into a host cell, culturing the transformed host cell under suitable conditions, and isolating and purifying the monoclonal antibody or its antigen-binding fragment in the host cell culture fluid.

16. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of a product for detecting CD4 protein.

17. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in preparing a cell sorting product.

18. A vector comprising the polynucleotide according to any one of claims 8 to 14.

19. A cell comprising the polynucleotide according to any one of claims 8 to 14, or the vector according to claim 18.

20. The cell according to claim 19, which is from a human or a non-human mammal.

21. The cell according to claim 19, which is derived from a human.

22. A conjugate, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and a substance conjugated to the antibody; the substance is a fluorescent dye, a chemiluminescent compound, a radioactive isotope, biotin or an antibody-conjugated enzyme.

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