An antibody against CD4 protein, its fragment and its application

Abstract

The present invention discloses an antibody against CD4 protein, its fragment and its application. The present invention provides an antibody against CD4 protein and its fragment, including H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region, and their sequences are shown in SEQ ID NO: 1, 2, and 3 respectively; including L-CDR1, L-CDR2, and L-CDR3 of the light chain variable region, and their sequences are shown in SEQ ID NO: 4, 5, and 6 respectively. The present invention also provides a nucleic acid molecule capable of encoding the antibody against CD4 protein and its fragment, a vector carrying the nucleic acid molecule, and a cell containing the nucleic acid molecule and / or the vector. The present invention also provides the related application and method of the antibody against CD4 protein and its fragment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an antibody against CD4 protein, its fragment and its application. 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. It plays a key role in the process of T cell recognition of antigens and initiation of immune responses. The CD4 molecule binds to MHC class II molecules on antigen-presenting cells to help T cells recognize and attack foreign pathogens such as viruses and bacteria. CD4 protein, also known as CD4 molecule or CD4 receptor, is a major surface glycoprotein expressed on the surface of T helper cells. It plays a key role in the process of T cell recognition of antigens and initiation of immune responses. The CD4 molecule binds to MHC-II molecules on antigen-presenting cells to help T cells recognize and attack foreign pathogens such as viruses and bacteria. CD4 + T cells are key players in the human immune system, capable of recognizing foreign antigens presented by antigen-presenting cells (APCs) and generating responses. Such responses can regulate the activities of other immune cells such as B cells or CD8 + T cells, or initiate new immune responses. CD4 + T cells can differentiate into various effector T cell subsets such as Th1, Th2, Th17 and Treg, etc., to perform different immune functions. CD4 + The activation and differentiation of CD4 T cells are crucial for the successful initiation of immune responses. CD4 antibodies can be used in techniques such as immunohistochemistry, immunoblotting, immunocytochemistry and immunoprecipitation to detect CD4 protein, and have multiple functions in basic research and clinical applications. In addition, CD4 antibodies play an important role in cell sorting and can also be used for flow cytometry analysis and detection of cell surface markers. Summary of the Invention

[0003] Among currently commercialized CD4 antibodies, none is sensitive and specific enough. Especially in the detection of cell surface CD4 protein and specific cell sorting, there are problems such as insufficient detection sensitivity and low sorting efficiency caused by non-specific binding and poor antibody affinity.

[0004] To solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The present invention provides an anti-CD4 protein antibody, a fragment or a fusion protein thereof, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an H-CDR1 with the amino acid sequence shown in SEQ ID NO: 1, an H-CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and an H-CDR3 with the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof that contains up to 3, 2 or 1 amino acid substitutions in the H-CDR; and the VL comprises an L-CDR1 with the amino acid sequence shown in SEQ ID NO: 4, an L-CDR2 with the amino acid sequence shown in SEQ ID NO: 5, and an L-CDR3 with the amino acid sequence shown in SEQ ID NO: 6, or a variant thereof that contains up to 3, 2 or 1 amino acid substitutions in the L-CDR.

[0006] Furthermore, the CDR division of the anti-CD4 protein antibody, its fragment or fusion protein is carried out according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.

[0007] Furthermore, the CDR division of the anti-CD4 protein antibody, its fragment or fusion protein is carried out according to the Kabat numbering system.

[0008] Furthermore, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region with the amino acid sequences shown in SEQ ID NOs: 4, SEQ ID NO: 5 and SEQ ID NO: 6;

[0009] Furthermore, the heavy chain variable region comprises one or more heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, and / or one or more light chain framework regions LFR1, LFR2, LFR3 and LFR4. The heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have homologous sequences with at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7-10, and the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have homologous sequences with at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 11-14;

[0010] Furthermore, the amino acid sequences of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 are respectively as shown in SEQ ID NOs: 7-10, and the amino acid sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 are respectively as shown in SEQ ID NOs: 11-14.

[0011] Furthermore, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or a homologous sequence having at least 85% sequence identity therewith; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or a homologous sequence having at least 85% sequence identity therewith;

[0012] Furthermore, the heavy chain variable region and the heavy chain constant region (CH1 constant region) comprise the amino acid sequence shown in SEQ ID NO: 17, or a homologous sequence having at least 85% sequence identity therewith; the light chain variable region and the light chain constant region comprise the amino acid sequence shown in SEQ ID NO: 18, or a homologous sequence having at least 85% sequence identity therewith.

[0013] Furthermore, the light chain variable region and the light chain constant region are the full length of the light chain.

[0014] Furthermore, the heavy chain variable region and the heavy chain constant region (CH1 constant region) are the part of the antibody heavy chain in the Fab fragment (antigen-binding fragment).

[0015] In some embodiments, the "percent sequence identity" can be calculated as follows: compare two optimally aligned sequences in a comparison window, determine the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to obtain the percent sequence identity.

[0016] Furthermore, it is a bispecific antibody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment, (dsFv)2, dual-specific dsFv, disulfide-stabilized bispecific antibody, single-chain antibody molecule, scFv dimer, multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody;

[0017] Furthermore, the source of the anti-CD4 protein antibody, its fragment, or fusion protein is a human or non-human mammal;

[0018] In some embodiments, the non-human mammal refers to a viviparous animal other than a human, including mouse, sheep, rabbit, horse, cow, pig, dog, monkey, etc.

[0019] Further, the source of the anti-CD4 protein antibody, its fragment or fusion protein is murine.

[0020] Further, the anti-CD4 protein antibody, its fragment or fusion protein further comprises a substitution or modification of one or more amino acid residues that does not affect the binding specificity.

[0021] In some embodiments, the VH region (heavy chain variable region) and the VL region (light chain variable region) can also be further divided into regions with high variability (called complementarity-determining regions (CDRs)), interspersed with relatively conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) in each heavy chain and light chain respectively form the antigen-binding site.

[0022] The present invention provides a nucleic acid molecule encoding the anti-CD4 protein antibody, its fragment or fusion protein described above.

[0023] Further, the nucleic acid molecule includes DNA, cDNA, mRNA, recombinant nucleic acid.

[0024] Further, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0025] In some specific embodiments, the nucleic acid molecule includes conservative nucleic acid substitutions. The conservative nucleic acid substitutions refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or in the case where the nucleic acid molecule does not encode an amino acid sequence, substantially the same or related sequences. In some specific embodiments, the nucleic acid molecule utilizes the degeneracy of the genetic code, and a large number of functionally identical nucleic acids encode most proteins.

[0026] The present invention provides a vector comprising the nucleic acid molecule described above.

[0027] Further, the vector includes retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses, plasmids.

[0028] In some embodiments, the choice of the specific vector will depend on the target cell and the conditions being treated.

[0029] The present invention provides a cell comprising the nucleic acid molecule described above and / or the vector described above.

[0030] Further, the cell includes eukaryotic cells and prokaryotic cells.

[0031] Further, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0032] Further, the eukaryotic cells include yeast cells, mammalian cells, and insect cells.

[0033] Further, the mammalian cells include humans, monkeys, mice, rats, hamsters, goats, sheep, cows, pigs, dogs, and cats.

[0034] Further, the eukaryotic cells include human cells, mouse cells, rat cells, hamster cells, goat cells, sheep cells, or any other cells.

[0035] In some embodiments, the cells further include artificially prepared cell lines.

[0036] The present invention provides any one of the following applications:

[0037] 1) Use of the anti-CD4 protein antibody, its fragment or fusion protein, the nucleic acid molecule, the vector, or the cell described above in the production of a drug for treating or preventing a disease or disorder caused by the CD4 protein;

[0038] 2) Use of the anti-CD4 protein antibody, its fragment or fusion protein, the nucleic acid molecule, the vector, or the cell described above in the production of a product for detecting the CD4 protein;

[0039] 3) Use of the anti-CD4 protein antibody, its fragment or fusion protein, the nucleic acid molecule, the vector, or the cell described above in the production of a cell sorting product;

[0040] Further, the product includes a kit, test strip, nucleic acid membrane strip, chip, system, or device.

[0041] Further, the cell sorting includes flow cytometry or cell sorting used in the detection of cell surface markers.

[0042] The present invention provides a pharmaceutical composition, which includes the anti-CD4 protein antibody, its fragment or fusion protein, the nucleic acid molecule, the vector, or the cell described above;

[0043] Further, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent, buffer, or excipient.

[0044] Furthermore, the pharmaceutical composition is administered systemically, specifically via the intranasal route, intravenous route, intraperitoneal route, intramuscular route, intradermal route, subcutaneous route, subdermal route, transdermal route, intracranial route, mucosal route, anal route, vaginal route, oral route, buccal route or they can be inhaled for delivery.

[0045] The present invention provides any one of the following methods:

[0046] 1) A method for expressing the anti-CD4 protein antibody, its fragment or fusion protein described above, the method comprising using the nucleic acid molecule, the vector or the cell described above to express the anti-CD4 protein antibody, its fragment or fusion protein described above, and finally classifying and purifying to obtain the product;

[0047] 2) A method for detecting CD4 protein in a sample, the method comprising the step of contacting the sample with the anti-CD4 protein antibody, its fragment or fusion protein described above;

[0048] 3) A method for cell sorting, the method comprising contacting the anti-CD4 protein antibody, its fragment or fusion protein described above with pre-sorted cells, and then separating the target cells by immobilization of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the result diagram of plasmid electrophoresis;

[0050] Figure 2 is the electrophoretic analysis diagram of CD4 protein expression;

[0051] Figure 3 is the result diagram of Ni affinity purification electrophoresis of recombinant human CD4 protein;

[0052] Figure 4 is the result diagram of ELISA titer detection of immunized mice;

[0053] Figure 5 is the result diagram of detection of anti-CD4 monoclonal antibody in affinity-purified ascites;

[0054] Figure 6 is the result diagram of identifying antibody subclass by strip method;

[0055] Figure 7 is the result diagram of detecting antibody sensitivity by indirect ELISA method. DETAILED DESCRIPTION OF THE INVENTION

[0056] As used herein, the terms "sequence identity" and "identity" are synonymous and refer to the "percent sequence identity" or "percent identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since gaps are not nucleotides, gaps present in the target sequence are not counted. Similarly, gaps present in the reference sequence are not counted since nucleotides from the target sequence are counted and nucleotides from the reference sequence are not. At least 60% sequence identity includes contiguous segments having sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the full length of the sequence.

[0057] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain may also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain CL. The constant domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions.

[0058] The term "nucleic acid" as used in the present invention is intended to include polymeric forms of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides and / or their analogs, which include DNA, RNA and DNA / RNA hybrids, and which also include DNA or RNA analogs.

[0059] As used herein, the term "vector", when the above-mentioned isolated nucleic acid molecule is ligated to the vector, can directly or indirectly connect the nucleic acid sequence to the regulatory elements on the vector, as long as these regulatory elements can regulate the translation and expression of the nucleic acid molecule, etc. Of course, these regulatory elements can come directly from the vector itself or can be exogenous, i.e., not from the vector itself.

[0060] As used herein, the terms "treatment" or "prevention" refer to any type of intervention or treatment administered to an individual, or the administration of an active agent to a subject, intended to reverse, mitigate, improve, inhibit, or slow down or prevent the progression, occurrence, exacerbation, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease.

[0061] As used herein, the terms "biological sample" or "sample" refer to a biological composition obtained from or sourced from a subject of interest and containing (e.g.) cells and / or other molecular entities that are identified and / or characterized based on physical, biochemical, chemical, and / or physiological characteristics. Biological samples include (but are not limited to) cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those skilled in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal mucus and sputum), peritoneal fluid, pleural fluid, thoracic fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lung, kidney, skin, or blood vessels of a subject.

[0062] Example 1 Recombinant Expression and Purification of Human CD4 Protein

[0063] 1. Construction of Expression Plasmid

[0064] 1.1 Expression Sequence Information

[0065] Protein sequence number: AAH25782, expression interval: Lys26 - Pro396, tag: 6×his-tag at the C-terminus, predicted molecular weight 42.1 kDa.

[0066] The expressed protein sequence (SEQ ID NO:19) is:

[0067] KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPHHHHHH。

[0068] Using HEK293 as the host, the gene sequence was optimized, and the optimized expressed gene sequence is:

[0069] SEQ ID NO:20

[0070]

[0071] SEQ ID NO:21

[0072]

[0073] 1.2 Vector construction and large-scale plasmid extraction

[0074] 1.2.1 Using pCDNA3.4 as the expression vector, with the Mouse Ig Kappa signal peptide: METDTLLLWVLLLWVPGSTGD, construct expression vectors with open reading frames of sequence 2 and sequence 3 respectively, named pTL-CD4-1 and pTL-CD4-2 respectively.

[0075] 1.2.2 Transform the above expression plasmids into Escherichia coli TOP10, and use the endotoxin-free plasmid large-scale extraction kit DP120-01 from Tiangen Biotech to extract the plasmids on a large scale. Elute the plasmids with endotoxin-free water, detect the concentration and endotoxin of the plasmids respectively. After sterile filtration, they can be used for transient expression.

[0076] 1.2.3 Large-scale plasmid extraction

[0077] The plasmid electrophoresis results are as Figure 1 shown.

[0078] 2. Transient expression

[0079] 2.1 Main materials

[0080] HEK293 cells (human embryonic kidney cells), self-preserved by our company; HEK293 serum-free medium (product number: AS-11), from Tongli Haiyuan; HEK-93 expression feeding medium (product number: AS-18), from Tongli Haiyuan; transfection reagent TA-293 (product number: K20001), from Zhuhai Kairui; recombinant protein expression enhancer KE-293 (product number: K30001), from Zhuhai Kairui; glutamine (product number: 21051024), from Gibco; expression plasmids: the plasmids extracted in step 1.1.2 (pTL-CD4-1, pTL-CD4-2). Cell culture flasks, from Corning; cell counter, from Contstar; cell culture shaker, from Eppendorf S41i; large-capacity centrifuge, from Hunan Xiangyi GL-21M.

[0081] 2.2 Methods

[0082] 2.2.1 Cell culture

[0083] Resuscitate HEK293 cells, and perform subculture when the cell culture density reaches 3 - 4×10 6 / mL. According to the cell density, add fresh 293 serum-free medium to dilute the cell density to 0.4 - 0.7×10 6 / mL, and culture in a cell culture shaker (37°C, CO 2 concentration 5%). Requirements: The volume of the medium does not exceed 1 / 5 of the volume of the culture flask.

[0084] 2.2.2 Transient transfection and feeding

[0085] 1) On the day of transfection, detect the cell density and viability, and adjust the cell density to 3×10 6 / mL;

[0086] 2) Take 500 µg of plasmid (pTL-CD4-1 plasmid, 1057.1 µg / mL, 475 µL), add it to a 50 mL centrifuge tube (tube A) containing 15 mL of 293 serum-free medium prepared in advance, and gently pipette to mix evenly; take 2.5 mL of TA-293, add it to a 50 mL centrifuge tube (tube B) containing 15 mL of 293 serum-free medium prepared in advance, and gently pipette to mix evenly;

[0087] 3) Mix the liquids in tube A and tube B, gently mix evenly, and let stand at room temperature for 10 min to prepare the transfection reagent-plasmid complex;

[0088] 4) Slowly add the transfection reagent-plasmid complex to the cells with the pre-adjusted density using a pipette, shaking the cell flask while adding to evenly disperse the complex. After completion, transfer the flask to a shaker and start culturing. Culture conditions: 5% CO 2 , 37 °C, 120 rpm;

[0089] 5) After culturing for 24 h (Day1), detect the cell density and viability, add 3 mL of KE-293 and 25 mL of feeding medium, and continue culturing;

[0090] 6) On Day3, detect the cell density and viability, add 25 mL of feeding medium, and continue culturing;

[0091] 7) On Day5 or when the cell viability is lower than 70%, end the expression, transfer the broth to a clean centrifuge bottle, centrifuge at 8000 rpm for 20 min at room temperature, and harvest the cell supernatant for purification.

[0092] 2.3 Results

[0093] The detection of cell status during the CD4 protein expression stage is shown in Table 1.

[0094] Table 1

[0095]

[0096] The electrophoretic analysis of CD4 protein expression is as Figure 2 shown.

[0097] 3. Purification

[0098] 3.1 Main materials

[0099] Metal chelation chromatography: GE, HisTrap HP (5 mL pre-packed column), 17524801; Desalting chromatography: Borglong, G-25 (100 mL), C0078; Imidazole: Sigma, 56749-1kg; Chromatography system, GE, AKTA Purifier 100;

[0100] 3.2 Method

[0101] 3.2.1 Metal chelation chromatography

[0102] 1) Sample treatment: The cell fermentation broth (cell feed solution) harvested by centrifugation was filtered through a 0.45 μm filter, and then 5 mL of eluent (i.e., 100% B solution: 50 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 8.0) was added to make the imidazole concentration in the feed solution 5 mM;

[0103] 2) Pre-packed column treatment: Flush with water at 5 mL / min for 10 CV; Flush the pre-packed column with the equilibration solution (i.e., 100% A solution: 50 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 8.0) until the UV280 value is stable at 5 mL / min;

[0104] 3) Loading: The retention time was 2 min, i.e., the flow rate was set at 2.5 mL / min, and the flow-through was collected;

[0105] 4) Equilibration: Flush with the equilibration solution at 5 mL / min until the baseline is stable;

[0106] 5) Washing: Flush with the washing solution (50 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 8.0) at 5 mL / min until the absorption is stable;

[0107] 6) Equilibration: Flush with the equilibration solution at 5 mL / min until the baseline is stable;

[0108] 7) Washing: Flush with the washing solution at 5 mL / min until the absorption is stable;

[0109] 8) Gradient elution: Elute at 5 mL / min with the imidazole concentration gradient from 20%, 30%, 50% to 100% in sequence, and collect the peaks;

[0110] 9) Regeneration: Flush with 0.2 M EDTA-Na2 solution at 5 mL / min for 10 column volumes;

[0111] 10) Storage: Flush with water at 5 mL / min for 10 column volumes, and finally soak the pre-packed column in 20% ethanol.

[0112] 3.2.2 G25 buffer exchange

[0113] 1) Column treatment: 5 mL / min, wash with water injection for 10 CV;

[0114] 2) CIP: 8 mL / min, wash with CIP solution for 10 CV;

[0115] 3) Equilibration: 8 mL / min, wash with desalting chromatography equilibration buffer (PBS, pH 7.4) until the baseline is stable;

[0116] 4) Sample loading: 5 mL / min, volume 25 mL;

[0117] 5) Equilibration: 8 mL / min, collect protein peak, 100 mAu - 1068 mAu - 150 mAu, volume 10 mL;

[0118] 6) Preservation: 8 mL / min, wash with water injection for 10 CV; 5 mL / min, wash with 20% ethanol for 10 CV.

[0119] 3.3 Results

[0120] SDS - PAGE electrophoresis was used to analyze the components eluted from the Ni column with an imidazole gradient. After one - step affinity purification, the purity of the eluate with 20%B was >90%. After buffer exchange with G25, the protein concentration was quantitatively detected by BCA to be 1.63 mg / mL. The results are as Figure 3 shown. From Figure 3 the results, it can be seen that we purified high - purity CD4 protein ( Figure 3 lane 1 of the elution).

[0121] Example 2 Immunize mice with CD4 protein and detect the serum antibody titer

[0122] 1. Immunize mice and monitor the serum titer of mice

[0123] 1.1 Main materials

[0124] Female BALB / c mice (6 - 8 weeks old), Sparrow Bioscience (Beijing) Biotechnology Co., Ltd.; Freund's complete adjuvant, Freund's incomplete adjuvant, mAb subclass identification kit, Sigma; goat anti - mouse IgG / HRP, TMB chromogenic reagent, Beijing ComWin Biotech Co., Ltd.

[0125] 1.2 Methods

[0126] 1.2.1 Animal immunization protocol: On day 0, 100 μg of recombinant human CD4 protein was taken, mixed with an equal volume of Freund's complete adjuvant and emulsified thoroughly, and then intraperitoneally injected into 6-8-week-old female BALB / c mice. On day 14, after thoroughly emulsifying 50 μg of CD4 protein with Freund's incomplete adjuvant, the mice were immunized again in the same way as before; on day 28, a booster immunization was carried out with the same operation as the second immunization; on day 35, blood was collected from the orbital cavity of the mice, serum was separated, and the antibody titer of the antiserum was detected by ELISA.

[0127] 1.2.2 Detection of serum antibody titer by ELISA

[0128] 1) Prepare an enzyme-linked immunosorbent assay (ELISA) plate, add 100 μL of CD4 protein (diluted to a concentration of 2 μg / mL with coating buffer) to each well, and incubate overnight at 4 °C;

[0129] 2) Discard the coated protein, add 300 μL of blocking solution (5% skim milk powder) to each well, and block at room temperature for 2 h;

[0130] 3) After discarding the blocking solution, wash the plate with TBST (TBS buffer containing 0.05% Tween-20), 2 min / time, for a total of 3 times;

[0131] 4) Dilute the serum with PBS buffer, perform a 4-fold serial dilution starting from 1:500, add the diluted serum to each well of the ELISA plate at 100 μL / well, and incubate at 37 °C for 2 h;

[0132] 5) Discard the serum in the wells, wash the plate with TBST in the same way as before, then add HRP-labeled goat anti-mouse antibody diluted 1:10000 to each well, and incubate at 37 °C for 1 h;

[0133] 6) After washing the plate in the same method as above, add freshly prepared TMB chromogenic solution to the wells and develop color in the dark;

[0134] 7) Read the absorbance value of each well at 450 nm, and take the ratio of OD sample / OD control (normal serum) ≥ 2.1 as ELISA positive.

[0135] 1.3 Results

[0136] Judged by ELISA, after three immunizations, the serum titers of the immunized mice all reached or exceeded 1:128000, meeting the cell fusion conditions. The specific ELISA titer detection results of the immunized mice are as Figure 4 shown.

[0137] Example 3 Screening of hybridoma cell lines capable of secreting anti-human CD4 antibodies

[0138] 1. Hybridoma cell fusion and subclonal screening

[0139] 1.1 Main materials

[0140] Mouse myeloma cell line SP2 / 0, stored in-house; fetal bovine serum, Hyclon; RPMI1640 medium, HAT, HT, fusogen PEG3350, etc., Gibco; goat anti-mouse IgG / HRP, Beijing ComWin Biotech Co., Ltd.; low molecular weight protein Marker, Thermo; primiparous Balb / c mice, female, 12 - 16 weeks old, Spiffbio Technology Co., Ltd.; liquid paraffin, Shanghai Sangon Biotech Co., Ltd.

[0141] 1.2 Methods

[0142] 1) Select mice with high serum antibody titers and collect spleen cells; at the same time, centrifuge to harvest SP2 / 0 cells. Mix spleen cells and SP2 / 0 cells at a ratio of 10:1, centrifuge to discard the supernatant, gently flick the bottom of the tube to disperse the cells, and add 1 mL of PEG3350 drop by drop. Gently shake the centrifuge tube to promote cell fusion. Let it stand for a while, add an appropriate amount of RPMI1640 medium (200 mL / L serum, 20 mL / L HAT), transfer the cell suspension to a 96-well culture plate pre-seeded with feeder cells, and culture it in a 37℃ CO 2 incubator.

[0143] 2) Use the ELISA method to screen positive wells from the fusion plate, and perform subcloning culture of hybridoma cells by the limiting dilution method. After 2 subclonings, the detected positive cells are passaged and amplified for culture.

[0144] 1.3 Results

[0145] The results of positive clones screened by ELISA are shown in Table 2.

[0146] Table 2

[0147]

[0148] The hybridoma cells were sequenced to obtain an anti-CD4 antibody, and the specific sequence is shown in Table 3.

[0149] Table 3

[0150]

[0151] 2. Preparation and identification of anti-human CD4 monoclonal antibody

[0152] 2.1 Main materials

[0153] Hybridoma cells, cells amplified and cultured in step 5; female, 12 - 16 weeks old, Speywood Biotechnology Co., Ltd.; liquid paraffin, Sangon Biotech (Shanghai) Co., Ltd.; MabSelectSure antibody affinity purification resin, GE; Quick Antibody Isotyping Kit (mouse), Thermo.

[0154] 2.2 Methods

[0155] 1) Amplify and culture hybridoma cells;

[0156] 2) For primiparous mice, inject liquid paraffin intraperitoneally at 0.5 mL / mouse. The immunization day is Day0;

[0157] 3) On Day7, collect the hybridoma cells amplified and cultured, adjust the density to 2×10^6 / mL, inject 0.5 mL of the cell suspension into the peritoneal cavity of each primiparous mouse. Start monitoring the status of the mice on Day11 and collect ascites in a timely manner;

[0158] 4) Purify the ascites with protein A affinity resin to obtain anti - human CD4 monoclonal antibody, and quantify it by BCA method.

[0159] 5) Use the Quick Antibody Isotyping Kit to detect the antibody subclass.

[0160] 6) Detect the antibody affinity by competitive ELISA: Coat the CD4 protein, block it with 5% non - fat milk powder at room temperature for 2 h, and wash with TBST; In a row of EP tubes, establish a concentration gradient of CD4 protein solution from 0.1 nmol / L - 1 μmol / L using the limited dilution method, add 0.5 μmol / L of the antibody solution to make the total volume 100 μl, incubate at room temperature for 30 min. Add 90 μl of the reaction mixture to the previously antigen - coated microplate (30 μl of 30% blocking solution was pre - added to the microplate), and incubate for 1 h. Wash the antigen plate thoroughly; Add the enzyme - labeled secondary antibody and incubate for 1 h; After washing the antigen plate thoroughly with TBST, add the TMB chromogenic solution, terminate the chromogenic reaction after 10 min, and read the OD450 of each well; Record the measured OD values, plot a fitting curve in combination with the antigen concentration of gradient dilution, and find the antigen concentration corresponding to the point where the signal is half of the strongest signal, which is the dissociation constant Kd.

[0161] 7) Detect the target specificity of the antibody by Western Blot: Collect CD4 +T cells were treated with cell lysate, and the protein lysate was harvested as test sample 1. NK cells amplified and cultured with the NK culture kit were collected and lysed in the same manner as the previous step to serve as test sample 2. The antigen for mouse immunization (recombinant human CD4 protein) was sample 3. After protein electrophoresis, membrane transfer, and blocking, the three samples were incubated with the antibody purified in the previous step as the primary antibody and anti-human IgG-Fc / HRP as the secondary antibody. After developing with the hypersensitive ECL luminescent solution, they were exposed in a darkroom to examine the specificity of the antibody.

[0162] 2.2.1 Affinity purification diagram of ascites antibody

[0163] After affinity purification, the purified anti-CD4 monoclonal antibody was obtained. The results are as Figure 5 shown.

[0164] 6.2.2 Antibody subclass detection results

[0165] The antibody subclass was identified by the test strip method. The typing test results of the anti-CD4 monoclonal antibody showed that this antibody belongs to the IgG1 subclass. The specific results are as Figure 6 shown.

[0166] 6.2.3 Antibody sensitivity and affinity detection results

[0167] The sensitivity of the antibody was detected by the indirect ELISA method, divided into 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 showed that the antibody could detect CD4 protein at 100 ng / L. The specific results are as Figure 7 shown.

Claims

1. An anti-CD4 protein antibody or a fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an H-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 1, an H-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 2, and an H-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprises an L-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 4, an L-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 5, and an L-CDR3 comprising an amino acid sequence as shown in SEQ ID NO:

6.

2. The anti-CD4 protein antibody or fragment thereof according to claim 1, wherein the heavy chain variable region comprises one or more heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, and / or one or more light chain framework regions LFR1, LFR2, LFR3 and LFR4, wherein the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have homologous sequences with at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7-10, and the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have homologous sequences with at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 11-14.

3. The anti-CD4 protein antibody or fragment thereof according to claim 1, wherein the amino acid sequences of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 7-10, respectively, and the amino acid sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 11-14, respectively.

4. The anti-CD4 protein antibody or fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or a homologous sequence thereof having at least 85% sequence identity; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or a homologous sequence thereof having at least 85% sequence identity.

5. The anti-CD4 protein antibody or fragment thereof according to claim 1, wherein the heavy chain variable region and the heavy chain constant region comprise the amino acid sequence shown in SEQ ID NO: 17, or a homologous sequence thereof having at least 85% sequence identity; the light chain variable region and the light chain constant region comprise the amino acid sequence shown in SEQ ID NO: 18, or a homologous sequence thereof having at least 85% sequence identity.

6. The anti-CD4 protein antibody or fragment thereof according to claim 1, which is a diabody, Fab, Fab', F(ab')2, Fv fragment, (dsFv)2, single-chain antibody molecule, or scFv dimer.

7. The anti-CD4 protein antibody or fragment thereof according to claim 1, wherein the anti-CD4 protein antibody or fragment thereof is derived from human or non-human mammals.

8. The anti-CD4 protein antibody or fragment thereof according to claim 1, further comprising one or more replacement or modification of amino acid residues that do not affect binding specificity.

9. A nucleic acid molecule encoding the anti-CD4 protein antibody or a fragment thereof according to any one of claims 1 to 8. A vector comprising the nucleic acid molecule according to claim 9.

11. A cell comprising the nucleic acid molecule of claim 9 and / or the vector of claim 10.

12. Use of the anti-CD4 protein antibody or a fragment thereof according to any one of claims 1 to 8 in producing a product for detecting CD4 protein.

13. Use of the anti-CD4 protein antibody or a fragment thereof according to any one of claims 1 to 8 in producing a cell sorting product. 14 . The use according to claim 13 , wherein the cell sorting comprises cell sorting used in flow cytometry or detection of cell surface markers.

15. A method for expressing the anti-CD4 protein antibody or fragment thereof according to any one of claims 1 to 8, the method comprising using the nucleic acid molecule according to claim 9, the vector according to claim 10 or the cell according to claim 7 to express the anti-CD4 protein antibody or fragment thereof according to claims 1 to 8, and finally classifying and purifying to obtain the product.

16. A method for detecting CD4 protein in a sample, comprising the step of contacting the sample with the anti-CD4 protein antibody or fragment thereof according to any one of claims 1 to 8.

17. A method for cell sorting, comprising contacting the anti-CD4 protein antibody or a fragment thereof according to any one of claims 1 to 8 with pre-sorted cells, and then isolating target cells by fixing the antibody.

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