Antibody of anti-CD4 protein, fragment or fusion protein of antibody and application of fragment or fusion protein

Abstract

The invention discloses an anti-CD4 protein antibody, a fragment or a fusion protein thereof and application thereof, and provides the anti-CD4 protein antibody, the fragment or the fusion protein thereof, the anti-CD4 protein antibody comprises H-CDR1, H-CDR2 and H-CDR3 of a heavy chain variable region, and the sequences of the H-CDR1, the H-CDR2 and the H-CDR3 are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; comprising L-CDR1, L-CDR2 and L-CDR3 of a light chain variable region, and the sequences of the L-CDR1, the L-CDR2 and the L-CDR3 are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The invention also provides a nucleic acid molecule capable of coding the antibody, the fragment or the fusion protein of the antibody, a carrier for carrying the nucleic acid molecule, and a cell containing the nucleic acid molecule and / or the carrier, and also provides related applications and methods of the antibody, the fragment or the fusion protein of the antibody.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an anti-CD4 protein antibody, a fragment or fusion protein thereof and applications 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. It plays a key role in the process of T cells recognizing antigens and initiating immune responses. CD4 molecules help T cells recognize and attack foreign pathogens, such as viruses and bacteria, by binding to MHC class II molecules on antigen-presenting cells. 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 cells recognizing antigens and initiating immune responses. CD4 molecules help T cells recognize and attack foreign pathogens, such as viruses and bacteria, by binding to MHC class II molecules on antigen-presenting cells. CD4 + T cells are key players in the human immune system, able to recognize foreign antigens presented by antigen-presenting cells (APCs) and respond. This response can regulate other immune cells, such as B cells or CD8 + T cell activity can also initiate new immune responses. + T cells can differentiate into a variety of effector T cell subsets, such as Th1, Th2, Th17 and Treg, to perform different immune functions. + The activation and differentiation of T cells are crucial for the successful initiation of immune responses. CD4 antibodies can be used in immunohistochemistry, immunoblotting, immunocytochemistry, and immunoprecipitation to detect CD4 proteins, 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 the 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 reasons such as non-specific binding and poor antibody affinity, which lead to insufficient detection sensitivity and low sorting efficiency.

[0004] In order 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, 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, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the H-CDR; 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, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the L-CDR.

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

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

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

[0009] Further, 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;

[0010] Furthermore, 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.

[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 thereto; 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 thereto;

[0012] Furthermore, the heavy chain variable region and 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 thereto; the light chain variable region and 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 thereto.

[0013] Furthermore, the light chain variable region and 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 parts of the antibody heavy chain in the Fab fragment (antigen binding fragment).

[0015] In some embodiments, the "percentage of sequence identity" can be calculated by comparing the two best aligned sequences in the window, determining the number of positions where 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) 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., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

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

[0017] Furthermore, the anti-CD4 protein antibody, its fragment or fusion protein is derived from human or non-human mammals;

[0018] In some embodiments, the non-human mammal refers to a viviparous animal other than humans, including mice, sheep, rabbits, horses, cows, pigs, dogs, monkeys, etc.

[0019] Furthermore, the anti-CD4 protein antibody, its fragment or fusion protein is derived from mice.

[0020] Furthermore, the anti-CD4 protein antibody, fragment or fusion protein thereof also includes one or more replacement or modification of amino acid residues that do not affect binding specificity.

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

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

[0023] Furthermore, the nucleic acid molecules include DNA, cDNA, mRNA, and recombinant nucleic acid.

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

[0025] In some specific embodiments, the nucleic acid molecules include conservative nucleic acid substitutions. The conservative nucleic acid substitutions refer to those nucleic acids that encode 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 molecules utilize 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 above-mentioned nucleic acid molecule.

[0027] Furthermore, the vector includes retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus, plasmid.

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

[0029] The present invention provides a cell, wherein the cell comprises the aforementioned nucleic acid molecule and / or the aforementioned vector.

[0030] Furthermore, the cells include eukaryotic cells and prokaryotic cells.

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

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

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

[0034] Furthermore, 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 also include artificially prepared cell lines.

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

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

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

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

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

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

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

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

[0044] Further, the pharmaceutical composition is administered systemically, specifically via 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 delivered by inhalation.

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

[0046] 1) A method for expressing the aforementioned anti-CD4 protein antibody, its fragment or fusion protein, which comprises using the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned cell to express the aforementioned anti-CD4 protein antibody, its fragment or fusion protein, 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, fragment thereof or fusion protein as described above;

[0048] 3) A method for cell sorting, which comprises contacting the aforementioned anti-CD4 protein antibody, its fragment or fusion protein with pre-sorted cells, and then isolating target cells by fixing the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0050] Figure 2 It is the electrophoresis analysis of CD4 protein expression;

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

[0052] Figure 4 This is a graph showing the ELISA titer test results of immunized mice;

[0053] Figure 5 This is a graph showing the results of anti-CD4 monoclonal antibody detection in affinity-purified ascites;

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

[0055] Figure 7 This is the result chart of antibody sensitivity detection by indirect ELISA method. DETAILED DESCRIPTION

[0056] The terms "sequence identity" and "identity" as used herein are synonymous and refer to the "percentage of sequence identity" or "percentage of identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences in the comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position in which the same nucleotides are present in both the target sequence and the reference sequence. Since the gap is not a nucleotide, the gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides are counted, and the nucleotides from the reference sequence are not counted, the gap present in the reference sequence is not counted. At least 60% sequence identity includes 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 total length of the sequence having a continuous segment with sequence identity.

[0057] The term "antibody" as used herein is generally an immunoglobulin molecule consisting 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 κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain may also include a "D" region of about 3 or more amino acids. 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 (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions.

[0058] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also including DNA or RNA analogs.

[0059] The term "vector" as used herein refers to the direct or indirect connection of the nucleic acid sequence with the regulatory elements on the vector when the above-mentioned isolated nucleic acid molecule is connected to the vector, as long as these regulatory elements can regulate the translation and expression of the nucleic acid molecule. Of course, these regulatory elements can be directly derived from the vector itself, or they can be exogenous, that is, not derived from the vector itself.

[0060] As used herein, the terms "treatment" or "prevention" refer to any type of intervention or treatment performed on an individual, or the administration of an active agent to a subject, with the intent to reverse, alleviate, ameliorate, inhibit, or slow or prevent the progression, occurrence, aggravation or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease.

[0061] As used herein, the term "biological sample" or "sample" refers to a biological composition containing cells and / or other molecular entities that are identified and / or differentiated, for example, based on physical, biochemical, chemical and / or physiological characteristics, obtained or derived from a subject of interest. 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, pleural fluid, saliva, urine, joint fluid, cerebrospinal fluid (CSF), thoracentesis fluid, peritoneal 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. Expression plasmid construction

[0064] 1.1 Expressed sequence information

[0065] Protein sequence number: AAH25782, expression range: Lys26-Pro396, tag: C-terminal with 6×his-tag, predicted molecular weight 42.1kDa.

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

[0067] KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQ LLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKK EGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPHHHHHHH.

[0068] HEK293 was used as the host to optimize the gene sequence. The optimized expression gene sequence is:

[0069] SEQ ID NO:20

[0070]

[0071] SEQ ID NO:21

[0072]

[0073] 1.2 Vector construction and plasmid extraction

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

[0075] 1.2.2 The above expression plasmids were transformed into Escherichia coli TOP10, and the plasmids were extracted using Tiangen Bio's endotoxin-free plasmid extraction kit DP120-01. The plasmids were eluted with endotoxin-free water, and the concentration and endotoxin of the plasmids were tested respectively. After sterile filtration, they can be used for transient expression.

[0076] 1.2.3 Large-scale plasmid extraction

[0077] Plasmid electrophoresis results are as follows Figure 1 shown.

[0078] 2. Transient expression

[0079] 2.1 Main Materials

[0080] HEK293 cells (human embryonic kidney cells), stored by our company; HEK293 serum-free medium (catalog number: AS-11), Tongli Haiyuan; HEK-93 expression feed medium (catalog number: AS-18), Tongli Haiyuan; transfection reagent TA-293 (catalog number: K20001), Zhuhai Kairui; recombinant protein expression enhancer KE-293 (catalog number: K30001), Zhuhai Kairui; glutamine (catalog number: 21051024), Gibco; expression plasmid: plasmid extracted in step 1.1.2 (pTL-CD4-1, pTL-CD4-2). Cell shaker, Corning; cell counter, Contstar; cell culture shaker, EppendorfS41i; large-capacity centrifuge, Hunan Xiangyi GL-21M.

[0081] 2.2 Methods

[0082] 2.2.1 Cell culture

[0083] 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 0.4-0.7×10 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.

[0084] 2.2.2 Transient Transformation and Feeding

[0085] 1) On the day of transfection, detect 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) and add it to a 50 mL centrifuge tube (tube A) pre-prepared with 15 mL of 293 serum-free medium, and gently pipette to mix; take 2.5 mL of TA-293 and add it to a 50 mL centrifuge tube (tube B) pre-prepared with 15 mL of 293 serum-free medium, and gently pipette to mix;

[0087] 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;

[0088] 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;

[0089] 5) After culturing for 24 h (Day 1), the cell density and viability were detected, 3 mL KE-293 and 25 mL feed medium were added, and the culture was continued;

[0090] 6) Day 3, check cell density and viability, add 25 mL of feed medium, and continue culturing;

[0091] 7) On Day 5 or when the cell viability is lower than 70%, the expression is terminated and the feed solution is transferred into a clean centrifuge bottle and centrifuged at 8000 rpm for 20 min at room temperature to harvest the cell supernatant for purification.

[0092] 2.3 Results

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

[0094] Table 1

[0095]

[0096] Electrophoretic analysis of CD4 protein expression Figure 2 shown.

[0097] 3. Purification

[0098] 3.1 Main Materials

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

[0100] 3.2 Methods

[0101] 3.2.1 Metal chelate chromatography

[0102] 1) Sample treatment: The cell fermentation liquid (cell feed liquid) harvested by centrifugation was filtered through 0.45 μm, and 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 liquid 5 mM;

[0103] 2) Pre-packed column treatment: 5 mL / min, water injection, 10 CV; flush the pre-packed column with equilibrium 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, 5 mL / min;

[0104] 3) Sample loading: The retention time is 2 min, i.e. the flow rate is set to 2.5 mL / min, and the flow-through is collected;

[0105] 4) Balance: 5 mL / min, flush with balance solution until the baseline is stable;

[0106] 5) Elution: 5 mL / min, eluent (50 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 8.0) until the absorption is stable;

[0107] 6) Balance: 5 mL / min, flush with balance solution until the baseline is stable;

[0108] 7) Eluent: 5 mL / min, rinse until the absorption is stable;

[0109] 8) Gradient elution: 5 mL / min, imidazole concentration from 20%, 30%, 50% to 100%, in order of gradient elution, and the peak was collected;

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

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

[0112] 3.2.2 G25 fluid change

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

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

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

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

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

[0118] 6) Storage: 8 mL / min, water flushing, 10 CV; 5 mL / min, 20% ethanol flushing, 10 CV.

[0119] 3.3 Results

[0120] 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. Figure 3 As shown. Figure 3 The results show that we have purified high-purity CD4 protein ( Figure 3 Elution lane 1).

[0121] Example 2 Immunization of mice with CD4 protein and detection of serum antibody titer

[0122] 1. Immunization of mice and monitoring of mouse serum titer

[0123] 1.1 Main Materials

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

[0125] 1.2 Methods

[0126] 1.2.1 Animal immunization procedure: On day 0, 100 μg of recombinant human CD4 protein was mixed with an equal volume of Freund's complete adjuvant and fully emulsified, and then intraperitoneally injected into 68-week-old female BALB / c mice. On day 14, 50 μg of CD4 protein was fully emulsified with Freund's incomplete adjuvant and immunized again in the same manner as before; on day 28, booster immunization was performed in the same manner as the second immunization; on day 35, blood was collected from the mouse orbits, serum was separated, and the titer of antiserum antibodies was detected by ELISA.

[0127] 1.2.2 ELISA method to detect serum antibody titer

[0128] 1) Prepare an ELISA plate and add 100 μL of CD4 protein (coating solution diluted to a concentration of 2 μg / mL) to each well and incubate at 4°C overnight.

[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) for 2 min each time, for a total of 3 times;

[0131] 4) Dilute the serum with PBS buffer, starting from 1:500 and diluting it 4 times. Add 100 μL / well of the diluted serum to each well of the ELISA plate and incubate at 37°C for 2 h.

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

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

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

[0135] 1.3 Results

[0136] According to ELISA, after three immunizations, the serum titer of the immunized mice reached or exceeded 1:128000, meeting the cell fusion conditions. Figure 4 shown.

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

[0138] 1. Hybridoma cell fusion and subclone screening

[0139] 1.1 Main Materials

[0140] Mouse myeloma cells SP2 / 0 were stored in our company; fetal bovine serum was from Hyclon; RPMI1640 medium, HAT, HT, fusion agent PEG3350, etc. were from Gibco; goat anti-mouse IgG / HRP was from Beijing Kangwei Century Biotechnology Co., Ltd.; low molecular weight protein marker was from Thermo; multiparous Balb / c mice, female, 12-16 weeks old, were from Spaf Biotechnology Co., Ltd.; liquid paraffin was from Shanghai Shenggong Biotechnology Co., Ltd.

[0141] 1.2 Methods

[0142] 1) Select mice with high serum antibody titers and take spleen cells; at the same time, harvest sp2 / 0 cells by centrifugation. Mix spleen cells and SP2 / 0 cells at a ratio of 10:1, centrifuge and discard the supernatant, flick the bottom of the tube to break up the cells, add 1mL PEG3350 dropwise, and gently shake the centrifuge tube to promote cell fusion. Let it stand for a while, add an appropriate amount of RPMI1640 medium (200mL / L serum, 20mL / L HAT), transfer the cell suspension to a 96-well culture plate pre-inoculated with feeder cells, and culture it in a 37℃ CO2 incubator.

[0143] 2) The positive wells were screened from the fusion plate using the ELISA method, and the hybridoma cells were subcloned and cultured using the limiting dilution method. After two subclones, the positive cells were passaged and expanded.

[0144] 1.3 Results

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

[0146] Table 2

[0147]

[0148]

[0149] The hybridoma cells were sequenced to obtain anti-CD4 antibodies, the specific sequences of which are shown in Table 3.

[0150] Table 3

[0151]

[0152]

[0153] 2. Preparation and identification of anti-human CD4 monoclonal antibodies

[0154] 2.1 Main Materials

[0155] Hybridoma cells, cells cultured in step 5; female, 12-16 weeks old, Spefocin Biotechnology Co., Ltd.; liquid paraffin, Shanghai Sangon Biotechnology Co., Ltd.; MabSelectSure antibody affinity purification medium, GE; rapid antibody typing kit (mouse), Thermo.

[0156] 2.2 Methods

[0157] 1) Proliferation and culture of hybridoma cells;

[0158] 2) Multiparous mice were intraperitoneally injected with liquid paraffin, 0.5 mL / mouse, and the day of immunization was designated as Day 0;

[0159] 3) On Day 7, the hybridoma cells were collected and cultured, and the density was adjusted to 2×10^6 / mL. 0.5 mL of cell suspension was injected into the peritoneum of each multiparous mouse. The status of the mice was monitored from Day 11, and ascites was collected at an appropriate time.

[0160] 4) The ascites was purified by protein A affinity filler to obtain anti-human CD4 monoclonal antibody, which was quantified by BCA method.

[0161] 5) Use rapid antibody typing kit to detect antibody subclasses.

[0162] 6) Competitive ELISA method to detect antibody affinity: Coat CD4 protein, block with 5% skim milk powder at room temperature for 2 hours, wash with TBST; in a row of EP tubes, use the limiting dilution method to establish a CD4 protein solution with a concentration gradient from 0.1nmo / L to 1μmol / L, add 0.5μmol / L antibody solution to make the total volume 100μl, incubate at room temperature for 30min, add 90μl reaction mixture to the microwells coated with antigens (30μl of 30% blocking solution is pre-added to the microwells), incubate for 1h. Wash the antigen plate thoroughly; add enzyme-labeled secondary antibody and incubate for 1h; after washing the plate thoroughly with TBST, add TMB colorimetric solution, stop the colorimetric reaction after 10min, and read the OD450 of each well; record the measured OD value, draw a fitting curve based on the gradient diluted antigen concentration, and find the antigen concentration corresponding to the point with half of the strongest signal, which is the dissociation constant Kd.

[0163] 7) Western Blot detection of antibody target specificity: CD4 +T cells were treated with cell lysis buffer, and the protein lysate was harvested as sample 1 to be tested; NK cells amplified and cultured by the NK culture kit were collected and lysed in the same way as above as sample 2 to be tested; the antigen (recombinant human CD4 protein) used for mouse immunization 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 development with ultrasensitive ECL luminescent solution, they were exposed in a dark room to examine the specificity of the antibody.

[0164] 2.2.1 Affinity purification of ascites antibody

[0165] After affinity purification, the purified anti-CD4 monoclonal antibody was obtained. Figure 5 shown.

[0166] 6.2.2 Antibody subclass detection results

[0167] The test strip method identified the antibody subclass, and the typing test results of the anti-CD4 monoclonal antibody showed that the antibody belonged to the IgG1 subclass. Figure 6 shown.

[0168] 6.2.3 Antibody sensitivity and affinity test results

[0169] The sensitivity of the antibody detected by indirect ELISA is divided into 1:100mg / L; 2:10mg / L; 3:1mg / L; 4:100μg / L; 5:10μg / L; 6:1μg / L; 7:100ng / L; 8:0ng / L. The results show that the antibody can detect 100ng / L of CD4 protein. The specific results are as follows Figure 7 shown.

Claims

1. An anti-CD4 protein antibody, a fragment or a fusion protein 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, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the H-CDR; 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, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the L-CDR.

2. The anti-CD4 protein antibody, fragment or fusion protein thereof according to claim 1, wherein the heavy chain variable region comprises: H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region of the amino acid sequences shown in SEQ ID NO: 1, 2 and 3; the light chain variable region comprises: L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region of the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; Preferably, 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; Preferably, the amino acid sequences of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 are shown as SEQ ID NOs: 7-10, respectively, and the amino acid sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 are shown as SEQ ID NOs: 11-14, respectively.

3. The anti-CD4 protein antibody, fragment or fusion protein thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or a homologous sequence thereof having at least 85% sequence identity; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16, or a homologous sequence thereof having at least 85% sequence identity; Preferably, the heavy chain variable region and heavy chain constant region comprise the amino acid sequence shown in SEQ ID NO: 17, or a homologous sequence having at least 85% sequence identity thereto; the light chain variable region and 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 thereto.

4. The anti-CD4 protein antibody, fragment or fusion protein according to claim 1, which is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment, (dsFv)2, dual-specific dsFv, disulfide-stabilized diabody, single-chain antibody molecule, scFv dimer, multi-specific antibody, camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody; Preferably, the anti-CD4 protein antibody, fragment or fusion protein thereof is derived from human or non-human mammals; Preferably, the anti-CD4 protein antibody, fragment or fusion protein thereof further comprises one or more replacement or modification of amino acid residues that do not affect binding specificity.

5. A nucleic acid molecule encoding the anti-CD4 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4. A vector comprising the nucleic acid molecule according to claim 5 .

7. A cell comprising the nucleic acid molecule of claim 5 and / or the vector of claim 6.

8. Any of the following applications: 1) Use of the anti-CD4 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the cell according to claim 7 in the production of a drug for treating or preventing a disease or condition caused by CD4 protein; 2) Use of the anti-CD4 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the cell according to claim 7 in the production of a product for detecting CD4 protein; 3) Use of the anti-CD4 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the cell according to claim 7 in producing a cell sorting product; Preferably, the cell sorting includes cell sorting used in flow cytometry or detection of cell surface markers.

9. A pharmaceutical composition, comprising the anti-CD4 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the cell according to claim 7; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, buffer or excipient.

10. Any of the following methods: 1) A method for expressing the anti-CD4 protein antibody, fragment thereof or fusion protein according to any one of claims 1 to 4, the method comprising using the nucleic acid molecule according to claim 5, the vector according to claim 6 or the cell according to claim 7 to express the anti-CD4 protein antibody, fragment thereof or fusion protein according to claims 1 to 4, and finally classifying and purifying to obtain the product; 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, fragment thereof or fusion protein according to any one of claims 1 to 4; 3) A method for cell sorting, comprising contacting the anti-CD4 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4 with pre-sorted cells, and then isolating target cells by fixing the antibody.

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