Monoclonal antibody against CD28 and cell

Abstract

The invention discloses an anti-CD28 monoclonal antibody and a cell, belongs to the technical field of biology, and provides amino acid sequences of CDR regions of the anti-CD28 monoclonal antibody, the amino acid sequences are respectively a CDR region of a heavy chain variable region and a CDR region of a light chain variable region, specifically, the amino acid sequences of the heavy chain variable regions CDR1, 2 and 3 as shown in SEQ ID NO: 1, 2 and 3, and the amino acid sequences of the light chain variable regions CDR1, 2 and 3 as shown in SEQ ID NO: 9, STS and SEQ ID NO: 10.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an anti-CD28 monoclonal antibody and a cell. Background Art

[0002] CD28 is a 45 kDa homodimer composed of two 44 kDa polypeptide chains connected by disulfide bonds. It is mainly expressed on the surface of activated T cells and belongs to the Ig superfamily. CD28 mainly acts as a receiver of T cell co-stimulatory signals, binding to CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells (APCs) to provide co-stimulatory signals to T cells and promote immune responses. CD28 signaling can stimulate the expression of IL-2 and its receptors, as well as the cytotoxicity of CD3-activated T cells. CD28 is essential for the proliferation and differentiation of T cells, and also plays a key role in autoimmune tolerance and the long-term balance of the immune system. In tumor immunotherapy (such as CAR-T cell therapy) and immune checkpoint inhibitor applications, CD28 co-stimulatory signals are essential for the sustained activation of T cells.

[0003] CD28 antibodies can significantly enhance T cell activity by simulating the second signal that activates T cells, and combined with CD3 antibodies to form a therapy that has a stronger tumor-killing effect. This combination therapy is particularly important in tumor immunotherapy because it can provide the co-stimulatory signal required for T cell activation and enhance the ability to clear tumor cells. In CAR-T cell therapy, CD28 co-stimulatory signals are essential for the sustained activation of T cells. CD28 antibodies can be co-cultured with CAR-T cells to enhance their ability to kill tumor cells. CD19-CD28 dual antibodies can provide effective and safe co-stimulatory signals for T cells, enhancing the effect of CAR-T therapy. CD28 antibodies are also used in flow cytometry to analyze the activation state and function of T cells. By using CD28 antibodies, researchers can deeply explore the state and function of T cells, providing strong support for the development of immunotherapy and disease treatment.

[0004] Currently, most of the commercialized CD28 antibodies are only used for scientific research. Due to the risks of intellectual property rights, product development in the fields of cell therapy, especially CAR-T cell therapy and cell sorting magnetic beads, requires CD28 antibodies with independent intellectual property rights. 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 a monoclonal antibody against CD28 protein or a functional portion thereof, wherein the monoclonal antibody or the functional portion thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in the following CDR1, CDR2 and CDR3 sequences, and the light chain variable region comprises an amino acid sequence as shown in the following CDR1, CDR2 and CDR3 sequences: The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 2; The amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 3; The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO:9; The amino acid sequence of CDR2 of the light chain variable region is STS; The amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:10.

[0007] Furthermore, the monoclonal antibody or its functional part also includes a heavy chain variable region framework region and a light chain variable region framework region, the heavy chain variable region framework region includes the amino acid sequence shown in SEQ ID NO:4, 5, 6, 7, and the light chain variable region framework region includes the amino acid sequence shown in SEQ ID NO:11, 12, 13, 14.

[0008] In some embodiments, the FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, 5, 6, and 7, respectively. The FR1, FR2, FR3, and FR4 of the light chain variable region framework region have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, 12, 13, and 14, respectively.

[0009] Furthermore, the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7 in the heavy chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region, respectively, and the amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14 in the light chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the light chain variable region framework region, respectively.

[0010] Further, the monoclonal antibody or a functional part thereof comprises a heavy chain variable region of SEQ ID NO:8 and a light chain variable region of SEQ ID NO:15.

[0011] In the present invention, the anti-CD28 monoclonal antibody or its functional part can be a murine, chimeric, primatized, humanized or fully human antibody.

[0012] In the present invention, the anti-CD28 monoclonal antibody or its functional part can be a multimeric, heterodimeric, monovalent, bivalent, tetravalent or single-chain antibody.

[0013] In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the imported CDR or FR sequences. These modifications are made to further improve and optimize antibody performance. In general, humanized antibodies may contain substantially all of at least 1, and generally 2 variable domains, wherein all or substantially all CDR regions correspond to CDR regions of non-human immunoglobulins, and all or substantially all FR residues are FR residues of human immunoglobulin consensus sequences. Humanized antibodies may also optionally contain at least a portion of an immunoglobulin constant region (Fc), generally a human immunoglobulin constant region.

[0014] In the present invention, the anti-CD28 monoclonal antibody or its functional part is modified, and the modification may be glycosylation, including N-glycosylation, O-glycosylation and C-glycosylation. Glycosylation modification includes fucosylation modification, galactosylation modification and terminal sialic acid modification.

[0015] Glycosylation sites in Fc-containing polypeptides (e.g., antibodies (e.g., IgG antibodies)) can be identified by standard techniques. Identification of glycosylation sites can be experimental or based on sequence analysis or modeling data. Consensus motifs, i.e., amino acid sequences recognized by various glycosyltransferases, have been described. For example, the consensus motif for N-linked glycosylation motifs is typically NXT or NXS, where X can be any amino acid except proline. Several algorithms for locating potential glycosylation motifs have been described. Therefore, to identify potential glycosylation sites within an antibody or Fc-containing fragment, the sequence of the antibody is examined, for example, by using publicly available databases, such as the website provided by the Center for Biological Sequence Analysis (see the NetNGlyc server for predicted N-linked glycosylation sites, and the NetOGlyc server for predicted O-linked glycosylation sites).

[0016] As used herein, the term "CD28" includes intact CD28, soluble CD28, and fusion proteins comprising a functionally active portion of CD28.

[0017] As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, for example and without limitation, IgA, IgD, IgE, IgG, and IgM, and combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals (e.g., humans, goats, rabbits, and mice) as well as non-mammalian species (e.g., shark immunoglobulins). Unless otherwise specifically indicated, the term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" (e.g., having a binding constant for a molecule of interest greater than at least 10 compared to the binding constant for other molecules in a biological sample) that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. 3 M -1 , at least 10 4 M -1 or at least 10 5 M -1 The term "antibody" also includes genetically engineered forms, such as chimeric antibodies (e.g., murine or humanized non-primate antibodies), heteroconjugate antibodies (e.g., bispecific antibodies).

[0018] In terms of antibody structure, immunoglobulins have heavy (H) chains and light (L) chains that are interconnected by disulfide bonds. There are two types of light chains: λ and κ. There are five major heavy chain types (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE. Each heavy chain and light chain includes a constant region and a variable region (the region is also referred to as a "domain"). The heavy chain and light chain variable regions specifically bind antigens together. The heavy chain and light chain variable regions include a "framework" region separated by three highly variable regions, which are also referred to as "complementarity determining regions" or "CDRs." The scope of the framework region and CDR has been determined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conservative within species. The framework region of an antibody, i.e., the combined framework region of the constituent light and heavy chains, predominantly adopts a β-sheet conformation, while the CDRs form loops that connect the β-sheet structure or, in some cases, form part of the β-sheet. Thus, the framework region acts to form a scaffold that serves to position the CDRs in the correct orientation through interchain non-covalent interactions.

[0019] The CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus, and are also usually determined by the chain in which the particular CDR is located. Thus, the VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while the VL CDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to CLDN 18.2 will have specific VH and VL region sequences, and thus specific CDR sequences. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0020] As used herein, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the antibody population are identical except for possible naturally occurring mutations that may be present in small amounts. The modifier "monoclonal" refers to the characteristic of an antibody obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma (murine or human) method first described by Kohler et al., Nature, 256:495 (1975), or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol. Biol., 222:581-597 (1991).

[0021] Antibodies with altered (e.g., reduced or eliminated) effector function can be produced by engineering or producing antibodies with variant constant, Fc, or heavy chain regions. Recombinant DNA techniques and / or cell culture and expression conditions can be used to produce antibodies with altered function and / or activity. For example, recombinant DNA techniques can be used to engineer one or more amino acid substitutions, deletions, or insertions in regions (e.g., Fc or constant regions) that affect antibody function, including effector function.

[0022] The present invention provides an isolated polynucleotide comprising a heavy chain coding sequence encoding the heavy chain variable region of the aforementioned monoclonal antibody or a functional portion thereof, and a light chain coding sequence encoding the light chain variable region of the aforementioned monoclonal antibody or a functional portion thereof.

[0023] Furthermore, the polynucleotide comprises the following nucleotide sequence: The nucleotide sequences shown in SEQ ID NOs: 16, 17, and 18 are used to encode CDR1, CDR2, and CDR3 of the heavy chain variable region described above, respectively; The nucleotide sequences shown in SEQ ID NO:24, AGCACATCC, and SEQ ID NO:25 are used to encode CDR1, CDR2, and CDR3 of the light chain variable region described above, respectively.

[0024] Furthermore, the polynucleotide also includes the following nucleotide sequence: The nucleotide sequences shown in SEQ ID NOs: 19, 20, 21, and 22 are used to encode the heavy chain variable region framework region described above; The nucleotide sequences shown in SEQ ID NOs: 26, 27, 28 and 29 are used to encode the light chain variable region framework region described above.

[0025] Further, the nucleotide sequences shown in SEQ ID NOs: 19, 20, 21, and 22 in the heavy chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region, respectively; The nucleotide sequences shown in SEQ ID NOs: 26, 27, 28, and 29 in the light chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the light chain variable region framework region, respectively.

[0026] Furthermore, the nucleotide sequence of the polynucleotide encoding the aforementioned monoclonal antibody or its functional part is shown in SEQ ID NO: 23, 30.

[0027] Furthermore, the polynucleotide also includes a nucleotide sequence encoding a heavy chain signal peptide domain, as specifically shown in SEQ ID NO:33.

[0028] The present invention provides a vector, which comprises the polynucleotide described above.

[0029] Furthermore, the vector includes a linear polynucleotide, a plasmid or a viral vector.

[0030] Furthermore, the viral vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0031] The term "vector" refers to any type of genetic construct comprising a nucleic acid encoding an RNA that can be transcribed. Possible vectors include, but are not limited to, transposons, cosmids, plasmids or modified viruses (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses and lentiviruses), as long as the vector is compatible with the host cell used. The vector is suitable for transforming host cells, which means that the vector contains the CD28 protein gene of the present invention and a regulatory sequence selected for expression based on the host cell, which is operably connected to the CD28 protein gene sequence. "Operably connected" means that the nucleic acid is connected to the regulatory sequence in a manner that allows the expression of the nucleic acid.

[0032] Suitable regulatory sequences can be derived from a variety of sources, including bacteria, fungi, viruses, mammals or insect genes. The selection of suitable regulatory sequences depends on the host cell selected as discussed below, and can be easily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: transcription promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, including translation initiation signals. In addition, depending on the selected host cell and the vector used, other sequences, such as replication origins, additional DNA restriction sites, enhancers and sequences that confer transcriptional induction ability can be incorporated into the expression vector.

[0033] The carrier of the present invention can also contain a selection marker gene, which helps to select a host cell transformed or transfected with the recombinant molecule of the present invention. The example of a selection marker gene is a gene encoding a protein that confers resistance to certain drugs, such as neomycin and hygromycin, beta-galactosidase, chloramphenicol acetyltransferase, firefly luciferase or an immunoglobulin or a portion thereof, such as the Fc portion of an immunoglobulin, preferably IgG. The transcription of the selection marker gene is monitored by changes in the concentration of a selection marker protein such as beta-galactosidase, chloramphenicol acetyltransferase or firefly luciferase.

[0034] The present invention provides a cell, wherein the cell comprises the aforementioned monoclonal antibody or a functional part thereof, the aforementioned isolated polynucleotide, or the aforementioned vector.

[0035] Furthermore, the cells include prokaryotic cells and eukaryotic cells.

[0036] Furthermore, the eukaryotic cells include mammalian cells, insect cells, flying animal cells, plant cells, and yeast cells.

[0037] Furthermore, the mammalian cells include bone marrow cells, immune cells, lymphocytes, and fibroblasts.

[0038] Furthermore, the cell is a hybridoma cell.

[0039] In the present invention, examples of cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as those produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), defective CHO cells, rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc.

[0040] The present invention provides an antibody complex, which comprises the above-mentioned monoclonal antibody or a functional part thereof, obtained by modification.

[0041] Furthermore, the modification includes modification using a detectable marker or an imaging agent.

[0042] In certain embodiments of the invention, the modified first amino acid residue of the anti-CD28 protein antibody containing the Fc region is connected to the functional part. In further embodiments, the functional part is a blocking part, a detectable part, or a diagnostic part, or a combination thereof. In certain embodiments, the blocking part can be, for example, a cysteine ​​adduct, a mixed disulfide, polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the detectable part can be, for example, a fluorescent part, a luminescent part or an isotope part. In the embodiment where a diagnostic part is used, the diagnostic part may be able to reveal the presence of a condition, a disease or a disorder.

[0043] In some embodiments, the detectable label may be a radioisotope, an enzyme, a dye, or biotin. In yet other embodiments, the antibodies of the invention are conjugated to an imaging agent that may be a labeling moiety. The labeling agent may be biotin, a fluorescent or luminescent moiety, a radioactive moiety, a histidine tag, or a peptide tag.

[0044] Detectable labels can be fluorescent dyes (e.g., fluorophores, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), Alexa Fluor® labels), near-infrared (NIR) dyes (e.g., Qdot® nanocrystals), colloidal metals, haptens, radioactive labels, biotin, and amplification agents such as streptavidin or enzymes such as horseradish peroxidase or alkaline phosphatase.

[0045] The present invention provides a method for preparing the monoclonal antibody or its functional part as described above, which comprises the steps of culturing the cells described above and extracting the monoclonal antibody or its functional part from the culture medium and / or cells.

[0046] The present invention provides the use of the aforementioned monoclonal antibody or its functional part, the aforementioned isolated polynucleotide, the aforementioned vector, the aforementioned cell, and the aforementioned antibody complex in the preparation of a product for detecting / diagnosing the level of CD28 molecules on the surface of T cells or the level of T cell activation.

[0047] The present invention provides a method for detecting CD28 protein in a sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the sample to be tested with the above-mentioned antibody or its antigen-binding portion, or contacting the sample to be tested with the above-mentioned antibody complex; detecting the formation of a product of CD28 protein and the above-mentioned antibody or its antigen-binding portion, or the above-mentioned antibody complex.

[0048] Advantages and beneficial effects of the present invention: 1. By optimizing the nucleotide sequence, the extracellular region of human CD28 protein was expressed in HEK293 cells.

[0049] 2. Use hybridoma fusion technology to screen out hybridoma cells that can secrete anti-human CD28 antibodies.

[0050] The hybridoma cells were sequenced to obtain the VL and VH region sequences of the antibody, and the anti-human CD28 monoclonal antibody was recombinantly expressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the result of plasmid extraction.

[0052] Figure 2 This is the result of SDS-PAGE electrophoresis.

[0053] Figure 3 It is a graph of potency monitoring results.

[0054] Figure 4 This is the result of affinity purification of ascites antibody.

[0055] Figure 5 This is a graph of antibody subclass detection results.

[0056] Figure 6 This is a graph of antibody sensitivity test results. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention clearer, embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0058] Example 1 Recombinant expression and purification of human CD28 protein 1. Expression plasmid construction 1.1 Expressed sequence information Protein sequence number: P10747, expression range: N19-P152, tag: C-terminal with 6×his-tag, predicted molecular weight 15.1 kDa.

[0059] Sequence 1 (target protein): (SEQ ID NO: 31) NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0060] HEK293 was used as the host to optimize the gene sequence. The optimized expression gene sequence is: Sequence 2 (optimized gene sequence): (SEQ ID NO: 32) Atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcaacaagatttggtgaagcagtcgcccatgcttgtagcgtacgacaatgcggtcaac cttagctgcaagtattcctacaatctcttctcaagggagttccgggcatcccttcacaaaggactggatagtgctgtggaagtctgtgttgtatatgggaattactcccagcagctt caggtttactcaaaaacggggttcaactgtgatgggaaattgggcaatgaatcagtgacattctacctccagaatttgtatgttaaccaaacagatatttacttctgcaaaattgaa gttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccctgataa.

[0061] 1.2 Vector construction and plasmid extraction 1.2.1 Using pCDNA3.4 as the expression vector and Mouse Ig Kappa signal peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 33), expression vectors with reading frames of sequence 2 were constructed and named pTL-CD28-1 respectively.

[0062] 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.

[0063] The results of plasmid extraction are as follows Figure 1 shown.

[0064] 2. Transient Expression 2.1 Main Materials 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-CD28-1). Cell shaker, Corning; cell counter, Contstar; cell culture shaker, Eppendorf S41i; large-capacity centrifuge, Hunan Xiangyi GL-21M.

[0065] 2.2 Methods 2.2.1 Cell culture HEK293 cells were revived and the cell culture density was 3.6 × 10 6 / mL, and then subculture. According to the cell density, add fresh 293 serum-free medium and dilute the cell density to 0.6-0.8×10 6 / mL, culture in a cell culture shaker (37℃, CO2 concentration 5%). Requirement: The volume of the culture medium should not exceed 1 / 5 of the volume of the culture flask.

[0066] 2.2.2 Transient Transformation and Feeding 1) On the day of transfection, detect cell density and viability and adjust the cell density to 3×106 / mL; 2) Take 1 mg of plasmid (pTL-CD28-1 plasmid, 1032.1 µg / mL, 970 µL) and add it to a 50 mL centrifuge tube (tube A) containing 25 mL of 293 serum-free medium. Mix by gently pipetting. Take 5 mL of TA-293 and add it to a 50 mL centrifuge tube (tube B) containing 25 mL of 293 serum-free medium. Mix by gently pipetting. 3) Mix the liquids in tube A and tube B, mix gently, and leave at room temperature for 5 minutes to prepare the transfection reagent-plasmid complex; 4) Use a pipette to slowly add the transfection reagent-plasmid complex to the cells at the pre-adjusted density, shaking the cell shaker while adding to evenly disperse the complex. After completion, move the shaker into a shaker and start culturing. The culture conditions are: 5% CO2, 37°C, 120 rpm; 5) Culture for 24 h (Day 1), detect cell density and viability, add 6 mL KE-293 and 50 mL feed medium, and continue culturing; 6) Day 3, detect cell density and viability, add 50 mL of feed medium, and continue culturing; 7) On Day 5 or when the cell viability is lower than 80%, 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.

[0067] 2.3 Results The monitoring results of the expression process are shown in Table 1.

[0068] Table 1

[0069] 3. Purification 3.1 Main Materials 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; 3.2 Methods 3.2.1 Metal chelate chromatography 1) Sample treatment: After the cell fermentation liquid (cell feed liquid) harvested by centrifugation was filtered through 0.45 μm, 5 mL of eluent (i.e., 100% B solution: 20 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; 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: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 8.0) until the UV280 value is stable, 5 mL / min; 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; 4) Balance: 5 mL / min, flush with balance solution until the baseline is stable; 5) Elution: 5 mL / min, eluent (20 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 8.0) until the absorption is stable; 6) Balance: 5 mL / min, flush with balance solution until the baseline is stable; 7) Eluent: 5 mL / min, rinse until the absorption is stable; 8) Gradient elution: 5 mL / min, imidazole concentration from 30%, 100%, in order of gradient elution, and the peak was collected; 9) Alkali treatment: flush with 0.1 M NaOH for 3 column volumes; 10) Storage: 5 mL / min, flush with water for 10 column volumes, and finally soak the pre-packed column in 20% ethanol.

[0070] 3.2.2 G25 fluid change 1) Column treatment: 5 mL / min, water injection, 10 CV; 2) CIP: 8 mL / min, CIP solution flushing 10 CV; 3) Equilibration: 8 mL / min, flush with desalting chromatography equilibration solution (PBS, pH 7.4) until the baseline is stable; 4) Sample loading: 5 mL / min, volume 20 mL; 5) Balance: 8 mL / min, collect protein peak, 200 mAu-954 mAu-200 mAu, volume 5 mL; 6) Washing: 8mL / min, water injection, 10CV; 7) Storage: 5 mL / min, 20% ethanol rinse, 10 CV.

[0071] 3.3 Results SDS-PAGE electrophoresis analysis of the components of Ni column imidazole gradient elution showed that the purity of the 30% B-2 elution product was >90% after one-step affinity purification. After G25 solution replacement, the protein concentration was 1.08 mg / mL by BCA quantitative detection. The specific results are as follows Figure 2 shown.

[0072] Example 2 Immunization of mice with CD28 protein 1. Immunization of mice and monitoring of mouse serum titer 1.1 Main Materials 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.

[0073] 1.2 Methods 1.2.1 Animal immunization procedure: On day 0, 100 μg of recombinant human CD28 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 CD28 protein was fully emulsified with Freund's incomplete adjuvant and immunized again in the same manner as before; on day 28, the same procedure as the second immunization was performed for booster immunization; on day 35, blood was collected from the mouse orbits, serum was separated, and the antiserum antibody titer was detected by ELISA. If the titer met the requirements, only 50 μg of CD28 protein was used for booster immunization on the same day, and hybridoma fusion experiments were carried out on Day 38; if it did not meet the requirements, the same procedure as the second immunization was performed on day 42 for another immunization, until the titer met the requirements, booster immunization was performed, and hybridoma fusion was carried out 3 days later.

[0074] 1.2.2 ELISA method to detect serum antibody titer 1) Prepare an ELISA plate and add 100 μL of CD28 protein (coating solution diluted to a concentration of 1 μg / mL) to each well and incubate at 4°C overnight; 2) Discard the coated protein, add 300 μL of blocking solution (5% skim milk powder) to each well, and block at room temperature for 1 hour; 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; 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. 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; 6) After washing the plate as described above, add freshly prepared TMB colorimetric solution to the wells and color in the dark; 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.

[0075] 1.3 Results The serum antibody titer is ≥1.28×10^5. The specific titer test is as follows: Figure 3 shown.

[0076] Example 3 Screening of hybridoma cell lines capable of secreting anti-human CD28 antibodies 1. Hybridoma cell fusion and subclone screening 1.1 Main Materials Mouse myeloma cells SP2 / 0, stored in our company; fetal bovine serum, Gibco; RPMI1640 medium, HAT, HT, fusion agent PEG3350, etc., Gibco; goat anti-mouse IgG / HRP, Beijing Kangwei Century Biotechnology Co., Ltd.; low molecular weight protein marker, Thermo; multiparous Balb / c mice, female, 12-16 weeks old, Sibei Fusheng Technology Co., Ltd.; liquid paraffin, Shanghai Shenggong Biotechnology Co., Ltd.

[0077] 1.2 Methods 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 culture 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.

[0078] 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.

[0079] 1.3 Results (taking the ELISA test results of the second subclone of the third positive clone in plate 3 as an example) Coating antigen: CD28, 1 μg / ml coating, coating volume 100 μl / well; detection wavelength: 450 nm.

[0080] The positive clones screened by ELISA are shown in Table 2.

[0081] Table 2

[0082] According to the ELISA results, we selected well B6 of plate 3 and gradually expanded and cultured the strains from the 96-well plate for subsequent experimental work.

[0083] 2. Preparation and identification of anti-human CD28 monoclonal antibodies 2.1 Main Materials Hybridoma cells, cells expanded and cultured in step 5 (named WT3-3); multiparous mice aged 12-16 weeks, from Spefco Biotechnology; liquid paraffin, from Shanghai Sangon Biotech Co., Ltd.; MabSelectSure antibody affinity purification medium, from GE; rapid antibody typing kit (mouse), from Thermo.

[0084] 2.2 Methods 1) Proliferation and culture of hybridoma cells; 2) Multiparous mice were intraperitoneally injected with liquid paraffin, 0.5 mL / mouse, and the day of immunization was designated as Day 0; 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 5, and ascites was collected at appropriate times. 4) The ascites was purified by protein A affinity filler to obtain anti-human CD4 monoclonal antibody, which was quantified by BCA method.

[0085] 5) Use rapid antibody typing kit to detect antibody subclasses.

[0086] 6) ELISA method to detect the sensitivity of antibodies to CD28 protein: coated with different concentrations of CD28 protein, blocked with 5% skim milk powder at room temperature for 2 hours, washed with TBST; added 100μl of 1μg / mL antibody solution to each well, incubated at room temperature for 1 hour; fully washed the antigen plate, added HRP-labeled goat anti-mouse IgG antibody (1:10000 dilution), incubated for 1 hour; after fully washing the plate with TBST, added TMB colorimetric solution, terminated the color reaction after 10 minutes, and read the OD450. The OD ratio > 2.1 was used as the positive standard to determine the sensitivity of the antibody to CD28 protein.

[0087] 2.3 Results The results of affinity purification of ascites antibody are as follows Figure 4 As shown. After affinity purification, the purified anti-CD28 monoclonal antibody (clone number marked as WT3-3) was obtained.

[0088] Antibody subclass test results Figure 5 Figure 2 shows the typing results of the anti-CD28 monoclonal antibody (clone number WT3-3), which belongs to the IgG1 subclass.

[0089] The detection sensitivity of anti-CD28 monoclonal antibody to CD28 protein is as follows Figure 6 As shown. 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 antibody can detect 20 ng / mL of CD28 protein.

[0090] Example 4 CDR region analysis of anti-human CD28 monoclonal antibody (WT3-3) 1. Experimental Materials WT3-3 cells, Trizol reagent (Invitrogen), Prime script 1 step RT-PCR Kit (Takara), pMD18T vector (Takara), AB3700 PCR instrument.

[0091] 2. Experimental methods 1. Based on the mouse heavy chain VH and light chain VL sequences, degenerate primers were designed and commissioned to Beijing Sanbo Yuanzhi Biological Co., Ltd. for synthesis. The primer sequences are shown below.

[0092] VH-F:CCGGAATTCGSARGTNMARCTGSAGSAGTCWGG.

[0093] VH-R: TGAGGAGACSGTGACYRDRGTYCCTTG.

[0094] VL-F:GAYATTGTGMTSACMCARWCTMCW.

[0095] VL-R:TTTGATYTCSARCTTKGTSCC.

[0096] 2. Extract total RNA from hybridoma cell WT3-3, use primer pairs VH-F+VH-R and VL-F+VL-R to amplify the heavy chain VH and light chain VL nucleic acid sequences of the antibody, respectively, and construct them on the pMD18T vector.

[0097] 3. The positive plasmid was sent for sequencing. The sequencing report was interpreted, and the VH and VL sequences of the WT3-3 antibody were known. According to the IMGT numbering rules of the antibody, the antibody light chain variable region CDR and heavy chain variable region CDR sequences were analyzed. The specific results are shown in Table 3.

[0098] Table 3

Claims

1. An anti-CD28 protein monoclonal antibody or a functional portion thereof, wherein the monoclonal antibody or the functional portion thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in the following CDR1, CDR2 and CDR3 sequences, and the light chain variable region comprises the amino acid sequence shown in the following CDR1, CDR2 and CDR3 sequences: The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 2; The amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 3; The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO:9; The amino acid sequence of CDR2 of the light chain variable region is STS; The amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:

10.

2. The monoclonal antibody or the functional portion thereof according to claim 1, further comprising a heavy chain variable region framework region and a light chain variable region framework region, wherein the heavy chain variable region framework region comprises the amino acid sequence shown in SEQ ID NOs: 4, 5, 6, and 7, and the light chain variable region framework region comprises the amino acid sequence shown in SEQ ID NOs: 11, 12, 13, and 14; Preferably, the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7 in the heavy chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region, respectively, and the amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14 in the light chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the light chain variable region framework region, respectively.

3. The monoclonal antibody or the functional part thereof according to claim 1, wherein the monoclonal antibody or the functional part thereof comprises the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO:

15.

4. An isolated polynucleotide comprising a heavy chain coding sequence encoding the heavy chain variable region of the monoclonal antibody or a functional portion thereof according to any one of claims 1 to 3, and a light chain coding sequence encoding the light chain variable region of the monoclonal antibody or a functional portion thereof according to any one of claims 1 to 3; Preferably, the polynucleotide comprises the following nucleotide sequence: The nucleotide sequences shown in SEQ ID NOs: 16, 17, and 18 are used to encode CDR1, CDR2, and CDR3 of the heavy chain variable region of any one of claims 1 to 3, respectively; The nucleotide sequences shown in SEQ ID NO:24, AGCACATCC, and SEQ ID NO:25 are used to encode CDR1, CDR2, and CDR3 of the light chain variable region of any one of claims 1 to 3, respectively; Preferably, the polynucleotide further comprises the following nucleotide sequence: The nucleotide sequences shown in SEQ ID NOs: 19, 20, 21, and 22 are used to encode the heavy chain variable region framework region according to any one of claims 1 to 3; The nucleotide sequences shown in SEQ ID NOs: 26, 27, 28, and 29 are used to encode the light chain variable region framework region according to any one of claims 1 to 3; Preferably, the nucleotide sequences shown in SEQ ID NOs: 19, 20, 21, and 22 in the heavy chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region, respectively; The nucleotide sequences shown in SEQ ID NOs: 26, 27, 28, and 29 in the light chain variable region framework region correspond to FR1, FR2, FR3, and FR4 of the light chain variable region framework region, respectively; Preferably, the nucleotide sequence of the polynucleotide encoding the monoclonal antibody or the functional part thereof according to any one of claims 1 to 3 is as shown in SEQ ID NO: 23, 30.

5. A vector comprising the polynucleotide according to claim 4; Preferably, the vector comprises a linear polynucleotide, a plasmid or a viral vector; Preferably, the viral vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

6. A cell comprising the monoclonal antibody or a functional part thereof according to any one of claims 1 to 3, the isolated polynucleotide according to claim 4, or the vector according to claim 5; Preferably, the cells include prokaryotic cells and eukaryotic cells; Preferably, the eukaryotic cells include mammalian cells, insect cells, flying animal cells, plant cells, and yeast cells; Preferably, the mammalian cells include bone marrow cells, immune cells, lymphocytes, and fibroblasts; Preferably, the cell is a hybridoma cell.

7. An antibody complex, comprising the monoclonal antibody or a functional part thereof according to any one of claims 1 to 3, obtained by modification; Preferably, the modification comprises modification with a detectable label or an imaging agent.

8. A method for preparing the monoclonal antibody or a functional portion thereof according to any one of claims 1 to 3, comprising the steps of culturing the cell according to claim 6 and extracting the monoclonal antibody or a functional portion thereof from the culture medium and / or the cell.

9. Use of the monoclonal antibody or functional part thereof according to any one of claims 1 to 3, the isolated polynucleotide according to claim 4, the vector according to claim 5, the cell according to claim 6, or the antibody complex according to claim 7 in the preparation of a product for detecting / diagnosing the level of CD28 molecules on the surface of T cells or the level of T cell activation.

10. A method for detecting CD28 protein in a sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the sample to be tested with the antibody or antigen binding portion thereof according to any one of claims 1 to 3, or contacting the sample to be tested with the antibody complex according to claim 7; detecting the formation of a product of the CD28 protein and the antibody or antigen binding portion thereof according to any one of claims 1 to 3, or the antibody complex according to claim 7.

Images