A monoclonal antibody against CD28 and cells

Abstract

The present invention discloses a monoclonal antibody against CD28 and cells, belonging to the field of biotechnology. The present invention provides the amino acid sequences of the CDR regions of the monoclonal antibody against CD28, namely the CDR regions of the heavy chain variable region and the light chain variable region, specifically the amino acid sequences of heavy chain variable region CDR1, 2, 3 shown in SEQ ID NO:1, 2, 3, and the amino acid sequences of light chain variable region CDR1, 2, 3 shown in SEQ ID NO:9, STS, SEQ ID NO:10.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a monoclonal antibody against CD28 and cells. Background Art

[0002] CD28 is a 45 kDa homodimer composed of two 44 kDa polypeptide chains linked by disulfide bonds. It is mainly expressed on the surface of activated T cells and belongs to the Ig superfamily. CD28 mainly serves as a receiver of T cell co-stimulatory signals, binds to CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APCs), provides co-stimulatory signals for T cells, and promotes immune responses. The signal transduction of CD28 can stimulate the expression of IL-2 and its receptor, as well as the cytotoxicity of CD3-activated T cells. CD28 is crucial 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 the application of immune checkpoint inhibitors, the CD28 co-stimulatory signal is crucial for the continuous activation of T cells.

[0003] CD28 antibodies can significantly enhance T cell activity by mimicking the second signal for activating T cells and form a combination therapy with CD3 antibodies, thereby having a stronger tumor-killing effect. This combination therapy is particularly important in tumor immunotherapy because it can provide the co-stimulatory signals required for T cell activation and enhance the ability to clear tumor cells. In CAR-T cell therapy, the CD28 co-stimulatory signal is crucial for the continuous activation of T cells. CD28 antibodies can be co-cultured with CAR-T cells to enhance their tumor cell-killing ability. CD19-CD28 bispecific antibodies can provide effective and safe co-stimulatory signals for T cells and enhance the effect of CAR-T therapy. CD28 antibodies are also used in flow cytometry to analyze the activation status and function of T cells. By using CD28 antibodies, researchers can deeply explore the status and function of T cells, providing strong support for the development of immunotherapy and disease treatment.

[0004] Most of the currently commercialized CD28 antibodies are only for scientific research use. Due to the risk of intellectual property rights, the development of products in the field of cell therapy, especially in CAR-T cell therapy, cell sorting magnetic beads, etc., 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, the monoclonal antibody or the functional portion thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences shown by the following CDR1, CDR2 and CDR3 sequences, and the light chain variable region comprising amino acid sequences shown by the following CDR1, CDR2 and CDR3 sequences:

[0007] The amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO:1;

[0008] The amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO:2;

[0009] The amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO:3;

[0010] The amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO:9;

[0011] The amino acid sequence of CDR2 of the light chain variable region is STS;

[0012] The amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO:10.

[0013] Furthermore, the monoclonal antibody or the functional portion thereof further comprises a heavy chain variable region framework region and a light chain variable region framework region, the heavy chain variable region framework region comprising amino acid sequences as shown in SEQ ID NO:4, 5, 6, 7, and the light chain variable region framework region comprising amino acid sequences as shown in SEQ ID NO:11, 12, 13, 14.

[0014] In some embodiments, FR1, FR2, FR3, FR4 of the heavy chain variable region framework region respectively have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, 6, 7. FR1, FR2, FR3, FR4 of the light chain variable region framework region respectively have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequences shown in SEQ ID NO:11, 12, 13, 14.

[0015] Further, the amino acid sequences shown in SEQ ID NO: 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 NO: 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.

[0016] Further, the monoclonal antibody or its functional portion comprises the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 15.

[0017] In the present invention, the anti-CD28 monoclonal antibody or its functional portion can be murine, chimeric, primatized, humanized or fully human antibody.

[0018] In the present invention, the anti-CD28 monoclonal antibody or its functional portion can be multimeric, heterodimeric, monovalent, bivalent, tetravalent or single-chain antibody.

[0019] In addition, a humanized antibody can contain residues not found in the recipient antibody and not found in the input CDR or FR sequences. These modifications are made to further improve and optimize antibody performance. In general, a humanized antibody can contain substantially all of at least 1, and generally 2 variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FR residues are FR residues of a human immunoglobulin consensus sequence. A humanized antibody optionally can also contain at least a portion of an immunoglobulin constant region (Fc), generally a human immunoglobulin constant region.

[0020] In the present invention, there are modifications on the anti-CD28 monoclonal antibody or its functional portion, and such modifications can be glycosylation, including N-glycosylation, O-glycosylation and C-glycosylation. In the glycosylation modification, it includes fucosylation modification, galactosylation modification, terminal sialic acid modification.

[0021] Glycosylation sites in Fc-containing polypeptides (such as antibodies (such as 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 is typically NXT or NXS, where X can be any amino acid other than proline. Several algorithms for localizing potential glycosylation motifs have been described. Thus, 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 predicting N-linked glycosylation sites and the NetOGlyc server for predicting O-linked glycosylation sites).

[0022] As used herein, the term "CD28" includes full-length CD28, soluble CD28, and fusion proteins comprising functional active portions of CD28.

[0023] As used herein, the term "antibody" refers generically to immunoglobulins or immunoglobulin-like molecules, including, for example and without limitation, IgA, IgD, IgE, IgG, and IgM and combinations thereof, as well as similar molecules produced during an immune response in any vertebrate, such as a mammal (e.g., human, goat, rabbit, and mouse) and non-mammalian species (e.g., shark immunoglobulins). Unless otherwise specifically stated, the term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) such that binding to other molecules is substantially excluded (e.g., antibodies and antibody fragments having a binding constant for the molecule of interest that is at least 10 3 M -1 , at least 10 4 M -1 or at least 10 5 M -1 greater than that for binding to other molecules in a biological sample). 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).

[0024] In terms of antibody structure, an immunoglobulin has heavy (H) and light (L) chains that are linked to each other by disulfide bonds. There are two types of light chains: λ and κ. There are five main heavy chain types (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain includes a constant region and a variable region (the regions are also referred to as "domains"). The variable regions of the heavy and light chains together specifically bind an antigen. The variable regions of the heavy and light chains include "framework" regions that are separated by three highly variable regions, which are also referred to as "complementary determining regions" or "CDRs". The ranges of the framework regions and CDRs have been determined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. 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 conserved within a species. The framework regions of an antibody, i.e., the framework regions that make up the binding of the light and heavy chains, mainly adopt a β-sheet conformation, while the CDRs form loops that connect the β-sheet structures or in some cases form part of the β-sheet. Thus, the framework regions serve to form a scaffold that is used to position the CDRs in the correct orientation by interchain non-covalent interactions.

[0025] The CDRs are mainly responsible for binding to the epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds 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 antibodies differ from one another in their CDRs, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are referred to as specific determining residues (SDRs).

[0026] In the present invention, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier "monoclonal" refers to the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be made by any particular method. For example, the monoclonal antibodies used according to the present invention can be prepared by the hybridoma (mouse or human) method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared by recombinant DNA methods (see, for example, US 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol. Biol., 222:581-597 (1991).

[0027] Antibodies with altered (e.g., reduced or eliminated) effector functions can be generated by engineering or generating antibodies with variant constant, Fc, or heavy chain regions. Recombinant DNA techniques and / or cell culture and expression conditions can be used to generate antibodies with altered functions and / or activities. For example, recombinant DNA techniques can be used to engineer one or more amino acid substitutions, deletions, or insertions in regions that affect antibody function (including effector functions), such as the Fc or constant region.

[0028] The present invention provides an isolated polynucleotide comprising a heavy chain coding sequence encoding the heavy chain variable region of the monoclonal antibody or a functional portion thereof described above, and a light chain coding sequence encoding the light chain variable region of the monoclonal antibody or a functional portion thereof described above.

[0029] Furthermore, the polynucleotide comprises the following nucleotide sequences:

[0030] The nucleotide sequences shown in SEQ ID NO:16, 17, 18 are respectively used to encode CDR1, CDR2, CDR3 of the heavy chain variable region described above;

[0031] The nucleotide sequences shown in SEQ ID NO:24, AGCACATCC, SEQ ID NO:25 are respectively used to encode CDR1, CDR2, CDR3 of the light chain variable region described above.

[0032] Furthermore, the polynucleotide further comprises the following nucleotide sequences:

[0033] The nucleotide sequences shown in SEQ ID NO:19, 20, 21, 22 are used to encode the framework region of the heavy chain variable region described above;

[0034] The nucleotide sequences shown in SEQ ID NO: 26, 27, 28, and 29 are used to encode the light chain variable region framework region described above.

[0035] Furthermore, the nucleotide sequences shown in SEQ ID NO: 19, 20, 21, and 22 in the heavy chain variable region framework region respectively correspond to FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region;

[0036] The nucleotide sequences shown in SEQ ID NO: 26, 27, 28, and 29 in the light chain variable region framework region respectively correspond to FR1, FR2, FR3, and FR4 of the light chain variable region framework region.

[0037] Furthermore, the nucleotide sequences of the polynucleotide encoding the monoclonal antibody or its functional part described above are as shown in SEQ ID NO: 23 and 30.

[0038] Furthermore, the polynucleotide also includes a nucleotide sequence encoding a heavy chain signal peptide domain, specifically as shown in SEQ ID NO: 33.

[0039] The present invention provides a vector, and the vector includes the polynucleotide described above.

[0040] Furthermore, the vector includes a linear polynucleotide, a plasmid, or a viral vector.

[0041] Furthermore, the viral vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0042] The term "vector" refers to any type of genetic construct that contains nucleic acid encoding 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), provided that the vector is compatible with the host cell used. The vector is suitable for transforming the host cell, which means that the vector contains the CD28 protein gene of the present invention and regulatory sequences selected for expression based on the host cell, which are operably linked to the CD28 protein gene sequence. "Operably linked" means that the nucleic acid is linked to the regulatory sequence in a manner that allows the nucleic acid to be expressed.

[0043] Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The choice of suitable regulatory sequences depends on the host cell selected as discussed below and can be readily accomplished by those of ordinary skill in the art. Examples of such regulatory sequences include: transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, including translation initiation signals. Additionally, depending on the host cell selected and the vector used, other sequences, such as origins of replication, additional DNA restriction sites, enhancers and sequences conferring transcriptional inducibility, can be incorporated into the expression vector.

[0044] The vectors of the present invention may also contain selectable marker genes, which facilitate the selection of host cells transformed or transfected with the recombinant molecules of the present invention. Examples of selectable marker genes are genes encoding proteins conferring resistance to certain drugs, such as neomycin and hygromycin, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase or immunoglobulins or portions thereof, such as the Fc portion of an immunoglobulin, preferably IgG. Transcription of the selectable marker gene is monitored by changes in the concentration of a selectable marker protein such as β-galactosidase, chloramphenicol acetyltransferase or firefly luciferase.

[0045] The present invention provides a cell comprising the monoclonal antibody or functional portion thereof described above, the isolated polynucleotide described above, or the vector described above.

[0046] Furthermore, the cell comprises a prokaryotic cell or a eukaryotic cell.

[0047] Furthermore, the eukaryotic cell comprises a mammalian cell, an insect cell, a winged animal cell, a plant cell, or a yeast cell.

[0048] Furthermore, the mammalian cell comprises a myeloid cell, an immune cell, a lymphocyte, or a fibroblast.

[0049] Furthermore, the cell is a hybridoma cell.

[0050] 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 Escherichia coli (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 generated from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), defective CHO cells, rat YB 2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc.

[0051] The present invention provides an antibody complex, which comprises the aforementioned monoclonal antibody or its functional part, and a complex obtained by modification.

[0052] Furthermore, the modification includes modification using a detectable marker or an imaging agent.

[0053] In certain embodiments of the present invention, the first amino acid residue of the modified anti-CD28 protein antibody containing an Fc region is linked to a 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 embodiments where a diagnostic part is used, the diagnostic part may be capable of revealing the presence of a condition, disease, or disorder.

[0054] In some embodiments, the detectable marker can be a radioisotope, an enzyme, a dye, or biotin. In other additional embodiments, the antibody of the present invention is conjugated to an imaging agent that can be a labeling part. The labeling agent can be biotin, a fluorescent or luminescent part, a radioactive part, a histidine tag, or a peptide tag.

[0055] Detectable labels can be fluorescent dyes (such as fluorophores, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), Alexa Fluor® labels), near-infrared (NIR) dyes (such as Qdot® nanocrystals), colloidal metals, haptens, radiolabels, biotin, and amplification reagents such as streptavidin or enzymes (such as horseradish peroxidase or alkaline phosphatase).

[0056] The present invention provides a method for preparing the monoclonal antibody or its functional portion as described above, which includes culturing the cells as described above and extracting the monoclonal antibody or its functional portion from the culture medium and / or cells.

[0057] The present invention provides the use of the monoclonal antibody or its functional portion as described above, the isolated polynucleotide as described above, the vector as described above, the cell as described above, and the antibody complex as described above in the preparation of products for detecting / diagnosing the level of CD28 molecules on the surface of T cells or the level of T cell activation.

[0058] The present invention provides a method for non-diagnostic and non-therapeutic detection of CD28 protein in a sample, the method comprising the steps of: contacting the sample to be tested with the antibody or its antigen-binding portion as described above, or contacting the sample to be tested with the antibody complex as described above; detecting the formation of a product of CD28 protein with the antibody or its antigen-binding portion as described above, or the antibody complex as described above.

[0059] Advantages and beneficial effects of the present invention:

[0060] 1. By optimizing the nucleotide sequence, the extracellular region of human CD28 protein was expressed in HEK293 cells.

[0061] 2. Using the hybridoma fusion technique, hybridoma cells capable of secreting anti-human CD28 antibodies were screened.

[0062] The hybridoma cells were sequenced to obtain the VL and VH region sequences of the antibody, and the recombinant anti-human CD28 monoclonal antibody was expressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a result diagram of large-scale plasmid extraction.

[0064] Figure 2 It is a result diagram of SDS-PAGE electrophoresis.

[0065] Figure 3 It is a result diagram of titer monitoring.

[0066] Figure 4 It is a result diagram of affinity purification of ascites antibody.

[0067] Figure 5It is a graph of antibody subclass detection results.

[0068] Figure 6 It is a graph of antibody sensitivity detection results. Detailed implementation manners

[0069] To make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, in which the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions from beginning to end. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0070] Example 1 Recombinant expression and purification of human CD28 protein

[0071] 1. Construction of expression plasmid

[0072] 1.1 Expression sequence information

[0073] Protein sequence number: P10747, expression interval: N19 - P152, tag: 6×his-tag at the C-terminus, predicted molecular weight 15.1 kDa.

[0074] Sequence 1 (target protein): (SEQ ID NO:31)

[0075] NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0076] Using HEK293 as the host, the gene sequence was optimized. The optimized expression gene sequence: Sequence 2 (optimized gene sequence): (SEQ ID NO:32)

[0077] Atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcaacaagattttggtgaagcagtcgcccatgcttgtagcgtacgacaatgcggtcaaccttagctgcaagtattcctacaatctcttctcaagggagttccgggcatcccttcacaaaggactggatagtgctgtggaagtctgtgttgtatatgggaattactcccagcagcttcaggtttactcaaaaacggggttcaactgtgatgggaaattgggcaatgaatcagtgacattctacctccagaatttgtatgttaaccaaacagatatttacttctgcaaaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccctgataa。

[0078] 1.2 Vector construction and large-scale plasmid extraction

[0079] 1.2.1 Using pCDNA3.4 as the expression vector and the Mouse Ig Kappa signal peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO:33), construct the expression vectors with open reading frames of Sequence 2, named pTL-CD28-1 respectively.

[0080] 1.2.2 Transform the above-mentioned 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. Wash the plasmids with endotoxin-free water, detect the concentration and endotoxin of the plasmids respectively. After sterile filtration, they can be used for transient transfection and expression.

[0081] The results of large-scale plasmid extraction are as Figure 1 shown.

[0082] 2. Transient transfection and expression

[0083] 2.1 Main materials

[0084] HEK293 cells (human embryonic kidney cells), self-preserved by our company; HEK293 serum-free medium (product number: AS-11), Tongli Haiyuan; HEK-93 expression feeding medium (product number: AS-18), Tongli Haiyuan; transfection reagent TA-293 (product number: K20001), Zhuhai Kairui; recombinant protein expression enhancer KE-293 (product number: K30001), Zhuhai Kairui; glutamine (product number: 21051024), Gibco; expression plasmid: the plasmid extracted in Step 1.1.2 (pTL-CD28-1). Cell culture flask, Corning; cell counter, Contstar; cell culture shaker, Eppendorf S41i; large-capacity centrifuge, Hunan Xiangyi GL-21M.

[0085] 2.2 Methods

[0086] 2.2.1 Cell culture

[0087] Resuscitate HEK293 cells and perform subculture when the cell culture density reaches 3.6×10 6 / mL. According to the cell density, add fresh 293 serum-free medium to dilute the cell density to 0.6 - 0.8×10 6 / mL and culture in a cell culture shaker (37°C, CO2 concentration 5%). Requirement: The volume of the medium does not exceed 1 / 5 of the culture flask volume.

[0088] 2.2.2 Transient transfection and feeding

[0089] 1) On the day of transfection, detect the cell density and viability, and adjust the cell density to 3×106 / mL;

[0090] 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) pre-prepared with 25 mL of 293 serum-free medium, and gently pipette to mix evenly; take 5 mL of TA-293 and add it to a 50 mL centrifuge tube (tube B) pre-prepared with 25 mL of 293 serum-free medium, and gently pipette to mix evenly;

[0091] 3) Mix the liquids in tube A and tube B, gently mix evenly, and place at room temperature for 5 min to prepare the transfection reagent-plasmid complex;

[0092] 4) Slowly add the transfection reagent-plasmid complex to the cells with the pre-adjusted density using a pipette, shaking the cell culture flask while adding to evenly disperse the complex. After completion, transfer the flask to the shaker and start culturing under the conditions: 5% CO2, 37°C, 120 rpm;

[0093] 5) Incubate for 24 h (Day 1), detect cell density and viability, add 6 mL of KE-293 and 50 mL of feeding medium, and continue the incubation.

[0094] 6) On Day 3, detect cell density and viability, add 50 mL of feeding medium, and continue the incubation.

[0095] 7) End the expression on Day 5 or when the cell viability is lower than 80%. Transfer the broth into a clean centrifuge bottle, centrifuge at 8000 rpm for 20 min at room temperature, and harvest the cell supernatant for purification.

[0096] 2.3 Results

[0097] The monitoring results of the expression process are shown in Table 1.

[0098] Table 1

[0099]

[0100] 3. Purification

[0101] 3.1 Main materials

[0102] Metal chelation chromatography: GE, HisTrap HP (5 mL pre-packed column), 17524801; Desalting chromatography: Boster, G-25 (100 mL), C0078; Imidazole: Sigma, 56749-1 kg; Chromatography system: GE, AKTA Purifier 100

[0103] 3.2 Method

[0104] 3.2.1 Metal chelation chromatography

[0105] 1) Sample treatment: The cell fermentation broth (cell broth) harvested by centrifugation is filtered through a 0.45 μm filter, and then 5 mL of eluent (i.e., 100% B solution: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 8.0) is added to make the imidazole concentration in the broth 5 mM.

[0106] 2) Pre-packed column treatment: Wash with water at a flow rate of 5 mL / min for 10 CV. Wash the pre-packed column with the equilibration 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 at a flow rate of 5 mL / min.

[0107] 3) Loading: The retention time is 2 min, that is, the flow rate is set to 2.5 mL / min, and the flow-through is collected.

[0108] 4) Equilibration: Wash with the equilibration solution at 5 mL / min until the baseline is stable.

[0109] 5) Elution: Rinse with the eluent (20 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 8.0) at 5 mL / min until the absorption is stable;

[0110] 6) Equilibration: Rinse with the equilibration solution at 5 mL / min until the baseline is stable;

[0111] 7) Elution: Rinse with the eluent at 5 mL / min until the absorption is stable;

[0112] 8) Gradient elution: Elute with a gradient of imidazole concentration from 30% to 100% at 5 mL / min in sequence, and collect the peaks;

[0113] 9) Alkali treatment: Rinse with 0.1 M NaOH for 3 column volumes;

[0114] 10) Preservation: Rinse with water at 5 mL / min for 10 column volumes, and finally soak the pre-packed column in 20% ethanol.

[0115] 3.2.2 Buffer exchange of G25

[0116] 1) Column treatment: Rinse with water at 5 mL / min for 10 CV;

[0117] 2) CIP: Rinse with the CIP solution at 8 mL / min for 10 CV;

[0118] 3) Equilibration: Rinse with the desalting chromatography equilibration solution (PBS, pH 7.4) at 8 mL / min until the baseline is stable;

[0119] 4) Sample loading: Load at 5 mL / min with a volume of 20 mL;

[0120] 5) Equilibration: Collect the protein peak at 8 mL / min, 200 mAu - 954 mAu - 200 mAu, with a volume of 5 mL;

[0121] 6) Washing of impurities: Rinse with water at 8 mL / min for 10 CV;

[0122] 7) Preservation: Rinse with 20% ethanol at 5 mL / min for 10 CV.

[0123] 3.3 Results

[0124] SDS-PAGE electrophoresis was used to analyze the components eluted with the imidazole gradient of the Ni column. After one-step affinity purification, the purity of the eluate of 30% B-2 was >90%. After buffer exchange with G25, the protein concentration was quantitatively detected by BCA to be 1.08 mg / mL. The specific results are as Figure 2 shown.

[0125] Example 2 Immunize mice with CD28 protein

[0126] 1. Immunized Mice and Monitoring of Serum Titers of Mice

[0127] 1.1 Main Materials

[0128] Female BALB / c mice (6 - 8 weeks old), Spbio (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.

[0129] 1.2 Methods

[0130] 1.2.1 Animal Immunization Procedure: On day 0, take 100 μg of recombinant human CD28 protein, mix it evenly with an equal volume of Freund's complete adjuvant and fully emulsify it, then intraperitoneally inject 6 - 8 week-old female BALB / c mice. On day 14, fully emulsify 50 μg of CD28 protein with Freund's incomplete adjuvant and immunize again in the same way as before; on day 28, perform a booster immunization with the same operation as the second immunization; on day 35, collect blood from the mouse orbits, separate the serum, and detect the antibody titer of the antiserum by ELISA. If the titer meets the requirements, only boost the immunization once with 50 μg of CD28 protein on the same day, and carry out the hybridoma fusion experiment on Day 38; if it does not meet the requirements, immunize again on day 42 with the same operation as the second immunization until the titer meets the requirements, then boost the immunization and carry out the hybridoma fusion 3 days later.

[0131] 1.2.2 Detection of Serum Antibody Titers by ELISA

[0132] 1) Prepare the enzyme-linked immunosorbent assay (ELISA) plate, add 100 μL of CD28 protein (diluted to a concentration of 1 μg / mL with the coating buffer) to each well, and incubate overnight at 4°C;

[0133] 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 h;

[0134] 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;

[0135] 4) Dilute the serum, dilute it with PBS buffer, start from 1:500 and perform 4-fold serial dilution, add the diluted serum to each well of the ELISA plate at 100 μL / well, and incubate at 37°C for 2 h;

[0136] 5) Discard the serum in the wells, wash the plate in the same way as before, then add the HRP-labeled goat anti-mouse antibody diluted 1:10000 to each well, and incubate at 37°C for 1 h;

[0137] 6) After washing the plate in the same method as above, add the freshly prepared TMB chromogenic solution to the wells and develop color in the dark;

[0138] 7) Read the absorbance values of each well at 450 nm, and take the ratio of OD sample / OD control (normal serum) ≥ 2.1 as ELISA positive.

[0139] 1.3 Results

[0140] The detected serum antibody titer ≥ 1.28×10^5, and the specific titer detection is as Figure 3 shown.

[0141] Example 3 Screening of hybridoma cell lines capable of secreting anti-human CD28 antibodies

[0142] 1. Hybridoma cell fusion and subclonal screening

[0143] 1.1 Main materials

[0144] Mouse myeloma cell line SP2 / 0, stored internally in our company; fetal bovine serum, Gibco; RPMI1640 medium, HAT, HT, fusogen PEG3350, etc., Gibco; goat anti-mouse IgG / HRP, Beijing ComWin Biotech Co., Ltd.; low molecular weight protein Marker, Thermo; multiparous Balb / c mice, female, 12 - 16 weeks old, Beijing SPF Biotechnology Co., Ltd.; liquid paraffin, Shanghai Sangon Biotech Co., Ltd.

[0145] 1.2 Methods

[0146] 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-inoculated with feeder cells, and culture in a 37°C CO2 incubator.

[0147] 2) Use the ELISA method to screen positive wells from the fusion plate, and perform subclonal culture of hybridoma cells by the limited dilution method. After 2 subclonings, the detected positive cells are passaged and amplified for culture.

[0148] 1.3 Results (taking the ELISA test results of the second subcloning of the third positive clone on plate 3 as an example)

[0149] Coated antigen: CD28, coated at 1 μg / ml, coating volume 100 μl / well; detection wavelength: 450 nm.

[0150] The positive clones screened by ELISA are shown in Table 2.

[0151] Table 2

[0152]

[0153]

[0154] According to the ELISA results, we selected well B6 on plate 3, gradually amplified and cultured it from the 96-well plate, and established a cell line for subsequent experimental work.

[0155] 2. Preparation and identification of anti-human CD28 monoclonal antibody

[0156] 2.1 Main materials

[0157] Hybridoma cells, the cells amplified and cultured in step 5 (named WT3-3); pregnant mice at 12-16 weeks old, Spf Bioscience, Inc.; liquid paraffin, Shanghai Sangon Biotech Co., Ltd.; MabSelectSure antibody affinity purification resin, GE; Quick Antibody Isotyping Kit (mouse), Thermo.

[0158] 2.2 Methods

[0159] 1) Amplify and culture hybridoma cells;

[0160] 2) Inject liquid paraffin intraperitoneally into pregnant mice at a dose of 0.5 mL per mouse. The immunization day is Day0;

[0161] 3) On Day7, collect the amplified and cultured hybridoma cells, adjust the density to 2×10^6 / mL, and inject 0.5 mL of the cell suspension into the peritoneal cavity of each pregnant mouse. Monitor the status of the mice starting from Day5 and collect ascites in a timely manner;

[0162] 4) Purify the ascites with protein A affinity resin to obtain anti-human CD4 monoclonal antibody, and quantify it by BCA method.

[0163] 5) Use the Quick Antibody Isotyping Kit to detect the antibody subclass.

[0164] 6) Detect the detection sensitivity of the antibody to CD28 protein by ELISA method: coat different concentrations of CD28 protein, block it with 5% skim milk powder at room temperature for 2 h, and wash it with TBST; add 100 μL of the antibody solution at 1 μg / mL to each well and incubate at room temperature for 1 h; wash the antigen plate thoroughly, add HRP-labeled goat anti-mouse IgG antibody (diluted 1:10000), and incubate for 1 h; after washing the plate thoroughly with TBST, add TMB chromogenic solution, terminate the chromogenic reaction after 10 min, and read OD450. Take OD ratio > 2.1 as the positive standard to determine the detection sensitivity of the antibody to CD28 protein.

[0165] 2.3 Results

[0166] The affinity purification results of ascites antibodies are as Figure 4 shown. After affinity purification, the purified anti-CD28 monoclonal antibody (clone number marked as WT3-3) was obtained.

[0167] The results of antibody subclass detection are as Figure 5 shown. The typing detection results of the anti-CD28 monoclonal antibody (clone number WT3-3) showed that this antibody belongs to the IgG1 subclass.

[0168] The detection sensitivity of the anti-CD28 monoclonal antibody to CD28 protein is as Figure 6 shown. 1: 100 mg / L; 2: 10mg / 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.

[0169] Analysis of CDR Regions of Anti-human CD28 Monoclonal Antibody (WT3-3) in Example 4

[0170] 1. Experimental Materials

[0171] WT3-3 cells, Trizol reagent (Invitrogen), Prime script 1 step RT-PCR Kit (Takara), pMD18T vector (Takara), AB3700 PCR instrument.

[0172] 2. Experimental Methods

[0173] 1. According to the mouse heavy chain VH and light chain VL sequences, degenerate primers were designed and synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. The primer sequences are as follows.

[0174] VH-F: CCGGAATTCGSARGTNMARCTGSAGSAGTCWGG.

[0175] VH-R: TGAGGAGACSGTGACYRDRGTYCCTTG.

[0176] VL-F: GAYATTGTGMTSACMCARWCTMCW.

[0177] VL-R: TTTGATYTCSARCTTKGTSCC.

[0178] 2. Extract the total RNA of hybridoma cell WT3-3, and use primer pairs VH-F + VH-R and VL-F + VL-R to amplify the nucleic acid sequences of the heavy chain VH and light chain VL of the antibody respectively, and construct them on the pMD18T vector.

[0179] 3. Send the positive plasmid for sequencing. Interpret the sequencing report, and know the VH sequence and VL sequence of the WT3-3 antibody. According to the IMGT numbering rules of the antibody, analyze the CDR sequences of the light chain variable region and heavy chain variable region of the antibody. The specific results are shown in Table 3.

[0180] Table 3

[0181]

[0182]

Claims

1. A monoclonal antibody against CD28 protein or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprises CDR1 with the amino acid sequence shown in SEQ ID NO:1, CDR2 with the amino acid sequence shown in SEQ ID NO:2, and CDR3 with the amino acid sequence shown in SEQ ID NO:3, and the light-chain variable region comprises CDR1 with the amino acid sequence shown in SEQ ID NO:9, CDR2 with the amino acid sequence STS, and CDR3 with the amino acid sequence shown in SEQ ID NO:

10.

2. The monoclonal antibody or the antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or the antigen-binding fragment thereof further comprises a heavy-chain variable region framework region and a light-chain variable region framework region, the heavy-chain variable region framework region comprises the amino acid sequences shown in SEQ ID NO:4, 5, 6, 7, and the light-chain variable region framework region comprises the amino acid sequences shown in SEQ ID NO:11, 12, 13, 14; The amino acid sequences shown in SEQ ID NO:4, 5, 6, 7 respectively correspond to FR1, FR2, FR3, FR4 of the heavy-chain variable region framework region, and the amino acid sequences shown in SEQ ID NO:11, 12, 13, 14 respectively correspond to FR1, FR2, FR3, FR4 of the light-chain variable region framework region.

3. The monoclonal antibody or the antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region with the amino acid sequence shown in SEQ ID NO:8 and a light-chain variable region with the amino acid sequence shown in SEQ ID NO:

15.

4. An isolated polynucleotide, which comprises a coding sequence encoding the heavy-chain variable region of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-3, and a coding sequence encoding the light-chain variable region of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-3.

5. The polynucleotide according to claim 4, wherein the polynucleotide comprises the following nucleotide sequences: The nucleotide sequences shown in SEQ ID NO:16, 17, 18 are respectively used for encoding CDR1, CDR2, CDR3 of the heavy-chain variable region of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-3; The nucleotide sequences shown in SEQ ID NO:24, AGCACATCC, SEQ ID NO:25 are respectively used for encoding CDR1, CDR2, CDR3 of the light-chain variable region of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-3.

6. The polynucleotide according to claim 4, wherein the polynucleotide further comprises the following nucleotide sequences: The nucleotide sequences shown in SEQ ID NO:19, 20, 21, 22 are respectively used for encoding FR1, FR2, FR3, FR4 of the heavy-chain variable region of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1-3; The nucleotide sequences shown in SEQ ID NO: 26, 27, 28, and 29 are respectively used to encode FR1, FR2, FR3, and FR4 of the light chain variable region of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3.

7. The polynucleotide according to claim 4, wherein the polynucleotide encodes the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3; The nucleotide sequence of the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment is as shown in SEQ ID NO: 23; The nucleotide sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment is as shown in SEQ ID NO:

30.

8. A vector, wherein the vector comprises the polynucleotide according to any one of claims 4-7.

9. The vector according to claim 8, wherein the vector comprises a linear polynucleotide, a plasmid, or a viral vector.

10. The vector according to claim 9, wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

11. A cell, wherein the cell comprises the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, the polynucleotide according to any one of claims 4-7, or the vector according to any one of claims 8-10.

12. The cell according to claim 11, wherein the cell comprises a prokaryotic cell or a eukaryotic cell.

13. The cell according to claim 12, wherein the eukaryotic cell comprises a mammalian cell, an insect cell, a flying animal cell, or a yeast cell.

14. The cell according to claim 13, wherein the mammalian cell comprises a myeloid cell, an immune cell, or a fibroblast.

15. The cell according to claim 13, wherein the mammalian cell comprises a lymphocyte.

16. The cell according to claim 11, wherein the cell is a hybridoma cell.

17. An antibody complex, wherein the antibody complex comprises the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, and a complex obtained by modification.

18. The antibody complex according to claim 17, wherein the modification comprises modification with a detectable label or an imaging agent.

19. A method for preparing the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, which comprises culturing the cell according to any one of claims 11-16, and extracting the monoclonal antibody or its antigen-binding fragment from the culture medium and / or the cells.

20. Use of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, the polynucleotide according to any one of claims 4-7, the vector according to any one of claims 8-10, the cell according to any one of claims 11-16, or the antibody complex according to claim 17 or 18 in the preparation of a product for detecting / diagnosing the level of CD28 molecule on the surface of T cells or the level of T cell activation.

21. 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 monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, or contacting the sample to be tested with the antibody complex according to claim 17 or 18; detecting the formation of a product of the CD28 protein and the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, or the antibody complex according to claim 17 or 18.

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