Monoclonal antibody against human cd147, expression vector, cell strain and application thereof

Abstract

The application discloses a monoclonal antibody against human CD147, a cell strain, an expression vector and application thereof. The antibody can be used for preparing an antibody conjugated drug, and can also be used for preparing a drug for diagnosing and treating a CD147 expression positive disease and a biotechnological product thereof.

Description

[0001] This case is a divisional application of application number CN201910796766.8, filed on August 27, 2019, with the subject matter of an invention application for an anti-human CD147 monoclonal antibody, expression vector, cell line and its application. Technical Field

[0002] This invention relates to the field of biotechnology. Specifically, this invention provides three humanized anti-CD147 antibodies, related cell lines, expression vectors, and their applications. Background Technology

[0003] CD147 has had several different names, including TCSF / EMMPRIN, M6, Basigin, and Neurothelin. It shares high homology with antigens from different species, such as mouse Basing / gp42, rat OX-47 / CE-9, and chicken HT7 / 5A11. The gene was ultimately designated Basigin by the Human Genome Nomenclature Committee (HUGO). The Human Leukocyte Differentiation Antigen Collaboration Group unified the various laboratory names under the name CD147, classifying it under the endothelial cell group. This molecule is a highly glycosylated transmembrane glycoprotein with a molecular weight of 50–60 kDa, belonging to the immunoglobulin superfamily (IgSF). In humans, CD147 consists of 269 amino acids, which can be divided into extracellular, transmembrane, and intracellular regions. The first 21 residues after N-terminal translation form the signal peptide; residues 22–205 constitute the extracellular region; residues 206–229 form the transmembrane region with a typical leucine zipper structure; and residues 230–269 form the intracellular region. CD147 has been confirmed to be overexpressed in many types of human solid tumors, such as lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, esophageal cancer, or gastric cancer.

[0004] Previous studies have shown that CD147 is an important functional membrane protein in tumor development, participating in various cancer-related phenomena. Furthermore, multiple retrospective studies have demonstrated a strong correlation between CD147 expression levels in tumor tissues and patient prognosis. In non-small cell lung cancer patients, elevated CD147 expression levels are closely associated with prognosis. Therefore, CD147 has become a novel target for cancer therapy, with the antibody drug "iodine-containing […]" being a potential candidate. 131 The successful development of "Metuximab Injection - Licartin" proves the safety and efficacy of drugs targeting this target.

[0005] Monoclonal antibodies (McAbs) are widely used in the diagnosis and treatment of many diseases due to their advantages such as high specificity, high affinity, low toxicity, low immunogenicity, long duration of action in vivo, and ability to exert therapeutic effects through the body's own immune system. They have become an effective approach for the development of new drugs. However, repeated injections of mouse-derived McAbs into humans can induce human anti-mouse antibody (HAMA) reactions, leading to systemic allergic toxicity and blocking the antibody's efficacy. Summary of the Invention

[0006] Based on the needs, shortcomings and defects of existing technologies, this invention provides a monoclonal antibody against human CD147, an expression vector, a cell line and their applications.

[0007] The monoclonal antibody against human CD147 provided by this invention has the following amino acid sequences for the heavy chain variable region and the light chain variable region:

[0008] WBP247.hAb12 antibody: The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:111 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:111, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:113 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:113.

[0009] WBP247.hAb4 antibody: The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:103 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:103, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:105 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:105.

[0010] Or it could be,

[0011] WBP247.hAb6 antibody: The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:107 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:107, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:109 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:109.

[0012] Furthermore, the nucleotide sequence of the heavy chain variable region of the WBP247.hAb12 antibody of the present invention contains specific antigen complementarity-determining regions CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the nucleotide sequence of the light chain variable region contains specific antigen complementarity-determining regions CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0013] The nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody of this invention are as follows:

[0014] WBP247.hAb12 antibody: The nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:110 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:110; the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO:112 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:112.

[0015] WBP247.hAb4 antibody: The nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:102 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:102; the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO:104 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:104.

[0016] Or it could be,

[0017] WBP247.hAb6 antibody: The nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:106 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:106; the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO:108 or a sequence with more than 90% homology to the sequence shown in SEQ ID NO:108.

[0018] On the other hand, the cell line expressing the above-mentioned monoclonal antibody provided by the present invention has the following name and accession number:

[0019] Cell line expressing WBP247.hAb12 antibody: named 247C-B4Z2-01-C-005, accession number: CCTCC NO.C2019147;

[0020] The cell line expressing WBP247.hAb4 antibody was named 247A-B9Z4-02-C-T9 and its accession number was CCTCCNO.C2019148.

[0021] Or it could be,

[0022] The cell line expressing the WBP247.hAb6 antibody was named 247B-B9Z4-01-C-T9, with accession number CCTCCNO.C2019149.

[0023] The present invention also provides an expression vector for the above-mentioned monoclonal antibody.

[0024] Furthermore, the present invention provides a method for preparing the above-mentioned monoclonal antibody. The provided method includes:

[0025] a) Obtain the DNA molecular sequence of the antibody according to claim 1, 2 or 3;

[0026] b) Construct an expression vector containing the DNA molecule described in step a) and the regulatory sequence for expressing the DNA molecule;

[0027] c) Transfect host cells, particularly mammalian cells, preferably CHO cells, with the expression vector described in step b); and culture them under culture conditions suitable for the host cells.

[0028] d) The monoclonal antibody described above was obtained through separation and purification steps.

[0029] The antibody of this invention can reduce the human antimouse antibody (HAMA) response caused by repeated injection of mouse-derived McAb into the human body, thus avoiding systemic allergic toxicity. Affinity analysis of the obtained antibody using ELISA showed that the EC50 value of the humanized antibody of this invention is approximately half that of the chimeric antibody WBP247.cAb1, and its affinity is approximately twice that of the chimeric antibody.

[0030] Preferably, the humanized antibody WBP247.hAb12 of the present invention has the heavy chain variable region amino acid sequence shown in SEQ ID NO:111 and the light chain variable region amino acid sequence shown in SEQ ID NO:113. The EC50 of the antibody prepared by expression is 0.002614 ug / ml, which is about twice that of the parental chimeric antibody WBP247.cAb1 (0.0382 nM). This antibody can specifically bind to solid tumors such as lung cancer and liver cancer.

[0031] In addition, the humanized antibodies WBP247.hAb4 and WBP247.hAb6 were stably screened. The EC50 values ​​of these two antibodies were 0.002864 ug / ml and 0.003022 ug / ml, respectively, indicating that these antibodies can specifically bind to solid tumors such as liver cancer.

[0032] In the construction of the antibodies described above, the light chain constant region is κ, and the heavy chain constant region is composed of IgG1. This invention also protects other antibody isotypes, such as IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. This invention also protects antigen-binding fragments of antibodies, including Fab, Fv, ScFv, and single-chain antibodies.

[0033] The antibody of this invention can be used in the preparation of drugs and biotechnology products for the diagnosis and treatment of CD147-positive diseases, as well as in detection reagents, imaging and laboratory diagnostics.

[0034] This invention also provides the application of the above-mentioned antibody in the preparation of antibody-drug conjugates. The drug is a maytansine derivative DM1, a microtubule polymerase inhibitor MMAE, or a radioactive isotope iodine. Attached Figure Description

[0035] Figure 1 SDS-PAGE analysis of humanized antibody clones 1–4; where, as shown in the attached figure, the left image is the non-reduction electrophoresis sample of purified protein, and the right image is the reduction electrophoresis sample of purified protein. From left to right: M is the protein marker; Lane 1: clone 1; Lane 2: clone 2; Lane 3: clone 3; Lane 4: clone 3 (prepared from different batches); Lane 5: WBP247.hAb4; Lane 6: WBP247.hAb4 (prepared from different batches);

[0036] Figure 2 SPR dissociation spectroscopy is used to determine the affinity of humanized antibodies. The horizontal axis represents the response time of the sample, and the vertical axis represents the signal value after binding. The lines in the figure are detection curves of the same sample at different concentration dilutions, indicating that there are specific changes in signal with changes in dosage.

[0037] Figure 3SDS-PAGE non-reducing electrophoresis analysis of humanized antibody clones 5–12; where Lane 1: clone 5 non-reduced; Lane 2: WBP247.hAb6 non-reduced; Lane 3: clone 7 non-reduced; Lane 4: clone 8 non-reduced; Lane 5: clone 9 non-reduced; Lane 6: clone 10 non-reduced; Lane 7: clone 11 non-reduced; Lane 8: clone 12 non-reduced;

[0038] Figure 4 SDS-PAGE reduction electrophoresis analysis of humanized antibody clones 5–12; Lane 1: clone 5 reduced; Lane 2: WBP247.hAb6 reduced; Lane 3: clone 7 reduced; Lane 4: clone 8 reduced; Lane 5: clone 9 reduced; Lane 6: clone 10 reduced; Lane 7: clone 11 reduced; Lane 8: clone 12 reduced;

[0039] Figure 5 SDS-PAGE reduction and non-reduction electrophoresis analysis of humanized antibody clones 13–16; Lane 1: clone 13 reduced; Lane 2: clone 14 reduced; Lane 3: clone 15 reduced; Lane 4: clone 16 reduced; Lane 5: clone 13 non-reduced; Lane 6: clone 14 non-reduced; Lane 7: clone 15 non-reduced; Lane 8: clone 16 non-reduced;

[0040] Figure 6 Gradient dilution curves of the three selected antibodies binding to the antigen;

[0041] Figure 7 Immunohistochemical screening diagram of full-length humanized antibodies; the attached diagram shows staining of WBP247.hAb5 (hepatocellular carcinoma), WBP247.hAb12 (32270) hepatocellular carcinoma and WBP247.hAb12 (32270) lung cancer from left to right, with the brown area indicating the antibody-positive area;

[0042] Figure 8 Image of expression vector pWX2.1-LC-B-247B4 plasmid;

[0043] Figure 9 Image of expression vector pWX2.1-LC-B-247B12 plasmid;

[0044] Figure 10 Image of expression vector pWX1.1-HC-Z-247B4 plasmid;

[0045] Figure 11 Image of the expression vector pWX1.1-HC-Z-247B6 plasmid;

[0046] Figure 12 Immunohistochemical staining images of CD147 humanized antibody on different tissues; the brown (dark) areas in the images are antibody-positive areas, and the white bars represent 100 μm;

[0047] Figure 13 In vitro killing experiment of humanized antibodies against tumor cells. Detailed Implementation

[0048] I. Terminology Explanation:

[0049] Immunoglobulins are a class of structurally related glycoproteins composed of two pairs of polypeptide chains: a pair of low-molecular-weight light chains (L) and a pair of high-molecular-weight heavy chains (H), all four chains linked together by disulfide bonds. Each heavy chain typically consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain typically consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region typically consists of a single domain, CL. VH and VL can be further subdivided into hypervariable regions (or highly variable regions in sequence and / or structurally defined loops), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, 1987). Typically, the amino acid residues in this region are numbered using the Kabat rule. (Sequences of Proteins of Immunological Interest, 5th Ed, Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (This numbering system is used in this paper for both heavy chain and light chain variable domains).

[0050] Humanized antibody: As used herein, this refers to an antibody derived from a non-human animal, typically a hamster, which has been modified to retain or substantially retain the antigen-binding properties of the parent antibody, but with reduced immunogenicity in the human body. Since the antibodies of this invention are defined by their structural and functional characteristics, "humanized antibody" can be used interchangeably with "antibody".

[0051] Complementarity-determining region (CDR): This refers to the characteristic sequence of multiple amino acids in an antibody that collectively defines the variable fragment (Fv) region of the immunoglobulin binding site, which determines the binding affinity and specificity of the target antigen CD147.

[0052] Frame regions (FRs): These are amino acid sequences inserted between CDRs. These portions of the antibody are used to hold the CDRs in place (allowing the CDRs to bind to the antigen). Both the light chain variable region and the heavy chain variable region contain frame regions (FRs) and typically three CDRs.

[0053] Constant region (CR): refers to the portion of the antibody molecule that confers effector function. In this invention, the constant regions of the humanized antibodies are all derived from human immunoglobulins. The heavy chain constant region can be selected from five isotypes: α, δ, ε, γ, or μ. Furthermore, various subclasses of the heavy chain (e.g., the IgG subclass of the heavy chain) can induce different effector functions; therefore, by selecting the desired heavy chain constant region, antibodies with the desired effector function can be produced. Preferred heavy chain constant regions are γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4), with γ2 (IgG2) being more preferred. The light chain constant region can be of type κ or λ, preferably type κ.

[0054] Chimeric Fab is created by recombining the light and heavy chain variable region genes of a functional antibody with the κ chain and heavy chain CH1 constant region genes of a human antibody, cloning them into an expression vector to construct a mouse-human chimeric Fab gene expression vector, and then transfecting it into host cells for expression. In 1994, the US FDA approved the first human-mouse chimeric Fab antibody, ReoPro (an antiplatelet receptor gIIb / IIIa), for antithrombotic therapy.

[0055] CDR-grafted antibodies are created by cloning all six CDRs from a murine monoclonal antibody into the corresponding frame regions (FRs) of a human antibody using methods such as PCR. Compared to chimeric antibodies, CDR grafting further reduces the amount of heterologous sequences in the antibody, thus decreasing antibody heterology.

[0056] Antibody affinity refers to the overall strength of the interaction between two molecules, such as an antibody and an antigen. Affinity characterizes the strength of binding between molecular pairs (e.g., antibody-antigen). The affinity of molecule X for ligand Y can be represented by the dissociation constant (KD), which is the concentration of Y required to occupy half of the binding site of X molecules present in solution. A smaller Kd indicates a stronger or higher affinity interaction and requires a lower ligand concentration to occupy the site.

[0057] The variable or constant regions of the immunoglobulin heavy or light chain can be linked as described by using standard recombinant DNA techniques to create polynucleotides that can be expressed in a suitable host (thus producing said immunoglobulin chain(s)), or the variable and constant regions can be linked by using peptide chemical synthesis.

[0058] The humanized antibody of this invention retains an important part of the binding properties of the parental murine antibody, which is the murine antibody HAb18, also known as the anti-human CD147 molecule. The establishment of this cell line can be found in *Monoclonal Antibody Communications*, 1989; 2:33-36, by Chen Zhinan, Liu Yanfang, Yang Jizhen, et al. (Prices already exist: 1. Light and heavy chain variable region genes of anti-human hepatocellular carcinoma monoclonal antibody HAb18 and their applications, Patent No.: ZL02114471.0, Publication (Announcement) No.: CN1381461A; and 2. VARIABLE REGION GENE OF HEAVY / LIGHT CHAIN ​​OF ANTI-HUMAN HEPATOMA MONOCLONAL ANTIBODYHAb18 AND USE THEREOF, (US Patent No.: US 7 638619). Specifically, this invention humanizes the anti-human CD147 murine parental antibody, retaining its ability to specifically bind to the parental antibody and recognize the antigen. Through optimization and screening, the obtained humanized antibodies exhibit the same or essentially the same antibody-binding affinity as the parent antibodies.

[0059] "Humanized antibodies" or "antibodies," as used in this invention, comprise the complete molecule as well as fragments of them capable of binding to epitope determinants, such as Fab, F(ab′)2, and Fv. These antibody fragments retain the ability to selectively bind to human CD147, and examples of these fragments include, but are not limited to, the following:

[0060] (1)Fab: Defined as a fragment containing a monovalent antigen-binding fragment of an antibody molecule, which is degraded by the enzyme papain to generate a complete light chain and a part of a heavy chain;

[0061] (2) Fab': Defined as a fragment containing a monovalent antigen-binding fragment of an antibody molecule. The whole antibody is treated with pepsin and then reduced to generate a portion of the complete light chain and heavy chain; each antibody molecule can yield two Fab' fragments.

[0062] (3)(Fab')2: defined as an antibody fragment obtained by treatment with the enzyme pepsin but without subsequent reduction;

[0063] (Fab)2 is a dimer of two Fab' segments linked together by two disulfide bonds;

[0064] (4) Fv: defined as a genetically engineered fragment containing a light chain variable region and a heavy chain variable region represented as two strands;

[0065] (4) Fv: Homology is a central concept in comparative biology. In molecular evolution research, homology generally refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules. Usually, homology must be tested by sequencing, but DNA-DNA or DNA-RNA hybridization can provide valuable judgment. (5) Coupling, also known as coupling reaction, is a process in which two organic chemical units undergo a certain chemical reaction to obtain an organic molecule. Depending on the type, it can be divided into cross-coupling and self-coupling reactions. Here, antibody coupling refers to the cross-linking of specific drug chemical reaction groups or small molecule drugs onto antibody molecules through specific reagent reactions, thereby improving the killing and therapeutic effects of antibodies or small molecule drugs.

[0066] This invention identified the CDR and FR regions of the light and heavy chain variable regions of the murine antibody HAb18 (ZL02114471.0) against human CD147 through bioinformatics analysis. Humanization design was performed using computer-aided antibody structure analysis, and molecular biology techniques, including phage display antibody library technology, were employed to humanize the framework region within the variable region. This yielded the gene sequence of the humanized variable region antibody against human CD147. A eukaryotic expression system containing the full-length antibody gene was constructed, and further expression in CHO host cells produced a humanized CDR-transplanted antibody against human CD147. Immunohistochemical and ELISA results showed that the selected humanized antibody against human CD147 maintained comparable affinity to the murine parent antibody and preserved the specificity of the parent antibody in recognizing the antigen.

[0067] The present invention will now be described in further detail with reference to the embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional molecular biology methods.

[0068] Unless otherwise specified, all materials and reagents used in the following examples can be obtained from commercial reagent companies.

[0069] II. Construction of a phage display vector containing the light and heavy chain variable regions of the murine antibody HAb18, which is an anti-human CD147 molecule. 1. Materials

[0070] The hybridoma cell line HAb18 was established in Monoclonal Antibody Communications, 1989; 2:33-36, by Chen Zhinan, Liu Yanfang, Yang Jizhen, et al. (Patent: CN1381461A).

[0071] PCR amplification primers for mouse-derived VH and VL of HAb18G: Among them, the primers for VH gene amplification are:

[0072] Upstream primer: HAb18-F, see SEQ ID NO:1 in the sequence listing;

[0073] Downstream primer 1: Linker-HAb18-R1, see SEQ ID NO:2 in the sequence listing.

[0074] Downstream primer 2: HAb18-linker-R2, see SEQ ID NO:3 in the sequence listing.

[0075] Among them, the primers for VL gene amplification are:

[0076] Upstream primer: HAb18-Linker-F1, see SEQ ID NO:4 in the sequence listing;

[0077] Downstream primer: HAb18-R, see SEQ ID NO:5 in the sequence listing.

[0078] 2. Methods and Results

[0079] 2.1 Total RNA extraction: Total RNA was extracted from hybridoma cells HAb18GC2 using the Total RNA Extraction Kit (OMEGA Total RNA R6834), and the integrity of the total RNA was detected by agarose gel electrophoresis.

[0080] 2.2 Reverse transcription of cDNA: Take 1 μg of the total RNA obtained in 2.1, and follow the instructions in TaKaRa... Synthesize the first strand of cDNA according to the instructions of the RTreagent Kit DRR037A reverse transcription kit, and store at -20℃ for later use.

[0081] 2.3 PCR amplification of VH and VL genes:

[0082] Using the cDNA prepared in 2.2 as a template, mouse-derived VH and VL gene fragments were amplified using VH and VL gene amplification primers, respectively. The amplification system was as follows: Instructions for High-Fidelity DNA Polymerase (NEB) preparation;

[0083] PCR reaction conditions: 94℃, 5 min; 94℃, 15 s, 54℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 10 min. The size of the amplified fragment was observed by 1% agarose gel electrophoresis.

[0084] The VH gene was first amplified using upstream primer HAb18-F and downstream primer 1 (Linker-HAb18-R1). After purification, the product was amplified again using HAb18-F and downstream primer 2 (HAb18-linker-R2) to obtain the VH gene. The VL gene was obtained by direct amplification using upstream primer HAb18-Linker-F1 and downstream primer HAb18-R.

[0085] 2.4 Amplification of the ScFv gene:

[0086] The VH gene fragment and VL gene fragment obtained in step 2.3 were mixed in equal molar amounts, and the ScFv gene was amplified using overlap-PCR. The amplification system is as follows:

[0087]

[0088]

[0089] PCR reaction conditions: 95℃, 5 min; 95℃, 15 s, 56℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 10 min. The target band was observed and recovered by 1% agarose gel electrophoresis.

[0090] 2.5 ScFv gene digestion, ligation with vector, and transformation: The amplified ScFv gene fragment and the pGEM-T vector (Promega) prepared above were digested with Nco I (NEB: C^CATGG) and Not I (NEB: GC^GGCCGC), respectively. 600 ng of the recovered fragment product from 2.4 and 3 μg of the pGEM-T vector were added, along with 1 μL of restriction endonuclease (NEB) and 5 μL of 10×CutSmart buffer. Water was added to a final volume of 50 μL, and the mixture was digested at 37°C for 1.5 h.

[0091] The enzyme digestion system is as follows:

[0092]

[0093] After digestion in a 37°C water bath, 5 μL of 10× loading buffer was added to terminate the reaction. The enzyme-digested bands were recovered by 1% agarose gel electrophoresis and quantified using UV spectroscopy.

[0094] The ligation reaction was performed using T4 phage DNA ligase, and the reaction system composition is as follows:

[0095]

[0096] The reaction was carried out overnight at 16°C. The ligation product was then transformed into TG1 competent cells. The cells were plated on LB agar plates and incubated overnight at 37°C. On the second day, 10 single clones were randomly selected and identified as positive clones using universal primers for the vector. These were recorded as pGEM-ScFv and sequenced for verification. Clones with correct sequencing results were stored at -40°C for later use.

[0097] III. CDR and FR sequence marking of anti-CD147 parental monoclonal antibody HAb18

[0098] The light and heavy chain variable region nucleotide sequences of the murine antibody HAb18, also known as HAb18G monoclonal antibody (CN021144710), encoding anti-CD147, were translated into their encoding amino acid sequences using the online software www.expasy.org. These marker sequences were identified according to the Kabat database principles and will be preserved in further humanization processes.

[0099] The amino acid sequences of CDR1, CDR2, and CDR3, the complementarity-determining regions (CDRs) identified in the light chain variable region of monoclonal antibody HAb18, are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, in the sequence listing. The amino acid sequences of CDR1, CDR2, and CDR3, the complementarity-determining regions (CDRs) identified in the heavy chain variable region of monoclonal antibody HAb18, are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, in the sequence listing.

[0100] IV. Selection of the FR (Frame Sequence) for Antibody Humanization

[0101] To select suitable human antibody framework sequences for transplantation onto mouse CDRs, the inventors constructed a human antibody sequence information database using antibody sequences derived from the Kabat protein database. Using Discovery Studio (VIOVIA, version 3.5), homology modeling and mechanical optimization techniques were employed to analyze and replace antibody molecular structures before and after humanization, ensuring that the replaced amino acids did not alter the overall VH and VL backbone structure, particularly the β-strand secondary structure, thus maintaining the original antibody affinity. The FR shuffling method, first proposed by Dall'Acqua, involves randomly substituting the FR region of a human antibody sequence with high homology to the FR region of the mouse antibody sequence to achieve humanization. This method reduces theoretical library capacity and irrelevant FR recombination.

[0102] The FR and CDR regions of the antibody variable region were determined using the Kabat database. Homology alignment was performed on the variable region sequence of the mouse antibody HAb18 in the antibody database. For the modification of FR1, FR2, and FR3 in VH, this invention referenced 12 FR1 germlines, 4 FR2 germlines, and 10 FR3 germlines, respectively. The VH-FR4 sequence is identical to the human IGHJ1*01 sequence and remains unchanged. For the modification of FR1, FR2, and FR3 in VL, 6, 9, and 7 germlines were referenced, respectively. The FR4 region of VH and VL was confirmed using JH and JK, respectively. Homology modeling was used to analyze the antibody molecular structure before and after humanization to ensure that the replaced amino acids did not alter the overall backbone structure of VH and VL, especially the β-strand secondary structure, thus maintaining the original antibody affinity. The design scheme is shown in Tables 1 and 2. The theoretical library size of the final designed humanized antibody library is 1.8 × 10^5.

[0103] Table 1V H Select the amino acid to change in the framework.

[0104]

[0105] Table 2 shows the amino acids to be replaced in the VL framework.

[0106]

[0107]

[0108] V. Construction and Screening of Phage Display CD147 Humanized Antibody Library

[0109] Based on the variable region sequence and replaceable amino acid sequence of the human antibody FR sequence obtained in (III) above, the combination of these sequences retains the CDR sequence (II) of the parental monoclonal antibody HAb18, i.e., the light chain variable region sequence contains the characteristic sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; the heavy chain variable region sequence contains the characteristic sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. In the FR frame region, according to the candidate sites in Tables 1 and 2, corresponding PCR primers were designed, and the designed full-length gene containing the mutation site was obtained by overlapping PCR. At the same time, codons commonly used in mammalian cells were partially replaced, and the obtained full-length gene was cloned into the phage vector pComb3Xss for the construction and screening of humanized antibody libraries. Finally, 16 effective humanized sequence combination clones of anti-CD147 light and heavy chain pairings were obtained.

[0110] 1. Primer design for CD147 humanized antibody library

[0111] Based on the selection analysis results of the humanized frame sequence FR of the antibody, a total of 70 primers for the antibody library were designed. All sequences begin with 247, such as 247-A1, 247-A2, etc., and are in the order of SEQ ID NO: 12 to SEQ ID NO: 81 in the sequence listing.

[0112] 2. Gene amplification protocol

[0113] Following the steps of overlapping-PCR, the specific protocol for amplifying the humanized antibody library is as follows:

[0114] Primer selection template length

[0115] first step

[0116]

[0117]

[0118] Step 2 247-A1,B1,F1,G1,A12 VH 404bp

[0119] 247-A13,A24 VL 394bp

[0120] Step 3

[0121] 247-A1,B1,F1,G1,A24 VH+VL 774bp

[0122] The target fragment was amplified under the following conditions: 95℃ for 3 min; 95℃ for 30 sec; 55℃ for 30 sec; 72℃ for 40 sec; 30 cycles, followed by a final extension at 72℃ for 10 min. After the PCR reaction, the PCR product was purified and recovered by 1% agarose gel electrophoresis.

[0123] 3. Construction of Phage Display Library

[0124] 3.1 Cloning of phage vectors

[0125] Humanized antibody library gene fragments amplified by PCR were recovered through double digestion with NcoI (NEB: C^CATGG) and Not I (NEB: GC^GGCCGC), under the same conditions as in condition (I) 2.5. After separation by 1% agarose gel electrophoresis, the digested fragments were purified using a Gel Extraction Kit (Omega Bio-tek). The purified fragments were then ligated with the NcoI / Not I double-digested phage vector pComb3Xss using T4 DNA ligase (TaKaRa). After deionization, the ligation was performed by electroporation into TG1 competent cells, which were then seeded onto LB agar plates for clonal selection. Forty-eight single clones were randomly selected, and positive colonies were identified using primers M13-48 and M13-47. The library volume was calculated, and the library was stored at -80℃ for later use.

[0126] 3.2 Preparation and titration of helper phages

[0127] Pick a single colony of XL-1Blue and inoculate it with 5 ml of SB-T + (20ug / ml) solution, incubate overnight at 37℃ with shaking. Inoculate 10ml of SB-T solution with a 1:500 dilution. + Incubate (20ug / ml) solution at 37°C with shaking for one hour. Inoculate a single plaque of phage M13K07 into 10ml of the above bacterial culture, incubate at 37°C with shaking for 2 hours, then add 200ml of SB-T. + (20ug / ml)K + Incubate (70ug / ml) solution overnight at 37°C with shaking. Centrifuge at 4000rpm for 15 minutes at 4°C, collect the supernatant to determine the phage titer, aseptically aliquot and store at 4°C.

[0128] 3.3 Phage rescue experiment

[0129] Scrape 6 mL of the electroporation bacteria from 3.1 and dilute it to 400 mL of SOB-GAT solution. Incubate at 30°C until A600 = 0.5. Add M13K07 and incubate at 37°C for 1 hour for superinfection (ideally with a multiinfection ratio of 5:1; the number of PFU of M13K07 added should be 5 × 10⁻⁶). 8(Bacteria / A600 units) × Multiple infection value (5) × A600 value (0.5) × Final bacterial volume (mL), centrifuge at 3500 RPM for 10 min at 4℃, resuspend the precipitate in the original volume of 2YT-AKT, and incubate overnight at 30℃ with medium speed and shaking. Centrifuge the rescue culture medium at 4000 rpm for 20 min at 4℃, take the supernatant, add 4% PEG8000 and 3% NaCl to precipitate the phage in an ice bath for 1 h, centrifuge at 15000g for 20 min at 4℃, resuspend the precipitate in 1-2 mL of sterile PBS (containing 1% BSA, 0.02 mol / L, pH 7.4), briefly centrifuge at low speed, and the supernatant is the rescued original phage antibody library (aliported and stored at 4℃ for later use).

[0130] 3.4 Selection of Phage Antibody Library

[0131] The antibody phage library obtained in 3.1 was subjected to antigen-specific panning using a solid-phase panning method. The antigen coating concentration was decreased sequentially in each round of panning, and positive / negative controls were included. The specific procedure was as follows: The antibody phage library preserved in 3.1 was revived in 60 ml of 2YT medium and cultured at 37°C in a shaker until OD600 = 0.3-0.4. M13KO7 helper phage (Invitrogen) was added; the cells were incubated statically at 37°C for 30 minutes, then incubated in a shaker for 60 minutes. The cells were centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in 60 ml of 50 μg / ml kanamycin (glucose-free) medium and cultured overnight in a shaker at 30°C. The phage library was precipitated by centrifugation at 12000 rpm for 10 minutes, and the supernatant was transferred to centrifuge tubes (30 ml / tube). 7.5 ml of PEG / NaCl was added to each centrifuge tube, mixed thoroughly, and placed on ice for 1 hour. Centrifuge at 12,000 rpm for 5 minutes and discard the supernatant. Resuspend the phage in 2.2 ml of PBS-5% BSA solution, centrifuge at 12,000 rpm for 5 minutes, and remove cell debris. Then, use the expressed CD147 molecules to coat the plate for affinity screening. After 5 rounds of panning (adsorption-elution-amplification), the antigen coating concentration decreased sequentially in each round (1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, 0.0001 μg / ml). The input / output ratio (recovery rate) of phages in each round of screening was calculated as an indicator of specific phage antibody enrichment. The calculation formula is: recovery rate (% yield) = (elution buffer volume × elution buffer titer × 100) / (input antibody library volume × antibody library titer). When the recovery rate was less than 10, the panning experiment was terminated. 768 clones were selected, induced to express, and the gene product containing humanized gene antibody heavy chain VH and light chain VL, namely ScFv antibody, was obtained for further ELISA detection.

[0132] VI. ELISA Analysis and Sequencing Analysis

[0133] Dilute CD147 to 1 μg / ml with coating buffer (200 mM Na2CO3 / NaHCO3, pH 9.2), add 50 μl to each well, and coat overnight at 4°C; discard the solution in the wells, wash 3 times with 1X PBS buffer, add 200 μl of blocking buffer (2% BSA / 1X PBS buffer) and block at room temperature for 1 h; wash 3 times with 200 μl of 1X PBS buffer; add 768 samples (8 96-well microplates) of cell culture supernatant containing ScFv antibody induced by (IV) 3.4 and negative control (50 μl / well), and incubate at room temperature for 2 h; wash 3 times with 200 μl of 1X PBS buffer; add Anti-c-MycAb(HRP)(Abcam) diluted (1:2500) with blocking buffer. Cat#ab19312 (50 μl / well), incubate at room temperature for 1 h; wash 6 times with 200 μl of 1XPBS buffer; add TMB substrate solution (50 μl / well) to each reaction well and react for 10 min; add stop solution (2M HCl, 50 μl / well) to terminate the reaction; read the absorbance at A450 nm using an ELISA reader;

[0134] Based on the ELISA results, 50 clone samples with A450 > 3.0 were selected for sequencing (sequencing was performed by Shanghai Boshan Biotechnology Co., Ltd.). DNA sequence analysis: The degree of humanization of the sequences was evaluated with reference to the Germline database of human antibodies and http: / / www.bioinf.org.uk / abs / shab / , and molecules with a high degree of humanization were sorted by affinity.

[0135] VII. SPR assay for the affinity of ScFv antibody

[0136] 7.1 SPR Combined Analysis Method

[0137] The affinity of the strong positive ELISA antibody in step (V) was determined using a ProteOn XPR36 (Bio-Rad, XPR36) instrument. The GLC chip (Bio-Rad, 1765011) was activated with 0.04M EDC + 0.01M sulfo-NHS (Bio-Rad). CD147 was diluted to 10mM with 10mM NaAc (pH 4.5) and injected onto the chip at a rate of 30 μL / min, allowing the antigen to couple with the activated chip via amino groups. Finally, the chip was inactivated with 1M ethanolamine-HCl (Bio-Rad); after rotating the chip 90 degrees, it was washed with buffer (PBS / 0.005% Tween 20) until the baseline stabilized. Cell culture supernatant containing the ScFv antibody induced by (IV) 3.4 was injected into each of the six horizontal channels at a rate of 30 μL / min. The sample binding time was 60 s and the dissociation time was 900 s; data analysis was performed using the kinetic-Langmuir model; clones with high affinity were selected for the construction of fully humanized antibodies.

[0138] 7.2 SPR determination of ScFv antibody affinity ranking

[0139] The binding of coated CD147 to cell culture supernatant containing ScFv antibody induced by (IV) 3.4 was monitored in real time using SPR, and the dissociation rate constant (K) was determined. off The results reflect the affinity between CD147 and the humanized ScFv antibody. Table 3 shows the results. Based on the changes in affinity, several molecules with good affinity were selected for the construction of fully humanized antibodies.

[0140] Table 3. Sequence analysis of the humanization degree of HAb18 and K off Sort

[0141]

[0142]

[0143]

[0144] 8. Construction and Detection of Full-Length Humanized Antibodies 8.1 Materials

[0145] Based on the aforementioned affinity ranking results, the first four clones (clones 26601, 26602, 27028, and 27044) with the highest affinity ranking of (vi)2, i.e., SPR assay fragment antibody ScFv antibody, were selected for the first round of full-length antibody construction and expression.

[0146] The four selected clones were inoculated and cultured overnight, and plasmids were extracted from each clone and sequenced to confirm the template sequence.

[0147] PCR primers: Humanized antibody VH gene amplification primers: upstream primer: 247-VH_F1, as shown in SEQ ID NO: 84 in the sequence listing; downstream primer: 247-VH_R1, as shown in SEQ ID NO: 85 in the sequence listing, the target product size is 392bp; Humanized antibody VL gene amplification primers: the first set of upstream primers: 247-33_F1, downstream primers: 247-33_R1, as shown in SEQ ID NO: 82 and SEQ ID NO: 83 in the sequence listing, respectively; Based on the sequencing results, the second set of upstream primers: 247-VL-2_F2; downstream primers: 247-vl_R1, corresponding to SEQ ID NO: 87 and SEQ ID NO: 86 in the sequence listing, respectively.

[0148] 8.2 PCR Amplification Process

[0149] After extracting plasmids and confirming the template sequence was correct via sequencing, PCR amplification of the target fragment was performed using the primers and templates described above. The amplification protocols are as follows:

[0150] Primer template target fragment length

[0151]

[0152] 8.3 Preparation of transient expression vectors

[0153] The light chain variable region gene or heavy chain variable region gene amplified in step 7.2 was double-digested with type II restriction endonucleases NgoMIV and SnaBI, respectively. After digestion, the DNA was purified using a DNA purification kit and ligated with the mammalian cell expression vector pCI-vector containing hIgG1 / k, digested with the same restriction endonucleases NgoMIV / SnaBI. The ligation product was transformed into TOP10 *E. coli* and plated on LB agar medium containing 100 μg / ml ampicillin. The obtained positive clones were cultured in LB liquid medium containing 100 μg / ml ampicillin, and plasmids were extracted and sequenced to obtain full-length eukaryotic expression vector clones 1-VH1–4 containing the humanized antibody VH gene and full-length eukaryotic expression vector clones 1-VL1–4 containing the humanized antibody VL gene, respectively. Sequencing revealed that the nucleotide sequence corresponding to the heavy chain variable region of clone 4 is as shown in SEQ ID NO:102; the nucleotide sequence corresponding to the light chain variable region is as shown in SEQ ID NO:104.

[0154] IX. Transient cell transfection and antibody expression and purification

[0155] 9.1 Transient transfection of cells with antibodies

[0156] Using Invitrogen's Freestyle Max Reagent transfection reagent, the full-length eukaryotic expression vector clones 1-VH1-4 of the VH gene obtained in (VII) 7.3 and the full-length eukaryotic expression vector clones 1-VL1-4 containing the humanized antibody VL gene were co-transfected into HEK293 cells (1.0 × 10⁻⁶ cells per ... 6 Cells were transfected (number of cells / ml) and placed in a shaker at 37°C in a 5% CO2 incubator at 120 rpm. Seven days after transfection, the cell supernatant was collected by centrifugation. The target full-length humanized antibody was isolated and purified from the cell culture supernatant using a Protein A affinity chromatography column. The protein concentration of the purified antibody was determined and used for further purification of the antibody (antibodies were designated as clones 1-4). The SPR (Symptom Ratio) of the antibody was also measured.

[0157] 9.2 SDS-PAGE analysis of antibodies

[0158] The standard laboratory SDS-PAGE analysis method was employed, involving the separate mixing of NuPAGE LDS sample buffer, NuPAGE reducing agent, and each sample, followed by incubation at 75°C for 10 minutes and centrifugation. The sample loading volume was 2 μg / well, and the gel was run at 200V for 35 minutes. After electrophoresis, the gel was rinsed three times for 5 minutes each time. Simply Blue Safestain staining was then added for one hour. After staining, the staining solution was discarded, and deionized water was added for destaining until the gel background was completely destained. The results are shown in the attached figure. Figure 1 .

[0159] The left image shows the non-reduction electrophoresis sample of purified protein, while the right image shows the reduction electrophoresis sample of purified protein. The six lanes in both images, from left to right, are: Lane 1: Clone 1; Lane 2: Clone 2; Lane 3: Clone 3; Lane 4: Clone 3 (prepared in different batches); Lane 5: Clone 4; Lane 6: Clone 4 (prepared in different batches); M stands for protein marker.

[0160] 10. Determination of affinity for humanized antibodies

[0161] SPR assay for affinity of humanized antibodies: The affinity of the first batch of four purified humanized antibodies was determined by SPR, using the same method as in (VI) 6.1. The results showed that the affinity of purified antibody clones 1, 2, 3, and 4 (1.9E-9, 2.63E-9, 1.61E-9, and 1.83E-9) was slightly lower than that of the parental chimeric antibody WBP247.cAb1 (3.94E-10). The results are shown in the figure. The binding and dissociation patterns of the antibodies are shown in the appendix. Figure 2 .

[0162] Table 4 SPR kinetic data of humanized antibodies

[0163]

[0164] XI. Immunohistochemical staining for the specific screening and identification of humanized antibodies

[0165] Although the affinity of the humanized antibody was determined using the SPR method, since the antigen used was a purified antigen prepared by in vitro expression, further immunohistochemical staining of the antibody was performed on the tumor tissue specimen maintained in the laboratory to further observe the binding specificity of the obtained humanized antibody.

[0166] The specific binding ability of the expressed humanized antibody clones 1-4 to tumor tissue was detected, and the immunohistochemical cross-reactivity of the antibodies was examined.

[0167] The specific procedures were as follows: routine xylene dewaxing, gradient alcohol dehydration, and tissue microarray hydration; 3% H2O2 blocking and inactivation of endogenous peroxidase; blocking with normal sheep serum working solution; using antibody clones 1-4 as primary antibodies, biotin-labeled rabbit anti-human Fc antibody as secondary antibodies, and horseradish peroxidase-labeled streptomycin avidin working solution as tertiary antibodies; DAB staining, hematoxylin counterstaining, dehydration and clearing, mounting, and microscopic examination. The histochemical screening results showed that only antibody clone 4, WBP247.hAb4, showed specific staining in malignant tumor tissues such as lung cancer and liver cancer, with staining levels of "++" or "+++"; while it rarely bound to normal tissues. The other three antibodies were negative. These results indicate that only one usable candidate humanized antibody, WBP247.hAb4, was screened through this round of expression. This antibody has similar antigen recognition specificity to the murine parent antibody. The humanized antibody was successfully constructed, but the number of usable humanized antibodies is extremely small, failing to meet the quantity requirements. Therefore, multiple rounds of expression and screening are needed.

[0168] XI. Repeated rounds of construction and immunohistochemical screening of full-length humanized antibodies

[0169] 11.1 Repeated rounds of construction and detection of full-length humanized antibodies

[0170] Based on the results provided by immunohistochemistry, since the specificity of the antibodies may change after humanization, the previous strategy was adjusted. First, the top 16 clones in affinity ranking for the SPR assay fragment antibody ScFv (as described in section (VI) 2) were used for full-length antibody construction and expression. The method was the same as in section 7.1, where the selected clones were used for vector construction and expression of the full-length antibodies. Specifically, the nucleotide sequence of clone 6 (WBP247.hAb6) is shown in SEQ ID NO:106; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:108. The nucleotide sequence of the heavy chain variable region of clone 12 (WBP247.hAb12) is shown in SEQ ID NO:110; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:112. Then, the obtained antibodies were purified according to section (VIII) 8.1 and transient cell transfection and antibody expression purification (antibodies were designated as clones 5-16). SDS-PAGE analysis was performed according to section 8.2, and the results are shown in the appendix. Figure 3 Appendix Figure 4 and attached Figure 5 .

[0171] 12. ELISA Analysis of Humanized Antibody Affinity

[0172] 200 ng of recombinant CD147 protein was coated onto an ELISA plate and incubated overnight at 4°C. Blocking was performed with 1X PBS / 2% BSA at room temperature for 1 hour. The purified antibody products from steps (viii) and subsequent preparations (clones 1-6) were serially diluted 1 μg / ml with blocking buffer at a 1:3.16 ratio, with 100 μl added to each well and incubated at room temperature for 1 hour. 100 μl of horseradish peroxidase-labeled goat anti-human IgG Fc HRP (Bethyl Cat#A80-304P) diluted 1:4000 was added and incubated at room temperature for 1 hour. TMB was added for color development, and the reaction was terminated with 2M H2SO4. Readings were taken at 450 nm using an ELISA reader. The ELISA results showed that the EC50 values ​​of the 16 humanized antibodies (clones 1-16) were approximately half that of the chimeric antibody WBP247.cAb1, and the affinity was approximately twice that of the chimeric antibody. (See Table 5.) Figure 6 ).

[0173] Table 5. Antibody-antigen CD147 binding EC 50 Measurement

[0174] chimeric cAb1 0.005733 0.0382 Clones 1 0.002725 0.0182 Clone 2 0.002944 0.0196 Clone 3 0.003076 0.0205 Clone 4 0.002864 0.0191 Clones 5 0.002811 0.0187 Clones 6 0.003022 0.0201 Clones 7 0.002507 0.0167 Clones 8 0.001701 0.0113 Clones 9 0.002099 0.0140 Clones 10 0.002061 0.0137 Clones 11 0.002362 0.0157 Clones 12 0.002614 0.0174 Clones 13 0.002527 0.0168 Clones 14 0.002509 0.0167 Clones 15 0.002432 0.0162 Clones 16 0.00325 0.0217

[0175] 13. Immunohistochemical screening of full-length humanized antibodies

[0176] To further detect the specific binding ability of the expressed humanized antibody clones 5-16 to tumor tissue and examine their immunohistochemical cross-reactivity, immunohistochemical staining was used to further screen the specificity of the humanized antibodies. The specific procedures were as follows: routine xylene dewaxing, gradient alcohol dehydration, and tissue microarray hydration; 3% H2O2 blocking and inactivation of endogenous peroxidase; blocking with normal sheep serum working solution; using antibody clones 5-16 as the primary antibody, biotin-labeled rabbit anti-human Fc antibody as the secondary antibody, and horseradish peroxidase-labeled streptomycin avidin working solution as the third antibody, DAB staining, hematoxylin counterstaining, dehydration and clearing, mounting, and microscopic examination. Positive result was defined as a brown staining of the cell membrane. Through this round of expression screening, two more usable candidate humanized antibodies, WBP247.hAb6 and clone 12, were obtained. Clone 12 can bind not only to liver cancer specimens but also to lung cancer specimens. Specific information is shown in Table 6 below.

[0177] Table 6. Specificity screening of humanized antibodies by immunohistochemical staining.

[0178]

[0179]

[0180] Through repeated histochemical staining, the binding specificity of the obtained humanized antibodies was determined. Three humanized monoclonal antibodies, namely clone 4 (WBP247.hAb4), clone 6 (WBP247.hAb6), and clone 12 (WBP247.hAb12), were initially screened for downstream stable cell lines.

[0181] XIV. Construction of high-efficiency expression vectors for humanized antibodies and screening of stable expression cell lines

[0182] Based on the results of the previous ELISA and immunization, the gene sequences of the three clones with the best affinity and specificity, namely clone 4, clone 6 and clone 12, namely “WBP247.hAb4, WBP247.hAb6 and WBP247.hAb12”, were selected for the construction of efficient expression vectors and the screening of stable cell lines.

[0183] Table 7. Sequence combination information for WBP247.hAb4, WBP247.hAb6, and WBP247.hAb12

[0184]

[0185] (Among them, the amino acid sequence of the light chain variable region of clone 12 is the sequence shown in SEQ ID NO:113, and the corresponding nucleotide sequence of the light chain variable region (VL) is SEQ ID NO:112; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO:111, and the corresponding nucleotide sequence of the heavy chain variable region (VH) is SEQ ID NO:110).

[0186] 14.1 Construction of the light chain expression vector pWX2.1-LC-B-247B4

[0187] In vitro amplification of the gene DNA (27989-VL) fragment was performed. Using this DNA as a template, the variable region fragment WBP247B4-VL was amplified using primers WX-893, WX-894, WX-895, and WX-900 (corresponding to SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:101 in the sequence listing). An EcoRI restriction enzyme site was introduced at the 5' end of WBP247B4-VL, and a BsiWI restriction enzyme site was introduced at the 3' end. The fragment and the expression vector pWX2.1 containing the light chain constant region gene were digested with EcoRI and BsiWI. After 1% agarose gel electrophoresis, the 5119 bp DNA fragment was recovered by gel excision. The purified DNA fragment was ligated with the fragment from plasmid pWX2.1 using 20 μl of T4 ligase at 16°C for 20 min. 10 μl of the ligation solution was then transformed into *E. coli* TOP10 competent cells. After colony PCR identification, enzyme digestion identification, and sequencing, a single correct colony was selected and cultured overnight at 37°C with shaking at 220 rpm in 200 ml LB medium. Plasmid was extracted in large quantities, and the final plasmid was named pWX2.1-LC-B-247B4 (5521 bp). Figure 8 (As shown).

[0188] 14.2 Construction of the light chain expression vector pWX2.1-LC-B-247B12

[0189] In vitro amplification of the gene DNA (32270-VL) fragment was performed. Using this DNA as a template, the variable region fragment WBP247B12-VL was amplified using primers WX-896, WX-897, WX-898, and WX-900 (corresponding to SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:101 in the sequence listing). An EcoRI restriction enzyme site was introduced at the 5' end of WBP247B12-VL, and a BsiWI restriction enzyme site was introduced at the 3' end. This fragment and the expression vector pWX2.1 containing the light chain constant region gene were digested with EcoRI and BsiWI. After 1% agarose gel electrophoresis, the 5119 bp DNA fragment was recovered by gel extraction. The gel-purified DNA fragment was ligated with the fragment from plasmid pWX2.1 and transformed into E. coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification, and sequencing, a single correct clone was selected and cultured in 200 ml of LB medium at 37°C with shaking at 220 rpm overnight. A large amount of plasmid was extracted, and the final plasmid was named pWX2.1-LC-B-247B12 (5521 bp). Figure 9 (As shown).

[0190] 14.3 Construction of the heavy chain expression vector pWX1.1-HC-Z-247B4

[0191] In vitro amplification of the gene DNA fragment (27989-VH) was performed. Using this DNA as a template, the variable region fragment WBP247B4-VH was amplified using primers WX-887, WX-888, WX-889, and WX-899 (corresponding to SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:100 in the sequence listing). An EcoRI restriction enzyme site was introduced at the 5' end of WBP247B4-VH, and an NheI restriction enzyme site was introduced at the 3' end. This fragment and the expression vector pWX1.1 containing the heavy chain constant region gene were digested with EcoRI and NheI. After 1% agarose gel electrophoresis, the 5202 bp DNA fragment was recovered by gel extraction. The gel-purified DNA fragment was ligated with the fragment from plasmid pWX1.1 and transformed into E. coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification, and sequencing, a single correct clone was selected and cultured in 200 ml of LB medium at 37°C with shaking at 220 rpm overnight. A large amount of plasmid was extracted, and the final plasmid was named pWX1.1-HC-Z-247B4 (5625 bp). Figure 10 (As shown).

[0192] 14.4 Construction of the heavy chain expression vector pWX1.1-HC-Z-247B6

[0193] In vitro amplification of the gene DNA (32338-VH) fragment was performed. Using this DNA as a template, the variable region fragment WBP247B6-VH was amplified using primers WX-890, WX-891, WX-892, and WX-899 (corresponding to SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO:100 in the sequence listing). An EcoRI restriction enzyme site was introduced at the 5' end of WBP247B6-VH, and an NheI restriction enzyme site was introduced at the 3' end. This fragment and the expression vector pWX1.1 containing the heavy chain constant region gene were digested with EcoRI and NheI. After 1% agarose gel electrophoresis, the 5202 bp DNA fragment was recovered by gel extraction. The gel-purified DNA fragment was ligated with the fragment from plasmid pWX1.1 and transformed into E. coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification, and sequencing, a single correct clone was selected and cultured in 200 ml of LB medium at 37°C with shaking at 220 rpm overnight. A large amount of plasmid was extracted, and the final plasmid was named pWX1.1-HC-Z-247B6 (5625 bp). Figure 11 (As shown).

[0194] Construction and screening of 14.5CHO stable expression cell lines

[0195] After constructing the vectors, WuXi AppTec was commissioned to co-transfect the above-mentioned humanized antibody light chain expression vectors and humanized antibody heavy chain expression vectors into CHO / DHFR cells according to (1) pWX2.1-LC-B-247B4, pWX1.1-HC-Z-247B4; (2) pWX2.1-LC-B-247B6, pWX1.1-HC-Z-247B4; (3) WX2.1-LC-B-247B12, pWX1.1-HC-Z-247B4. Appropriate concentrations of culture medium were added for screening and passage, and single clones were selected to obtain stable CHO expression cell lines.

[0196] Subsequently, Minipool batch feeding experiments were conducted to measure the titers. After four rounds of batch feeding optimization, cell lines stably expressing WBP247.hAb12 antibody (named: cell line 247C-B4Z2-01-C-005 expressing mehozumab monoclonal antibody (CCTCC NO. C2019147), cell line 247A-B9Z4-02-C-T9 expressing WBP247.hAb4 (CCTCC NO. C2019148), and cell line 247B-B9Z4-01-C-T9 expressing WBP247.hAb6 (CCTCC NO. C2019149) were obtained. All of these cell lines are currently deposited at the China Center for Type Culture Collection (CCCHC) in Wuhan, with a deposit date of July 16, 2019.

[0197] XV. In vitro tumor cell killing experiment of humanized anti-CD147 antibody

[0198] Methods: Human lung cancer cells NCI-H520 in the logarithmic growth phase were obtained, digested with trypsin, and prepared into a single-cell suspension, with the concentration adjusted to 1×10⁻⁶. 4 Cells / ml. 2000 cells (200 μl cell suspension) were seeded into 96-well plates and incubated overnight at 37°C with 5% CO2 until the cells adhered and resumed logarithmic proliferation.

[0199] Following standard laboratory preparation methods for ADC drugs, three humanized antibodies were labeled with DM1 to obtain WBP247.hAb12-DM1, WBP247.hAb4-DM1, and WBP247.hAb6-DM1. These were diluted with serum-free RPMI 1640 medium to the following concentrations: 1000, 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μg / mL. 20 μl of each concentration was added to the corresponding well, with six replicates per concentration. A negative antibody control (human IgG) and a background assay (cell-free medium only) were also included. The mixture was incubated at 37°C for 48 h with 5% CO2. Then, 10 μL of CCK-8 chromogenic solution was added to each well. After incubation at 37°C for 2 h, the absorbance was read at 450 nm using a full-wavelength microplate reader. Results are as follows: Figure 13 As shown, the screened humanized antibodies all exhibited good killing effects against tumor cells (see attached diagram). Figure 13 This indicates that the prepared human antibodies all possess good tumor specificity and targeting. Based on this effect, those skilled in the art can infer that the antibodies of the present invention can be used to prepare drugs and their biotechnology products for diagnosing and treating CD147-positive diseases, as well as diagnostic reagents, imaging and laboratory diagnostic reagents.

Claims

1. A monoclonal antibody against human CD147, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:107 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

109.

2. A gene encoding the anti-human CD147 monoclonal antibody of claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the anti-human CD147 monoclonal antibody is shown in SEQ ID NO:106, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

108.

3. A cell line expressing the monoclonal antibody of claim 1, characterized in that, The cell line has the following preservation number: CCTCC NO. C2019149.

4. The antibody expressed by the cell line of claim 3.

5. The expression vector for the monoclonal antibody of claim 1.

6. The method for preparing the antibody according to claim 1, characterized in that the method... include: a) Obtain the DNA molecular sequence of the antibody according to claim 1; b) Construct an expression vector containing the DNA molecule described in step a) and the regulatory sequence for expressing the DNA molecule; c) Transfect host cells with the expression vector described in step b); culture them under suitable culture conditions for the host cells; d) Obtain the monoclonal antibody of claim 1 through separation and purification steps.

7. The use of an antibody-drug conjugate of claim 1 in the preparation of a drug for treating liver cancer or lung cancer, wherein the antibody-drug conjugate is a maytansine derivative DM1 conjugated with the antibody of claim 1 or a microtubule polymerase inhibitor MMAE conjugated with the antibody of claim 1.

8. The use of the antibody of claim 1 in the preparation of a drug for diagnosing CD147-positive diseases, wherein the CD147-positive disease is liver cancer or lung cancer.

Images