CN110642947B - Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof - Google Patents

Abstract

The invention discloses an anti-human CD147 monoclonal antibody, a cell line, an expression vector and applications thereof. The antibody of the invention can be used to prepare antibody-coupled drugs, and can also be used to prepare drugs for diagnosing and treating CD147-positive diseases and biotechnological products thereof.

Description

Anti-human CD147 monoclonal antibody, expression vector, cell line and application thereof

technical field

The invention relates to the field of biotechnology, specifically, the invention provides three anti-CD147 humanized antibody anti-human, related cell lines, expression vectors and applications thereof.

Background technique

CD147 has a variety of different names, including TCSF/EMMPRIN, M6, Basigin, Neurothelin, and has high homology with antigens from different species such as mouse Basing/gp42, rat OX-47/CE-9 and chicken HT7/5A11 The gene was finally identified as Basigin by the Human Gene Nomenclature Committee HUGO; while the Human Leukocyte Differentiation Antigen Cooperative Group unified the names from various laboratories as CD147, which belonged to the endothelial cell group. The molecule is a highly glycosylated, transmembrane glycoprotein with a molecular weight of 50-60kD, which is a member of the immunoglobulin superfamily (IgSF). In humans, CD147 consists of 269 amino acids, which can be divided into extracellular region, transmembrane region and intracellular region. The first 21 residues after N-terminal translation are signal peptides, 22-205 constitute the extracellular region, 206-229 are transmembrane regions with a typical leucine zipper structure, and C-terminal 230-269 is the intracellular region. CD147 has been shown 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.

Previous studies have shown that CD147 molecule is an important functional membrane protein in the process of tumor development and is involved in various cancer-related phenomena. Moreover, multiple retrospective studies have also shown that the expression intensity of CD147 molecule in tumor tissue is closely related to the prognosis of tumor patients. There is a close correlation between them. In patients with non-small cell lung cancer, the increased expression level of CD147 is closely related to the prognosis of the patients. Therefore, the CD147 molecule has become a new target for tumor treatment, and the successful development of the antibody drug "Iodine [ ¹³¹ I] Metuximab Injection - Licartin" has proved the safety and effectiveness of this target drug .

Monoclonal antibody (McAb) has been widely used in the diagnosis and treatment of many diseases due to its high specificity, high affinity, low toxicity and side effects, low immunogenicity, long acting time in the body, and the ability to exert curative effects on the body's own immune system. An effective way to develop new drugs. However, repeated injections of mouse McAbs into the human body will induce human anti mouse antibody (human anti mouse antibody, HAMA) reactions in patients, causing systemic allergic toxic reactions and blocking the efficacy of antibodies.

Contents of the invention

Based on the requirements, deficiencies and defects of the prior art, the present invention provides anti-human CD147 monoclonal antibody, expression vector, cell line and application thereof.

In the anti-human CD147 monoclonal antibody provided by the present invention, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are:

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 with the sequence shown in SEQ ID NO: 111, and the amino acid sequence of the light chain variable region is as follows: A sequence shown in SEQ ID NO: 113 or having a homology of more than 90% with the sequence shown in SEQ ID NO: 113;

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 with 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 with the sequence shown in SEQ ID NO: 105;

or for,

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 with 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 with the sequence shown in SEQ ID NO: 109.

Further, the nucleotide sequence of the heavy chain variable region of the WBP247.hAb12 antibody of the present invention contains the sequences of the specific antigen complementarity determining regions CDR1, CDR2 and CDR3 respectively as SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 17 and SEQ ID NO: ID NO: 18, the sequences of CDR1, CDR2 and CDR3 of the specific antigen complementarity determining region included in the nucleotide sequence of the light chain variable region are SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 .

The nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody of the present invention are:

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

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

or for,

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 with the sequence shown in SEQ ID NO: 106; the nucleus of the light chain variable region The nucleotide sequence is the sequence shown in SEQ ID NO: 108 or a sequence with more than 90% homology with the sequence shown in SEQ ID NO: 108.

On the other hand, the cell line expressing the above-mentioned monoclonal antibody provided by the present invention, the name and storage number of the cell line are:

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

Cell line expressing WBP247.hAb4 antibody: named 247A-B9Z4-02-C-T9, deposited

The number is: CCTCC NO. C2019148;

or for,

Cell line expressing WBP247.hAb6 antibody: named 247B-B9Z4-01-C-T9, deposit number: CCTCCNO. C2019149.

The present invention also provides the expression vector of the above-mentioned monoclonal antibody.

Further, the present invention provides the preparation method of the above-mentioned monoclonal antibody. The methods provided include:

a) obtaining the DNA molecular sequence of the antibody described in claim 1, 2 or 3;

b) constructing an expression vector, which contains the DNA molecule described in step a) and the regulatory sequence for expressing the DNA molecule;

c) using the expression vector described in step b) to transfect host cells, especially mammalian cells, preferably CHO cells; culturing under suitable culture conditions for the host cells;

d) Obtaining the above-mentioned monoclonal antibody through separation and purification steps.

The antibody of the present invention can reduce the human antimouse antibody (human antimouse antibody, HAMA) reaction caused by repeated injection of the mouse McAb into the human body, and avoid systemic allergic toxic reaction. The affinity of the obtained antibody was analyzed by ELISA analysis method. The EC50 value of the humanized antibody of the present invention is about half of that of the chimeric antibody WBP247.cAb1, and the affinity is about one time higher than that of the chimeric antibody.

Preferably, the amino acid sequence of the heavy chain variable region of the humanized antibody WBP247.hAb12 of the present invention is 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, The EC50 of the antibody prepared by expression was 0.002614 ug/ml, which was about double 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.

In addition, the humanized antibodies WBP247.hAb4 and WBP247.hAb6 were stably screened. After testing, the EC50 of the two antibodies prepared by expression were 0.002864 ug/ml and 0.003022 ug/ml respectively. This antibody can be specific to solid tumors such as liver cancer combined.

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

The antibody of the present invention can be used in the preparation of medicaments for diagnosing and treating CD147-positive diseases, as well as its biotechnology products, detection reagents, imaging, inspection and diagnosis, and the like.

The present invention also provides the application of the above-mentioned antibody in the preparation of antibody-coupled drugs. The drug is a maytansine derivative DM1, a tubulin polymerase inhibitor MMAE or a radionuclide iodine.

Description of drawings

Fig. 1 SDS-PAGE analysis of humanized antibody clones 1 to 4; where: the left image is the non-reducing electrophoresis sample of the purified protein, and the right image is the reducing electrophoresis sample of the purified protein. From left to right: M is protein marker; Lane1: clone 1; Lane2: clone 2; Lane3: clone 3; Lane4: clone 3 (prepared in different batches); Lane5: WBP247.hAb4; Lane6: WBP247.hAb4 ( prepared in different batches);

Fig. 2 SPR dissociation spectrum to measure the affinity of humanized antibody, in which the abscissa represents the response time of the sample, and the ordinate represents the signal value after binding. Each line in the figure is the detection curve of different concentration dilutions of the same sample, indicating that the There is a specific change in signal as the dose changes;

Figure 3 SDS-PAGE non-reducing electrophoresis analysis of humanized antibody clones 5-12; Lane1: clone 5

Lane2: WBP247.hAb6 non-reduced; Lane3: Clone 7 non-reduced; Lane4: Clone 8 non-reduced; Lane5: Clone 9 non-reduced; Lane6: Clone 10 non-reduced; Lane7: Clone 11 non-reduced ; Lane8: clone 12 non-reduced;

Figure 4 SDS-PAGE reduction electrophoresis analysis of humanized antibody clones 5-12; Lane1: clone 5 reduced; Lane2: WBP247.hAb6 reduced; Lane3: clone 7 reduced; Lane4: clone 8 reduced; Lane5: clone 9 reduced; Lane6: clone 10 reduced ; Lane7: clone 11 reduced; Lane8: clone 12 reduced;

Figure 5 SDS-PAGE reduction and non-reduction electrophoresis analysis of humanized antibody clones 13-16; Lane1: clone 13 reduced; Lane2: clone 14 reduced; Lane3: clone 15 reduced; Lane4: clone 16 reduced; Lane5: clone 13 non-reduced ; Lane6: clone 14 non-reduced; Lane7: clone 15 non-reduced; Lane8: clone 16 non-reduced;

The three strains of antibodies screened in Figure 6 are combined with the antigen-binding gradient dilution curve;

Figure 7 Immunohistochemical screening of the full-length humanized antibody; from left to right shown in the accompanying drawings are WBP247. hAb5 (liver cancer), WBP247. hAb12 (32270) liver cancer and WBP247. The brown area indicates the positive area of antibody staining;

Figure 8 The plasmid map of the expression vector pWX2.1-LC-B-247B4;

Fig. 9 plasmid map of expression vector pWX2.1-LC-B-247B12;

Fig. 10 plasmid map of expression vector pWX1.1-HC-Z-247B4;

Fig. 11 plasmid map of expression vector pWX1.1-HC-Z-247B6;

Figure 12 Immunohistochemical staining of CD147 humanized antibody on different tissues; the brown (dark) area in the figure is the positive area of antibody staining, and the white Bar is 100 μm;

Figure 13 In vitro killing experiment of humanized antibody against tumor cells.

Detailed ways

1. Terminology Explanation:

Immunoglobulin: A class of structurally related glycoproteins consisting 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), through which all four chains pass Disulfide bonds link each other. Each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically consists of one domain, CL. VH and VL can be further subdivided into hypervariable regions (or hypervariable regions of hypervariability in sequence and/or in the form of structurally defined loops), also known as complementarity determining regions (CDRs), interspersed with less conserved regions , called the framework region (FR). Each VH and VL typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, 1987 ). Typically, the numbering of amino acid residues in this segment can be numbered by Kabat's rules. Sequences of Proteins of Immunological Interest, 5thEd, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD. (1991) (this paper uses this numbering system description method for the heavy chain variable domain or the light chain variable domain).

Humanized antibody: as used herein, refers to an antibody derived from a non-human antibody, typically a murine, that has been engineered so that it retains or substantially retains the antigen-binding properties of the parent antibody, However, its immunogenicity in humans is reduced. Since the antibodies of the invention are defined by their structural and functional characteristics, "humanized antibody" is used interchangeably with "antibody".

Complementarity Determining Region (CDR): Refers to the characteristic sequence of an antibody containing multiple amino acids, which jointly define the binding affinity and specificity of the variable fragment (Fv) region of the immunoglobulin binding site to the target antigen CD147.

Framework Region (FR): Refers to the amino acid sequence inserted between the CDRs. These parts of the antibody serve to hold the CDRs in place (allowing the CDRs to bind the antigen). Both light and heavy chain variable regions comprise framework regions (FRs) and typically three CDRs.

Constant Region (CR): Refers to the portion of an antibody molecule that confers effector function. The constant regions of the humanized antibodies in the present invention are all derived from human immunoglobulins. The heavy chain constant region can be selected from five isotypes: alpha, delta, epsilon, gamma or mu. Further, various subclasses of heavy chains (eg, IgG subclasses of heavy chains) can elicit different effector functions, and thus, by selecting desired heavy chain constant regions, antibodies with desired effector functions can be produced. Preferred heavy chain constant regions are γ1 (IgG1), γ2 (IgG2), γ3 (IgG3) and γ4 (IgG4), more preferably γ2 (IgG2). The light chain constant region can be of the kappa or lambda type, preferably the kappa type.

Chimeric Fab is to recombine functional antibody light and heavy chain variable region genes with human antibody κ chain and heavy chain CH1 constant region genes respectively, and clone them into an expression vector to construct a mouse-human chimeric Fab gene expression vector , and then transferred into host cells for expression. The US FDA approved the first human-mouse chimeric Fab antibody ReoPro (anti-platelet receptor gIIb/IIIa) in 1994 for antithrombotic therapy.

The CDR-grafted antibody is to clone all six CDRs of the mouse monoclonal antibody to the corresponding framework region (FR) of the human antibody by PCR or other methods to construct a new antibody. Compared with chimeric antibodies, CDR grafting further reduces the content of heterologous sequences in antibodies and reduces antibody heterogeneity.

Antibody affinity (avidity): refers to the total strength of the interaction between two molecules, such as between an antibody and an antigen. Avidity characterizes the strength of the binding between a molecular pair (eg, antibody-antigen). The affinity of a molecule X for a ligand Y can be expressed by the dissociation constant (KD), which is the concentration of Y required to occupy the binding sites of half of the X molecules present in solution. A smaller Kd indicates a stronger or higher affinity interaction, and a lower concentration of ligand is required to occupy the site.

The variable or constant regions of an immunoglobulin heavy or light chain can be joined as described by using standard recombinant DNA techniques to create an immunoglobulin chain that can be expressed in a suitable host (thus producing the immunoglobulin chain (a or more)), or variable and constant regions linked by synthesis using peptide chemistry.

The humanized antibody of the present invention preserves an important part of the binding properties of the parental murine antibody, which is the murine antibody HAb18 called anti-human CD147 molecule. See "Monoclonal Antibody Letters" for the establishment of this cell line ,1989;2:33-36, Chen Zhinan, Liu Yanfang, Yang Jizhen, etc. (existing patents: 1. Anti-human liver cancer monoclonal antibody HAb18 light and heavy chain variable region genes and their application, patent number: ZL02114471.0, published ( Announcement) No.: CN1381461A; and 2 VARIABLEREGION GENE OF HEAVY/LIGHT CHAIN OF ANTI-HUMAN HEPATOMA MONOCLONAL ANTIBODYHAb18 AND USE THEREOF, (US) Patent No.: US 7 638 619). In particular, the humanized transformation of the anti-human CD147 murine parent antibody of the present invention retains the ability of specifically binding to the parent antibody to recognize the antigen. Through optimization and screening, the obtained humanized antibody exhibits the same or substantially the same antibody binding affinity (affinity) or avidity (avidity) as the parental antibody.

"Humanized antibody" or "antibody", as used in the present invention, includes intact molecules as well as fragments thereof which are capable of binding epitopic determinants, such as Fab, F(ab')2 and Fv. These antibody fragments retain the ability to selectively bind to human CD147. Examples of these fragments include, but are not limited to the following:

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

(2) Fab': defined as a fragment containing a monovalent antigen-binding fragment of an antibody molecule, the whole antibody is treated with pepsin, followed by reduction, thereby generating a complete light chain and a part of the heavy chain; two Fabs can be obtained per antibody molecule 'fragment;

(3) (Fab') ₂ : defined as an antibody fragment obtained by treatment with the enzyme pepsin without subsequent reduction; (Fab) ₂ is a dimer of two Fab' fragments held together by two disulfide bonds ;

(4) Fv: defined as a genetically engineered fragment containing a light chain variable region and a heavy chain variable region expressed as two chains;

(4) Fv: Homology is a central concept in comparative biology. In the study of molecular evolution, homology generally refers to the relationship between the nucleotide sequences of two nucleic acid molecules or between two protein molecules. The degree of similarity between amino acid sequences. Usually, homology must be checked by sequence determination, but DNA-DNA or DNA-RNA hybridization can provide valuable judgments (5) Coupling (Coupling), also known as coupling reaction, coupling reaction, oxidation coupling, is The process of obtaining an organic molecule by a certain chemical reaction of two organic chemical units (moiety). According to different types, it can be divided into cross-coupling and self-coupling reactions. Antibody coupling here refers to the cross-linking of specific medicinal chemical reaction groups or small molecule drugs to antibody molecules through specific reagent reactions, thereby improving the killing and therapeutic effects of antibodies or small molecule drugs.

The present invention clarifies the CDR and FR region genes of the light and heavy chain variable regions of the mouse-derived antibody HAb18 (ZL02114471.0) against human CD147 molecules through bioinformatics analysis, and uses computer-aided antibody structure for humanization design. Phage display antibody library technology and other molecular biology methods were used to humanize the framework region of the variable region, and the humanized antibody variable region gene sequence of the anti-human CD147 molecule was obtained, and the full-length antibody molecule was constructed. The eukaryotic expression system of the gene was further expressed in CHO host cells to prepare the anti-human CD147 humanized CDR-grafted antibody. The results of immunohistochemistry and ELISA showed that the screened humanized antibody against human CD147 molecule maintained an affinity comparable to that of the murine parental antibody, and maintained the specificity of the parental antibody's recognition of the antigen.

The present invention will be described in further detail below in conjunction with examples. It should be understood that these examples are for illustrative purposes only, and are not intended to limit the present invention. The experimental methods used in the following examples are conventional molecular biology methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial reagent companies unless otherwise specified.

2. Construction of phage display vectors for the light and heavy chain variable regions of the murine antibody HAb18 containing anti-human CD147 molecules

1. Materials

Hybridoma cell line HAb18, the establishment of this cell line is described in "Monoclonal Antibody Communication", 1989; 2:33-36, Chen Zhinan, Liu Yanfang, Yang Jizhen, etc. (patent: CN1381461A).

PCR amplification primers of murine VH and VL of HAb18G: wherein, VH gene amplification primers:

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

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

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

Wherein, VL gene amplification primers:

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

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

2. Methods and Results

2.1 Total RNA extraction: The total RNA in the hybridoma cells HAb18GC2 was extracted according to the total RNA extraction kit (OMEGA Total RNA R6834), and the integrity of the total RNA was detected by agarose gel electrophoresis.

2.2 Reverse transcription of cDNA: Take 1 μg of the total RNA obtained in 2.1, synthesize the first strand of cDNA according to the instructions of the TaKaRa PrimeScript® RTreagent Kit DRR037A reverse transcription kit, and store it at -20°C for future use.

2.3 PCR amplification of VH gene and VL gene:

Using the cDNA prepared in 2.2 as a template, use VH gene amplification primers and VL gene amplification primers to amplify the mouse-derived VH gene fragment and VL gene fragment respectively. The amplification system follows the instructions of Phusion® High-Fidelity DNA Polymerase (NEB) configuration;

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

Among them, the VH gene was first amplified with the upstream primer HAb18-F and the downstream primer 1 Linker-HAb18-R1. After the product was purified, the VH gene was amplified again with HAb18-F and the downstream primer 2 HAb18-linker-R2. VL gene was directly amplified by upstream primer HAb18-Linker-F1 and downstream primer HAb18-R.

2.4 Amplification of ScFv gene:

Mix the VH gene fragment and VL gene fragment obtained in 2.3 with the same molar number, and use Overlap-PCR to amplify the ScFv gene. The amplification system is as follows:

PCR reaction conditions: 95°C, 5min; 95°C, 15s, 56°C, 30s, 72°C, 1min, 35 cycles; 72°C, 10min. 1% agarose gel electrophoresis to observe and recover the target band.

2.5 Digestion of ScFv gene, ligation with vector and transformation: Nco I (NEB:C^CATGG) and Not I (NEB:GC ^GGCCGC) for enzyme digestion. Take 600ng and 3μg of the recovered fragment product of 2.4 and pGEM-T vector respectively, add 1μL of restriction endonuclease (NEB), 5μL of 10×CutSmart buffer, add water to 50μL, and digest at 37°C for 1.5h.

The enzyme digestion system is as follows:

After digestion in a water bath at 37°C, 5ul 10× loading buffer was added to terminate the reaction. 1% agarose gel electrophoresis to recover enzyme-cut bands, UV quantification.

Use T4 bacteriophage DNA ligase for ligation reaction, the composition of the reaction system is as follows:

React overnight at 16°C. The ligated product was transformed into TG1 competent cells. Spread on LB agar plates and incubate overnight at 37°C. On the second day, 10 single clones were randomly selected, identified as positive colonies with vector universal primers, recorded as pGEM-ScFv, and sequenced for verification. The clones with correct sequencing were stored at -40°C for future use.

3. CDR and FR sequence calibration of anti-CD147 parental monoclonal antibody HAb18

Use www.expasy.org online software to translate the nucleotide sequence of the light and heavy chain variable regions of the anti-CD147 murine antibody HAb18, also known as HAb18G monoclonal antibody (CN021144710), into its encoded amino acid sequence, according to Kabat The database is calibrated in principle, and these iconic sequences will be retained in further humanized transformation.

The amino acid sequences of CDR1, CDR2 and CDR3 of the CDR1, CDR2 and CDR3 marked in the variable region sequence of the light chain of the monoclonal antibody HAb18 are respectively shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing. The amino acid sequences of CDR1, CDR2 and CDR3 identified in the heavy chain variable region sequence of the monoclonal antibody HAb18 are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 in the sequence listing, respectively.

4. Selection of framework sequence FR for antibody humanization transformation

In order to select a suitable human antibody framework sequence on which mouse CDRs are grafted, the inventors used the human antibody sequence information database constructed from the antibody sequences provided by the Kabat protein database; using DiscoveryStudio (VIOVIA, version 3.5), through the same Source modeling and mechanical optimization technology analyze and replace the antibody molecular structure before and after humanization to ensure that the replaced amino acids will not change the overall skeleton structure of VH and VL, especially will not destroy the β-strand secondary structure, thereby Maintain the original affinity of the antibody. The FR shuffling method was first proposed by Dall'Acqua. The FR region of the human antibody sequence with high homology to the FR region of the mouse antibody sequence is selected to randomly replace the mouse antibody to realize humanization. This method reduces the theoretical library capacity and irrelevant FR recombination.

The FR regions and CDR regions of antibody variable regions were determined according to the Kabat database. In the antibody database, the sequence of the variable region of the murine antibody HAb18 was used for homology comparison. For the modification of FR1, FR2, and FR3 of VH, the present invention refers to 12 germlines of FR1, 4 germlines of FR2 and 10 germlines of FR3. For the transformation of FR1, FR2, and FR3 of VL, 6 germlines, 9 germlines, and 7 germlines were referred to; JH and JK were used to confirm the FR4 regions of VH and VL, respectively. Through homology modeling, the molecular structure of the antibody before and after humanization is analyzed to ensure that the replaced amino acids cannot change the overall skeleton structure of VH and VL, especially the secondary structure of β-strand, so as to maintain the original affinity of the antibody . The design scheme is shown in Table 1 and Table 2. The theoretical library capacity of the final designed humanized antibody library is 1.8×10^5.

Table 1 Amino acids selected for replacement in V _H framework

Table 2 Amino acids selected for replacement in the VL framework

5. Construction and screening of phage display CD147 humanized antibody library

According to the human antibody FR sequence variable region sequence and its replaceable amino acid sequence obtained according to the above (3), the combination of these sequences retains the CDR sequence of the parent monoclonal antibody HAb18 (2), that is, the light chain variable region sequence contains SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 characteristic sequences; the heavy chain variable region sequence contains SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 characteristic sequences; in the FR framework region, according to For the candidate sites in Table 1 and 2, design corresponding PCR primers and overlap PCR to obtain the designed full-length gene containing the mutation site; at the same time, select codons commonly used in mammalian cells for partial replacement, and obtain the full-length All the genes were cloned into the phage vector pComb3Xss to construct and screen the humanized antibody library. Finally, 16 combined clones of effective humanized sequence paired anti-CD147 light and heavy chains were obtained.

1. Primer design for CD147 humanized antibody library

According to the selection and analysis results of the framework sequence FR for antibody humanization transformation, a total of 70 primers for the antibody library were designed, all sequences beginning with 247, such as 247-A1, 247-A2, etc., in order of SEQ ID NO in the sequence list :12 to SEQID NO:81.

2. Gene Amplification Protocol

According to the steps of Overlapping-PCR, the specific humanized antibody library amplification scheme is as follows:

Primer Selection Template Length

first step

247-A1,A2,A3,A4VH-FR1-A122bp

247-B1,B2,B3,A4VH-FR1-B122bp

247-B1,B2,C3,A4VH-FR1-C122bp

247-B1,B2,D3,A4VH-FR1-D122bp

247-B1,B2,E3,E4VH-FR1-E122bp

247-F1,B2,F3,A4VH-FR1-F122bp

247-G1,G2,A3,A4VH-FR1-G122bp

247-H1,H2,H3,A4VH-FR1-H122bp

247-A5,A6VH-FR2-A87bp

247-B5,B6VH-FR2-B87bp

247-C5,C6VH-FR2-C87bp

247-A7,A8,A9,A10VH-FR3-A150bp

247-A7,B8,B9,B10VH-FR3-B150bp

247-A7,C8,B9,C10VH-FR3-C150bp

247-A7,C8,D9,B10VH-FR3-D150bp

247-A7,C8,E9,E10VH-FR3-E150bp

247-A7,F8,B9,B10VH-FR3-F150bp

247-A7,G8,B9,B10VH-FR3-G150bp

247-A11, A12VH-FR484bp

247-A13,A14,A15,A16VL-FR1-A139bp

247-A13,B14,A15,A16VL-FR1-B139bp

247-A13,C14,A15,A16VL-FR1-C139bp

247-A13,D14,A15,A16VL-FR1-D139bp

247-A17, A18VL-FR2-A80bp

247-A17,B18VL-FR2-B80bp

247-A17,C18VL-FR2-C80bp

247-A17,D18VL-FR2-D80bp

247-A17,E18VL-FR2-E80bp

247-F17,F18VL-FR2-F80bp

247-A19,A20,A21,A22VL-FR3-A140bp

247-B19,B20,B21,B22VL-FR3-B140bp

247-B19,B20,C21,B22VL-FR3-C140bp

247-B19,D20,D21,B22VL-FR3-D140bp

247-B19,E20,E21,B22VL-FR3-E140bp

247-A23, A24VL-FR478bp

second step

247-A1,B1,F1,G1,A12VH404bp

247-A13, A24VL394bp

third step

247-A1,B1,F1,G1,A24VH+VL774bp

Amplify the target fragment; the reaction conditions are 95°C, 3min; 95°C, 30sec; 55°C, 30sec; 72°C, 40sec; 30 cycles, and finally 72°C extension, 10min. After the PCR reaction, the PCR product was purified and recovered by 1% agarose gel electrophoresis.

3 Phage display library construction

3.1 Cloning of phage vector

Use Nco I (NEB:C^CATGG) and Not I (NEB:GC^GGCCGC) for double enzyme digestion to recover the PCR-amplified humanized antibody library gene fragments, the specific conditions are the same as condition (1) 2.5, after 1 After separation by % agarose electrophoresis, the digested fragments were purified with GelExtraction Kit (Omega bio-tek). Then, the purified enzyme-digested fragment was ligated with the Nco I/Not I double-digested phage vector pComb3Xss with T4 DNA ligase (TaKaRa Company), and after deionization, electrotransformed TG1 competent cells and inoculated them on LB culture plates. Clone screening. Randomly pick 48 single clones, and use M13-48 and M13-47 primers to identify positive clone colonies; count the storage capacity, and store the library at -80°C for future use.

3.2 Preparation and titration of helper phage

Pick a single colony of XL-1 Blue to inoculate 5m1 SB-T ⁺ (20ug/ml) solution, and cultivate overnight at 37°C with shaking. Dilute 1:500 and inoculate into 10ml SB-T ⁺ (20ug/ml) solution, shake at 37°C for one hour. Pick a single phage M13K07 virus plaque and insert it into the above 10ml bacterial solution, culture with shaking at 37°C for 2 hours, then add 200ml of SB-T ⁺ (20ug/ml) K ⁺ (70ug/ml) solution, and culture with shaking at 37°C overnight. Centrifuge at 4000 rpm at 4°C for 15 minutes, take the supernatant to measure the titer of phage, aliquot it aseptically and store it at 4°C.

3.3 Phage rescue experiment

Scrape 6 mL of electroporated bacteria from 3.1 and dilute to 400 mL SOB-GAT solution, culture at 30°C until A600=0.5, add M13K07 and let stand at 37°C for 1 hour for superinfection (if the multi-infection value reaches 5:1, add M13K07 The number of pfu: 5×10 ⁸ bacteria/A600 unit)×multiple infection value (5)×A600 value (0.5)×final volume of bacteria (mL), centrifuge at 3500RPM at 4°C for 10min, and resuspend the pellet with the original volume of 2YT-AKT , 30°C medium-speed overnight shaking culture. Rescue the culture solution and centrifuge at 4000rpm for 20min at 4°C, take the supernatant and add 4% PEG8000 and 3%NaC1 to precipitate the phage in an ice bath for 1h, centrifuge at 15000g at 4°C for 20min, add 1-2mL sterilized PBS (containing 1%BSA, 0.02mol/L , pH 7.4) to resuspend the pellet, briefly centrifuge at low speed, and the supernatant is the rescued original phage antibody library (aliquoted and stored at 4°C for later use).

3.4 Panning of phage antibody library

Perform antigen-specific panning on the antibody phage library obtained in 3.1 by solid-phase panning. The antigen-coating concentration of each round of panning was decreased successively, and a positive/negative control was set. The specific operation is as follows: Recovery 3.1 The saved antibody phage library is cultured in 60ml of 2YT medium in a shaker at 37°C until OD600=0.3-0.4. Add M13KO7 helper phage (Invitrogen); incubate at 37°C for 30 minutes, and shake for 60 minutes. Centrifuge at 1500 rpm/10 minutes, discard the supernatant, resuspend the cells with 60ml of 50ug/ml kanamycin (glucose-free) medium, and culture overnight in a shaker at 30°C; centrifuge at 12,000 rpm/10 minutes to precipitate phage Library, transfer the supernatant to a centrifuge tube, 30ml/tube. Add 7.5ml PEG/NaCl to each centrifuge tube, mix well, and place on ice for 1h. Centrifuge at 12000 rpm for 5 minutes, discard the supernatant; resuspend the phage with 2.2ml of a solution containing PBS-5%BSA, centrifuge at 12000 rpm for 5 minutes to remove cell debris. Afterwards, the expressed CD147 molecule was used to coat the plate for affinity screening, and after five rounds of panning (adsorption-elution-amplification), the antigen coating concentration of each round of panning decreased successively (1ug/ml, 0.1ug /ml, 0.01ug/ml, 0.001ug/ml, 0.0001ug/ml). Calculate the phage input/output ratio (recovery rate) for each round of screening as an index for the enrichment of specific phage antibodies. The calculation formula is: recovery rate (%yield) = (volume of eluate x eluate titer x 100) /(input antibody library volume×antibody library titer), panning was carried out until the recovery rate was less than 10, the panning experiment was terminated, 768 clones obtained were picked and induced to express, and the heavy chain VH of the antibody containing the humanized gene was obtained And the gene product of light chain VL, that is, ScFv antibody, for further Elisa detection.

6. ELISA analysis and sequencing analysis

Dilute CD147 to 1 μg/ml with coating buffer (200mM Na ₂ CO ₃ /NaHCO ₃ , pH 9.2), add 50 μl to each reaction well, and coat at 4°C overnight; discard the solution in the reaction well and wash with 1 X Wash 3 times with PBS buffer, add 200 μl blocking buffer (2% BSA/1XPBS buffer) to block for 1 hour at room temperature; wash 3 times with 200 μl 1XPBS buffer; add the cell culture supernatant containing ScFv antibody induced by (4) 3.4 768 samples (8 96-well microplates) and negative control (50 μl/well), incubated at room temperature for 2 hours; washed 3 times with 200 μl 1X PBS buffer; added Anti-c diluted with blocking buffer (1:2500) -Myc Ab(HRP) (Abcam Cat# ab19312, 50μl/well), incubate at room temperature for 1h; wash 6 times with 200μl of 1XPBS buffer; add TMB substrate solution (50μl/well) to each reaction well for 10min; Add a stop solution (2M HCl, 50 μl/well) to stop the reaction; read the absorbance at A450nm with an ELISA plate reader;

According to the results of ELISA, 50 clone samples with A450>3.0 were selected for sequencing (the sequencing work was completed by Shanghai Boshang Biotechnology Co., Ltd.). DNA sequence analysis: Refer to the germline database of human antibodies and http://www.bioinf.org.uk/abs/shab/ to evaluate the degree of humanization of the sequence, and at the same time perform affinity ranking for molecules with a better degree of humanization.

7. Determination of the affinity of ScFv antibody by SPR

7.1 SPR binding assay method

Use the ProteOn XPR36 (Bio-Rad, XPR36) instrument to perform the affinity determination of the strong positive antibody of the ELISA antibody in step (5). The GLC chip (Bio-Rad, 1765011) was activated with 0.04 M EDC+0.01 M sulfo-NHS (Bio-Rad). Dilute CD147 to 10 mM with 10 mM NaAc (pH 4.5), and inject it onto the chip at a speed of 30 ul/min to couple the antigen to the activated chip through amino groups. Finally, the chip was inactivated with 1 M ethanolamine-HCl (Bio-Rad); the chip was rotated 90 degrees and washed with buffer solution (PBS/ 0.005% Tween 20) until the baseline stabilized. The cell culture supernatant containing the ScFv-containing antibody induced by (4) 3.4 was injected on 6 horizontal channels respectively. The injection speed was 30 μl/min. The sample binding time was 60s, and the dissociation time was 900s; data analysis was performed using the Kinetic-Langmuir model; clones with high affinity were selected for the construction of complete humanized antibodies.

7.2 SPR determination of affinity ranking of ScFv antibodies

SPR was used to monitor the binding of coated CD147 to the cell culture supernatant containing ScFv antibody induced by (4) 3.4 in real time, and the affinity between CD147 and humanized ScFv antibody was reflected by measuring the dissociation rate constant (K _off ). The results are shown in Table 3. According to the change of affinity, multiple molecules with better affinity were selected to construct a complete humanized antibody.

Table 3 HAb18 humanization degree sequence analysis and _Koff sorting

8. Construction and detection of full-length humanized antibody

8.1 Materials

According to the results of the aforementioned affinity sorting, first pick the top 4 clones (namely, clones 26601, 26602, 27028 and 27044) of (6) 2, namely the SPR assay fragment antibody ScFv antibody, for the first round of full-length antibody construction and expression.

That is, the four clones selected first were inoculated and cultured overnight, the plasmids were extracted respectively, and the template sequence was confirmed by sequencing.

PCR primer: humanized antibody VH gene amplification primer: upstream primer: 247-VH_F1, such as SEQ ID NO: 84 in the sequence listing; downstream primer: 247-VH_R1, such as 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, which are respectively SEQ ID NO: 82 and SEQ ID NO: 83 in the sequence listing; according to the sequencing results , the second set of upstream primers: 247-VL-2_F2; downstream primers: 247-vl_R1, respectively corresponding to SEQ ID NO: 87 and SEQ ID NO: 86 in the sequence listing.

8.2 PCR amplification process

Plasmids were extracted separately, and after the sequence of the template was confirmed to be correct, the target fragments were amplified by PCR using the above primers and templates respectively. The amplification schemes were as follows:

Primer template target fragment length

247-VH_F1&247-VH_R125280-VHPw247-VH392bp

247-33_F1&247-33_R1WBP247-hIGKV1-33Pw247-hIGKV1-33-VL361bp

247-33_F1&247-33_R1WBP247-hIGKV1-39Pw247-hIGKV1-39-VL 361bp

247-VL-2_F2&247-vl_R126364-VLPw247-mut16-VL361bp

247-VL-2_F2&247-vl_R126372-VLPw247-mut18-VL361bp

8.3 Preparation of transient expression vector

Add the light chain variable region gene or heavy chain variable region gene amplified in step 7.2 to type II restriction endonucleases NgoMIV and SnaBI for double digestion, and then purify it with a DNA purification kit, and use the same Mammalian cell expression vector pCI-vector containing hIgG1/k digested with restriction endonuclease NgoMIV/SnaBI was ligated. The ligation product was transformed into TOP10 Escherichia coli, spread on LB agar medium containing 100 μg/ml ampicillin, and the positive clones obtained were cultured in LB liquid medium containing 100 μg/ml ampicillin, and plasmid extraction and sequencing were performed to obtain The full-length eukaryotic expression vector clone 1-VH1-4 containing the humanized antibody VH gene and the full-length eukaryotic expression vector clone 1-VL1-4 containing the humanized antibody VL gene were respectively. After sequence determination, the nucleotide sequence corresponding to the variable region of the heavy chain of clone 4 is shown in SEQ ID NO: 102; the nucleotide sequence corresponding to the variable region of the light chain is shown in SEQ ID NO: 104.

9. Transient cell transfection and antibody expression and purification

9.1 Cell Transient Transfection of Antibody

The full-length eukaryotic expression vector clone 1-VH1-4 of the VH gene obtained in (7) 7.3 and the full-length eukaryotic expression vector clone 1- VL1-4 were paired and co-transfected into HEK293 cells (1.0× ¹⁰⁶ /ml), and the transfected cells were placed on a shaker at 37°C and placed in an incubator with a CO ₂ concentration of 5% for shaking culture , the shaking table rotates at 120 rpm. After 7 days of transfection, the transfected cell supernatant was collected by centrifugation, and the protein A affinity chromatography column was used to separate and purify the target full-length humanized antibody from the cell culture supernatant, and the protein concentration of the expressed and purified antibody was determined and used for The further purified antibodies (the antibodies are respectively marked as clones 1-4) were tested for affinity SPR.

9.2 SDS-PAGE analysis of antibodies

Use the routine SDS-PAGE analysis method in the laboratory, that is, mix NuPAGE LDS samplebuffer, NuPAGE Reducing Agent and each sample separately, and centrifuge in a 75°C water bath for 10 minutes. The sample load is 2 μg/well, and the voltage of 200V runs for 35 minutes. After the electrophoresis is completed, take the gel and rinse it three times, 5 minutes each time. Add the staining solution SimplyBlueSafestain for one hour, discard the staining solution and add deionized water to decolorize until the gel background is completely decolorized; the observation results are shown in Figure 1.

Among them, the left picture is the non-reducing electrophoresis sample of the purified protein, and the right picture is the reducing electrophoresis sample of the purified protein. The six lanes in the left and right figures are from left to right: Lane1: Clone 1; Lane2: Clone 2; Lane3: Clone 3; Lane4: Clone 3 (prepared in different batches); Lane5: Clone 4; Lane6: Clone 4 (different Batch preparation); M is protein marker.

10. Determination of humanized antibody affinity

SPR determination of humanized antibody affinity: use SPR to measure the affinity of the first 4 purified humanized antibodies, the method is the same as (6) 6.1, the results show the affinity of purified antibody clone 1, clone 2, clone 3 and clone 4 (1.9E-9, 2.63E-9, 1.61E-9 and 1.83E-9) slightly lower than the parental chimeric antibody WBP247.cAb1 (3.94E-10), the results are shown in the figure, where the binding solution of the antibody See attached figure 2 for the atlas.

Table 4 SPR kinetic data of humanized antibody

11. Specific screening and identification of humanized antibodies by immunohistochemical staining

Although the affinity of the humanized antibody was determined by the SPR method, since the antigen used was a purified antigen expressed in vitro, in order to further observe the binding specificity of the obtained humanized antibody, tumors maintained in the laboratory Tissue samples were further immunohistochemically stained for the above antibodies.

The specific binding ability of the expressed series of humanized antibody clones 1-4 to tumor tissues was detected, and the immune tissue cross-reaction of the antibodies was investigated.

The specific operations are as follows: conventional xylene dewaxing, gradient alcohol dehydration, and hydration of tissue chips; 3% H ₂ O ₂ blocking and inactivation of endogenous peroxidase; normal sheep serum working solution blocking; antibody clones 1-4 as the primary antibody, biotin-labeled rabbit anti-human Fc antibody as the secondary antibody, horseradish peroxidase-labeled streptavidin working solution as the third antibody, developed with DAB, counterstained with hematoxylin, sealed after dehydration and transparency film, microscopic examination. The results of histochemical screening showed that only antibody clone 4, namely WBP247.hAb4, was specifically stained in malignant tumor tissues such as lung cancer and liver cancer, and the staining degrees were all "++" or "+++"; Combine less. The other three antibodies were negative. The above results show that only one available candidate humanized antibody WBP247.hAb4 was screened through this round of expression. Very few, failed to meet the quantitative requirements, so multiple rounds of expression and screening are required.

11. Multiple rounds of full-length humanized antibody construction and immunohistochemical screening

11.1 Repeated rounds of full-length humanized antibody construction and detection

According to the results provided by immunohistochemistry, due to possible changes in the specificity of the antibody after humanization, the previous strategy was adjusted, and the first 16 of the above (6) 2, namely, the affinity of the fragment antibody ScFv antibody was determined by SPR All clones were constructed and expressed with full-length antibodies; the method was the same as in 7.1 above, and vectors with full-length antibodies were constructed and expressed for the selected clones. Wherein, the nucleotide sequence corresponding to clone 6, namely WBP247.hAb6, is shown in SEQ ID NO:106; the corresponding nucleotide sequence of the light chain variable region is shown in SEQ ID NO:108. The nucleotide sequence of clone 12, WBP247.hAb12, corresponds to the variable region of the heavy chain, such as SEQ ID NO: 110; the nucleotide sequence corresponding to the variable region of the light chain, such as SEQ ID NO: 112. Afterwards, purify the obtained antibodies according to the above-mentioned (8) 8.1 and transient transfection of cells and antibody expression and purification (antibodies are respectively marked as clones 5-16), and perform SDS-PAGE analysis according to the method in the above-mentioned 8.2, and the results are shown in Figure 3 , Accompanying drawing 4 and accompanying drawing 5.

12. ELISA analysis of humanized antibody affinity

Coat 200ng of recombinant CD147 protein onto a microtiter plate and let stand overnight at 4°C; block with 1XPBS/2%BSA for 1 hour at room temperature. The purified antibody product clones 1-6 prepared in step (8) and subsequent preparations were serially diluted with blocking solution 1:3.16 starting from 1 ug/ml, and 100 μl was added to each well, and allowed to stand at room temperature for 1 hour. Add 100μl 1:4000 dilution of horseradish peroxidase-labeled goat anti-human secondary antibody Goat anti-human IgG Fc HRP (Bethyl Cat#A80-304P), and let stand at room temperature for 1 hour. TMB was added for color development, and the reaction was terminated with 2M H ₂ SO ₄ . Read at 450nm with a microplate reader. From the ELISA results, it can be seen that the EC50 value of the 16 humanized antibodies screened from clone 1 to clone 16 is about half that of the chimeric antibody WBP247.cAb1, and the affinity is about twice that of the chimeric antibody. (See Table 5, Figure 6).

Table 5 Determination of EC ₅₀ of antibody binding to antigen CD147

13. Immunohistochemical screening of full-length humanized antibody

In order to further detect the specific binding ability of the expressed series of humanized antibody clones 5-16 to tumor tissue, investigate the immune tissue cross-reaction of the antibody, and further screen the specificity of the humanized antibody by immunohistochemical staining . The specific operation is as follows: Step 10: conventional xylene dewaxing, gradient alcohol dehydration, and hydration of the tissue chip; 3% H ₂ O ₂ blocking and inactivation of endogenous peroxidase; normal sheep serum working solution blocking; antibody clone 5 ～16 is the primary antibody, biotin-labeled rabbit anti-human Fc antibody is the secondary antibody, horseradish peroxidase-labeled streptavidin working solution is the third antibody, DAB color development, hematoxylin counterstaining, dehydrated and transparent After sealing, microscopic examination. Positive judgment: the cell membrane is positive when it is stained brown. Through this round of expression screening, two usable candidate antibody WBP247.hAb6 and clone 12 were obtained again, and clone 12 could not only bind to liver cancer specimens, but also bind to lung cancer specimens. The specific information is shown in Table 6 below:

Table 6 Specific screening of humanized antibodies by immunohistochemical staining

Through multiple histochemical staining, the binding specificity of the obtained humanized antibody was preliminarily screened for three humanized monoclonal antibodies, clone 4 (WBP247.hAb4), clone 6 (WBP247.hAb6) and clone 12 ( WBP247.hAb12) for downstream screening of stable cell lines.

14. Construction of high-efficiency expression vectors for humanized antibodies and screening of stable expression cell lines

According to the previous ELISA and immunization results, select the three clones with the best affinity and specificity, that is, clone 4, clone 6 and clone 12, namely "WBP247.hAb4, WBP247.hAb6 and WBP247.hAb 12" gene sequences for high-efficiency expression vectors Construction and screening of stable cell lines.

Table 7 Sequence combination information of WBP247.hAb4, WBP247.hAb6 and WBP247.hAb12

(Wherein, 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 heavy chain variable region The amino acid sequence is the sequence shown in SEQ ID NO: 111, and the corresponding heavy chain variable region (VH) nucleotide sequence is shown in SEQ ID NO: 110).

14.1 Construction of light chain expression vector pWX2.1-LC-B-247B4

Gene DNA (27989-VL) fragment amplification was carried out in vitro, using the DNA as a template, 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) to amplify the variable region fragment WBP247B4-VL, and introduce an EcoRI restriction enzyme site at the 5' end of WBP247B4-VL, and introduce a BsiWI restriction enzyme at the 3' end The cutting site, the fragment and the expression vector pWX2.1 containing the light chain constant region gene were digested with EcoRI and BsiWI, and the 5119bp DNA fragment was recovered after 1% agarose gel electrophoresis. The gel-cut purified DNA fragment was ligated with the fragment from the plasmid pWX2.1, and reacted with 20 μl of T4 ligase system at 16°C for 20 min, and 10 μl of the ligation solution was used to transform Escherichia coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification and sequencing, pick a correct single clone in 200ml LB medium, shake culture overnight at 37°C, 220rpm, extract a large number of plasmids, and finally obtain the plasmid named pWX2.1-LC-B -247B4 (5521bp) (shown in Figure 8).

14.2 Construction of light chain expression vector pWX2.1-LC-B-247B12

Amplify gene DNA (32270-VL) fragments in vitro, use the DNA as a template, and use 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) to amplify the variable region fragment WBP247B12-VL, and introduce an EcoRI restriction enzyme site at the 5' end of WBP247B12-VL, and introduce a BsiWI restriction enzyme at the 3' end The cutting site, the fragment and the expression vector pWX2.1 containing the light chain constant region gene were digested with EcoRI and BsiWI, and the 5119bp DNA fragment was recovered after 1% agarose gel electrophoresis. The DNA fragment purified by cutting gel was ligated with the fragment from plasmid pWX2.1, and transformed into Escherichia coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification and sequencing, pick a correct single clone in 200ml LB medium, shake culture overnight at 37°C, 220rpm, extract a large number of plasmids, and finally obtain the plasmid named pWX2.1-LC-B -247B12 (5521bp) (as shown in Figure 9).

14.3 Construction of heavy chain expression vector pWX1.1-HC-Z-247B4

Gene DNA (27989-VH) fragment amplification was carried out in vitro, using the DNA as a template, 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) to amplify the variable region fragment WBP247B4-VH, and introduce an EcoRI restriction enzyme site at the 5' end of WBP247B4-VH, and introduce NheI restriction enzyme at the 3' end Cutting site, the fragment and the expression vector pWX1.1 containing the heavy chain constant region gene were digested with EcoRI and NheI, and a 5202bp DNA fragment was recovered after 1% agarose gel electrophoresis. The DNA fragment purified by cutting gel was ligated with the fragment from plasmid pWX1.1, and transformed into Escherichia coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification, and sequencing, pick a correct single clone in 200ml LB medium, 37°C, 220rpm overnight shaking culture, a large number of plasmids are extracted, and the final plasmid is named pWX1.1-HC-Z -247B4 (5625bp) (shown in Figure 10).

14.4 Construction of heavy chain expression vector pWX1.1-HC-Z-247B6

Amplify gene DNA (32338-VH) fragments in vitro, use the DNA as a template, and use 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) to amplify the variable region fragment WBP247B6-VH, and introduce an EcoRI restriction enzyme site at the 5' end of WBP247B6-VH, and introduce an NheI restriction enzyme at the 3' end Cutting site, the fragment and the expression vector pWX1.1 containing the heavy chain constant region gene were digested with EcoRI and NheI, and a 5202bp DNA fragment was recovered after 1% agarose gel electrophoresis. The DNA fragment purified by cutting gel was ligated with the fragment from plasmid pWX1.1, and transformed into Escherichia coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification, and sequencing, pick a correct single clone in 200ml LB medium, 37°C, 220rpm overnight shaking culture, a large number of plasmids are extracted, and the final plasmid is named pWX1.1-HC-Z -247B6 (5625bp) (shown in Figure 11).

14.5 Construction and Screening of CHO Stable Expression Cell Lines

After constructing the vector, WuXi AppTec entrusted WuXi AppTec to carry out the above humanized antibody light chain expression vector and humanized antibody heavy chain expression vector respectively 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 Co-transfect into CHO/DHFR cells, add appropriate concentration of medium for screening and passage, and select a single clone to obtain CHO stable expression cell lines.

Afterwards, Minipool batch feeding experiment was carried out to measure the titer. After four rounds of fed-batch optimization, the cells stably expressing WBP247.hAb12 antibody (named: cell line 247C-B4Z2-01-C-005 (CCTCC NO. C2019147) and WBP247.hAb4 were obtained, respectively. Strain 247A-B9Z4-02-C-T9 (CCTCC NO.C2019148) and cell line 247B-B9Z4-01-C-T9 (CCTCC NO. C2019149) expressing WBP247. The culture preservation center, the preservation date is July 16, 2019.

15. Tumor cell killing experiment of humanized anti-CD147 antibody in vitro

Methods: Human lung cancer cells NCI-H520 in the logarithmic growth phase were digested with trypsin to make a single cell suspension, and the concentration was adjusted to 1×10 ⁴ cells/ml. 2000 cells (200 μl cell suspension) were inoculated into a 96-well plate, incubated overnight at 37°C in 5% CO ₂ , waiting for the cells to adhere to the wall and restore the state of logarithmic proliferation.

According to the conventional ADC drug laboratory preparation method, the three humanized antibodies were labeled with DM1 to obtain WBP247.hAb12-DM1, WBP247.hAb4-DM1 and WBP247.hAb6-DM1. Dilute with serum-free RPMI1640 medium to the following gradients: 1000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 μg/mL; add 20 μl of each concentration into corresponding wells, and design 6 replicates for each concentration. hole. At the same time design the negative antibody control (human IgG) and the background (no cells only culture medium). Incubate at 37°C for 48 hours in 5% CO ₂ , and add 10 μL of CCK-8 chromogenic solution to each well. Incubate at 37°C for 2 h, and read the absorbance at 450 nm with a full-wavelength microplate reader. The results are shown in Figure 13. All the screened humanized antibodies had a good killing effect on tumor cells (Figure 13), indicating that all the prepared human antibodies had good tumor specificity and targeting. According to this effect, those skilled in the art can infer that the antibody of the present invention can be used for the preparation of diagnosing and treating CD147 expression-positive diseases (drugs and biotechnology products, detection reagents, imaging and diagnostic reagents).

Nucleotide sequence listing electronic file

<110> Fourth Military Medical University of Chinese People's Liberation Army

<120> Anti-human CD147 monoclonal antibody, expression vector, cell line and its application

<210>1

<211>37

<212>DNA

<213>

<220>HAb18-F

<400>1

CCCAGCCGGCCATGGCCGAAGTGAAGCTTGAGGAGTC

<210>2

<211>50

<212>DNA

<213>

<220>Linker-HAb18-R1

<400>2

TACCACCCACCACCAGAACCGCCACCACCTGAAGAGACAGTGACCAGAGTC

<210>3

<211>45

<212>DNA

<213>

<220>HAb18-linker-R2

<400>3

ACCGCCACTACCACCGCCACCGCTACCACCACCACCAGAACCGCC

<210>4

<211>51

<212>DNA

<213>

<220>HAb18-Linker-F1

<400>4

GGCGGTGGTAGTGGCGGTGGCGGTGCTAGCAGTATTGTGATGACCCAGACT

<210>5

<211>39

<212>DNA

<213>

<220>HAb18-R

<400>5

GTTTTTGTTCTGCGGCCGCTTTGATTTCCAGCTTTGTCC

<210>6

<211>11

<212>PRT

<213>

<220>L-CDR1

<400>6

KASQSVINDVA

<210>7

<211>7

<213>PRT

<213>

<220>L-CDR2

<400>7

YASNRNT

<210>8

<211>9

<214>PRT

<213>

<220>L-CDR3

<400>8

QQDYSPPFT

<210>9

<211>5

<215>PRT

<213>

<220>H-CDR1

<400>9

DAWMD

<210>10

<211>19

<216>PRT

<213>

<220>H-CDR2

<400>10

EIRSKANNHAPYYTESVKG

<210>11

<211>6

<217>PRT

<213>

<220>H-CDR3

<400>11

DSTATH

<210>12

<211>39

<212>DNA

<213>

<220>247-A1

<400>12

CCCAGCCGGCCATGGCCGAAGTGCAGCTTGAGGAGTCTG

<210>13

<211>42

<212>DNA

<213>

<220>247-A2

<400>13

GGATCCTCCAGGTTGCACCAAGCCTCCTCCAGACTCCTCAAAG

<210>14

<211>42

<212>DNA

<213>

<220>247-A3

<400>14

CCTGGAGGATCCCTGAGGCTGTCTTGTGCCGCCTCTGGATTC

<210>15

<211>34

<212>DNA

<213>

<220>247-A4

<400>15

GTCCATCCAGGCGTCACTAAAAGTGAATCCAGAG

<210>16

<211>50

<212>DNA

<213>

<220>247-A5

<400>16

CGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGG

<210>17

<211>50

<212>DNA

<213>

<220>247-A6

<400>17

GGTGCATGATTATTAGCTTTGCTTCTAATTTCAGMAACCCACTCAAGTCC

<210>18

<211>48

<212>DNA

<213>

<220>247-A7

<400>18

AATAATCATGCACCATACTATACTGAGTCTGTGAAAGGGAGGTTCACC

<210>19

<211>49

<212>DNA

<213>

<220>247-A8

<400>19

GCAGGTAGGTAATACTTTTGGAATCATCTCGTGAGATGGTGAACCTCCC

<210>20

<211>41

<212>DNA

<213>

<220>247-A9

<400>20

GTATTACCTACCTGCAAATGAACAGCTTAAGAGCTGAAGAC

<210>21

<211>50

<212>DNA

<213>

<220>247-A10

<400>21

GGTAGCCGTGCTATCCCTGGCACAGTAATACACGGCCRTGTCTTCAGCTC

<210>22

<211>48

<212>DNA

<213>

<220>247-A11

<400>22

TAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTC

<210>23

<211>50

<212>DNA

<213>

<220>247-A12

<400>23

CCGCCACTACCACCGCCACCGCTACCACCACCACCTGAAGAGACAGTGAC

<210>24

<211>46

<212>DNA

<213>

<220>247-A13

<400>24

TAGCGGTGGCGGTGGTAGTGGCGGTGGCGGTGCTAGCGACATTCAG

<210>25

<211>49

<212>DNA

<213>

<220>247-A14

<400>25

CTGAGGCGCTCASGGAGGAGGGGGACTGGGTCATCTGAATGTCGCTAGC

<211>38

<212>DNA

<213>

<220>247-A15

<400>26

TGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGC

<210>27

<211>45

<212>DNA

<213>

<220>247-A16

<400>27

AGCTACATCATTAATCACACTCTGACTGGCCTTGCAGGTTATGGT

<210>28

<211>40

<212>DNA

<213>

<220>247-A17

<400>28

TGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGG

<210>29

<211>50

<212>DNA

<213>

<220>247-A18

<400>29

TGCGATTGGATGCATAGTATATCAGCAGTTTAGGGGGCTGCCCTGGCTTC

<210>30

<211>43

<212>DNA

<213>

<220>247-A19

<400>30

CATCCAATCGCAACACTGGAGTTCCTGATCGCTTCAGCGGCAG

<210>31

<211>43

<212>DNA

<213>

<220>247-A20

<400>31

GATTTTGAGAGTGAAATCCGTCCCGGMTCCACTGCCGCTGAAG

<210>32

<211>50

<212>DNA

<213>

<220>247-A21

<400>32

CTCTCAAAATCAGCCGGGTGGAGGCTGAAGACGTGGGGGTTTATTACTGT

<210>33

<211>39

<212>DNA

<213>

<220>247-A22

<400>33

CGTGAATGGAGGACTATAATCCTGCTGACAGTAATAAAC

<210>34

<211>44

<212>DNA

<213>

<220>247-A23

<400>34

CCTCCATTCACGTTCGGCCAGGGGACAAAAGCTGGAAATCAAGC

<210>35

<211>48

<212>DNA

<213>

<220>247-A24

<400>35

ATCCTCTTCTGAGATGAGTTTTTTGTTCTGCGGCCGCTTTGATTTCCAG

<210>36

<211>39

<212>DNA

<213>

<220>247-B1

<400>36

CCCAGCCGGCCATGGCCGAAGTGCAGCTTGTGGAGTCTG

<210>37

<211>42

<212>DNA

<213>

<220>247-B2

<400>37

GGATCCTCCAGGTTGCACCAAGCCTCCTCCAGACTCCCAAG

<210>38

<211>42

<212>DNA

<213>

<220>247-B3

<400>38

CCTGGAGGATCCCTGAAACTGTCTTGTGCCGCCTCTGGATTC

<210>39

<211>50

<212>DNA

<213>

<220>247-B4

<400>39

CGCCTGGATGGACTGGGTCTGCCAGGCCCCAGAGAAGGGACTTGAGTGGG

<210>40

<211>50

<212>DNA

<213>

<220>247-B5

<400>40

GGTGCATGATTATTAGCTTTGCTTCTAATTTCAGCAACCCACTCAAGTCC

<210>41

<211>51

<212>DNA

<213>

<220>247-B6

<400>41

TTGCAGGTACAGTCTGTTTTTGGAGTTATCTCGTGAGATGGTGAACCTCCC

<210>42

<211>50

<212>DNA

<213>

<220>247-B7

<400>42

TGTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGCCGTGTATTAC

<210>43

<211>39

<212>DNA

<213>

<220>247-B8

<400>43

GGTAGCCGTGCTATCCCTGGCACAGTAATACACGGCAGT

<210>44

<211>46

<212>DNA

<213>

<220>247-B9

<400>44

CTGAGGCGCTCAGGGAGGAGGGGGACTGGGTCATCTGAATGTCGCT

<210>45

<211>50

<212>DNA

<213>

<220>247-B10

<400>45

TGCGATTGGATGCATAGTATATCAGCAGCCTAGGGGCCTGCCCTGGCTTC

<210>46

<211>43

<212>DNA

<213>

<220>247-B11

<400>46

CATCCAATCGCAACACTGGAGTTCCTAGCCGCTTCAGCGGCAG

<210>47

<211>43

<212>DNA

<213>

<220>247-B12

<400>47

GATGGTGAAAGTGAAATCCGTCCCGCTTCCACTGCCGCTGAAG

<210>48

<211>50

<212>DNA

<213>

<220>247-B13

<400>48

CTTTCACCATCAGCAGCCTCCAGCCCGAAGACATCGCAACCTATTACTGT

<210>49

<211>41

<212>DNA

<213>

<220>247-B14

<400>49

CGTGAATGGAGGACTATAATCCTGCTGACAGTAATAGGTTG

<210>50

<211>42

<212>DNA

<213>

<220>247-C3

<400>50

CCTGGAGGATCCCTGAGGCTGTCTTGTBCCGCCTCTGGATTC

<210>51

<211>50

<212>DNA

<213>

<220>247-C5

<400>51

CGCCTGGATGGACTGGGTCCGCCAGCCCCCAGGCAAGGGACTTGAGTGGG

<210>52

<211>50

<212>DNA

<213>

<220>247-C6

<400>52

GGTGCATGATTATTAGCTTTGCTTCTAATTTTCAGAAACCCACTCAAGTCC

<210>53

<211>51

<212>DNA

<213>

<220>247-C8

<400>53

TTGCAGGTACAGGGTGTTTTTGGAGTTATCTCGTGAGATGGTGAACCTCCC

<210>54

<211>38

<212>DNA

<213>

<220>247-C10

<400>54

GGTAGCCGTGCTATCCYTGGCACAGTAATACACGGCAG

<210>55

<211>49

<212>DNA

<213>

<220>247-C14

<400>55

CTGAGGCGCTCAGGRAGGAGGGGGACTGGGTCAGCTGAATGTCGCTAGC

<210>56

<211>50

<212>DNA

<213>

<220>247-C18

<400>56

TGCGATTGGATGCATAGTATATCAGCAGTTTAGGGGYCTTCCCTGGCTTC

<210>57

<211>50

<212>DNA

<213>

<220>247-C21

<400>57

CTTTCACCATCAGCAGCCTCGAGGCTGAAGACGCCGCAACCTATTACTGT

<210>58

<211>42

<212>DNA

<213>

<220>247-D3

<400>58

CCTGGAGGATCCCTGAGGCTGTCTTGTGCCGGCTCTGGATTC

<210>59

<211>50

<212>DNA

<213>

<220>247-D9

<400>59

TGTACCTGCAAATGAACARCTTAAGAGCTGAAGGCACTGCCGTGTATTAC

<210>60

<211>49

<212>DNA

<213>

<220>247-D14

<400>60

CTGAGGCGCTCAGGGAGGAGGGGGACTGGGTCACCTGAATGTCGCTAGC

<210>61

<211>50

<212>DNA

<213>

<220>247-D18

<400>61

TGCGATTGGATGCATAGTATATCAGCAGTTTAGGGACCTTCCCTGGCTTC

<210>62

<211>43

<212>DNA

<213>

<220>247-D20

<400>62

GATGGTGAGAGTGAAATCCGTCCCGCTTCCACTGCCGCTGAAG

<210>63

<211>50

<212>DNA

<213>

<220>247-D21

<400>63

CTCTCACCATCAGCAGCCTCCAGCCCGAAGACKTCGCAACCTATTACTGT

<210>64

<211>42

<212>DNA

<213>

<220>247-E3

<400>64

CCTGGAGGATCCCTGAGGCTGTCTTGTGCCGCCTCTGGATTC

<210>65

<211>36

<212>DNA

<213>

<220>247-E4

<400>65

GTCCATCCAGGCGTCACTCACAGTGAATCCAGAGGC

<210>66

<211>50

<212>DNA

<213>

<220>247-E9

<400>66

TGTACCTGCAAATGAACAACTTAAGAGCTGAAGGCACTGCCGCCTATTAC

<210>67

<211>38

<212>DNA

<213>

<220>247-E10

<400>67

GGTAGCCGTGCTATCCCTGGCACAGTAATAGGCGGCAG

<210>68

<211>50

<212>DNA

<213>

<220>247-E18

<400>68

TGCGATTGGATGCATAGTATATCACCAGCACAGGAKMCTGCCCTGGCTTC

<210>69

<211>43

<212>DNA

<213>

<220>247-E20

<400>69

GATGGTGAGAGTGAAATCCGTCCCGCTTCCACTGCCGCTGAAG

<210>70

<211>50

<212>DNA

<213>

<220>247-E21

<400>70

CTCTCACCATCAGCTGCCTCCAGAGCGAAGACTTCGCAACCTATTACTGT

<210>71

<211>40

<212>DNA

<213>

<220>247-F1

<400>71

GCCCAGCCGGCCATGGCCCAGGTGCAGCTTGTGGAGTCTG

<210>72

<211>42

<212>DNA

<213>

<220>247-F3

<400>72

CCTGGAGGATCCCTGAGGCTGTCTTGTTCCGCCTCTGGATTC

<210>73

<211>51

<212>DNA

<213>

<220>247-F8

<400>73

TTGCAGGTACAGGCTGTTTTTGGCGTTATCTCGTGAGATGGTGAACCTCCC

<210>74

<211>40

<212>DNA

<213>

<220>247-F17

<400>74

TGTGATTAATGATGTAGCTTGGTACCTGCAGAAGCCAGGG

<210>75

<211>50

<212>DNA

<213>

<220>247-F18

<400>75

TGCGATTGGATGCATAGTATATCAGCAGCTGAGGAGACTGCCCTGGCTTC

<210>76

<211>40

<212> DNA

<213>

<220>247-G1

<400>76

GCCCAGCCGGCCATGGCCSAAGTGCAGCTTGTGGAGTCTG

<210>77

<211>42

<212>DNA

<213>

<220>247-G2

<400>77

GGATCCTCCAGGCTTCACCAAGCCTCCTCCAGACTCCCAAG

<210>78

<211>51

<212>DNA

<213>

<220>247-G8

<400>78

TTGCAGGTACAGGGTGTTTTTGGCGTTATCTCGTGAGATGGTGAACCTCCC

<210>79

<211>40

<212>DNA

<213>

<220>247-H1

<400>79

GCCCAGCCGGCCATGGCCSAAGTGCAGCTTCTGGAGTCTG

<210>80

<211>42

<212>DNA

<213>

<220>247-H2

<400>80

GGATCCTCCAGGTTKCACCAAAGCCTCCTCCAGACTCCAGAAG

<210>81

<211>42

<212>DNA

<213>

<220>247-H3

<400>81

CCTGGAGGATCCCTGAGGCTGTCTTGTGCCGCCTCTGGATTC

<210>82

<211>40

<212>DNA

<213>

<220>247-33_F1

<400>82

GGCTCCCCGGGGCGCGCTGTGACATCCAGATGACCCAGTC

<210>83

<211>40

<212>DNA

<213>

<220>247-33_R1

<400>83

GATGGTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC

<210>84

<211>41

<212>DNA

<213>

<220>247-VH_F1

<400>84

CTCTCCACAGGTGTACACTCCCAAGTGCAGCTTGTGGAGTC

<210>85

<211>41

<212>DNA

<213>

<220>247-VH_R1

<400>85

GATGGGCCCTTGGTCGACGCTGAAGAGACAGTGACCAGAGT

<210>86

<211>40

<212>DNA

<213>

<220>247-vl_R1

<400>86

GATGGTGCAGCCACCGTACGTTTGATTTCCAGCTTTGTCC

<210>87

<211>40

<212>DNA

<213>

<220>247-VL-2_F2

<400>87

GGCTCCCCGGGGCGCGCTGTGACATTCAGATGACCCAGTC

<210>88

<211>52

<212>DNA

<213>

<220>WX-887

<400>88

TTGTTGTTATTTTACAAGGTGTCCAGTGTCAAGTGCAGCTTGTGGAGTCTGG

<210>89

<211>43

<212>DNA

<213>

<220>WX-888

<400>89

AGTTTGGGCTGAGCTGGGTTTTCCTTGTTGTTATTTTACAAGG

<210>90

<211>55

<212>DNA

<213>

<220>WX-889

<400>90

TCTAGAGTCGACGAATTCGCCGCCACCATGGAGTTTGGGCTGAGCTGGGTTTTCC

<210>91

<211>44

<212>DNA

<213>

<220>WX-890

<400>91

TATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTTGTGGAGTCTG

<210>92

<211>50

<212>DNA

<213>

<220>WX-891

<400>92

GGAGTTTGGGCTGAGCTGGATTTTCCTTGCTGCTATTTTAAAAGGTGTCC

<210>93

<211>48

<212>DNA

<213>

<220>WX-892

<400>93

TCTAGAGTCGACGAATTCGCCGCCACCATGGAGTTTGGGCTGAGCTGG

<210>94

<211>46

<212>DNA

<213>

<220>WX-893

<400>94

GCTCTGGCTCTCAGGTGCCAGATGTGACATTCAGATGACCCAGTCC

<210>95

<211>50

<212>DNA

<213>

<220>WX-894

<400>95

GAGGGTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCTCAGGTG

<210>96

<211>50

<212>DNA

<213>

<220>WX-895

<400>96

TCTAGAGTCGACGAATTCGCCGCCACCATGGACATGAGGGTCCCTGCTCA

<210>97

<211>48

<212>DNA

<213>

<220>WX-896

<400>97

CTACTCTGGCTCCGAGGTGCCAGATGTGACATTCAGATGACCCAGTCC

<210>98

<211>48

<212>DNA

<213>

<220>WX-897

<400>98

GGGTCCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG

<210>99

<211>55

<212>DNA

<213>

<220>WX-898

<400>99

TCTAGAGTCGACGAATTCGCCGCCACCATGGACATGAGGGTCCCCGCTCAGCTCC

<210>100

<211>32

<212>DNA

<213>

<220>WX-899

<400>100

GGTGCTAGCTGAAGAGACAGTGACCAGAGTCC

<210>101

<211>29

<212>DNA

<213>

<220>WX-900

<400>101

CACCGTACGTTTGATTTCCAGCTTTGTCC

<210>102

<211>351

<212>DNA

<213> Humanization

<220> Nucleotide sequence of humanized antibody WBP247.hAb4 heavy chain variable region (VH)

<400>102

CAAGTGCAGCTTGTGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCCTGCGGCTGTCTTGTGTTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGGTTGCCGAAATTAGAAGCAAAGCTAATAATCATGCACCATACTACTGAGTCTGTGAAAGGGAGGT TCACCATCTCACGAGATGACTCCAAAAACCCCTCTACCTGCAAATGAACAGCTTAAAGACCGAAGACACTGCCGTGTATTACTGTGCCAGGGATAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

<210>103

<211>117

<212>PRT

<213> Humanization

<220> amino acid sequence of humanized antibody WBP247.hAb4 heavy chain variable region (VH)

<400>103

QVQLVESGGGLVQPGGSLRLSCVASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHAPYYTESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDSTATHWGQGTLVTVSS

<210>104

<211>321

<212>DNA

<213> Humanization

<220> Nucleotide sequence of humanized antibody WBP247.hAb4 light chain variable region (VL)

<400>104

GACATTCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGGTGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGCCCCTAAACTGCTGATATACTATGCATCCAATCGCAACACTGGAGTTCCTAGCCGCTTCAGCGGCAGTGGAAGCGGGACGGATTTCACTTT CACCATCAGCAGCCTCCAGCCCGAAGACATCGCAACCTATTACTGTCAGCAGGATTATAGTCCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAA

<210>105

<211>107

<212>PRT

<213> Humanization

<220> Amino acid sequence of humanized antibody WBP247.hAb4 light chain variable region (VL)

<400>105

DIQMTQSPSSLSASVGDRVTITCKASQSVINDVAWYQQKPGQPPKLLIYYASNRNTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDYSPPFTFGQGTKLEIK

<210>106

<211>351

<212>DNA

<213> Humanization

<220> Nucleotide sequence of humanized antibody WBP247.hAb6 heavy chain variable region (VH)

<400>106

GAAGTGCAGCTTGTGGAGTCTGGAGGAGGCTTGGTGAAGCCTGGAGGATCCCTGAGGCTGTCTTGTGCCGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGGTTGCTGAAATTAGAAGCAAAGCTAATAATCATGCACCATACTACTGAGTCTGTGAAAGGGAGGT TCACCATCTCACGAGATGATTCCAAAAAGTATTACCTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGCCGTGTATTACTGTGCCAGGGATAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

<210>107

<211>117

<212>PRT

<213> Humanization

<220> amino acid sequence of humanized antibody WBP247.hAb6 heavy chain variable region (VH)

<400>107

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHAPYYTESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCARDSTATHWGQGTLVTVSS

<210>108

<211>321

<212>DNA

<213> Humanization

<220> Nucleotide sequence of humanized antibody WBP247.hAb6 light chain variable region (VL)

<400>108

GACATTCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGGTGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGCCCCTAAACTGCTGATATACTATGCATCCAATCGCAACACTGGAGTTCCTAGCCGCTTCAGCGGCAGTGGAAGCGGGACGGATTTCACTTT CACCATCAGCAGCCTCCAGCCCGAAGACATCGCAACCTATTACTGTCAGCAGGATTATAGTCCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAA

<210>109

<211>107

<212>PRT

<213> Humanization

<220> Amino acid sequence of humanized antibody WBP247.hAb6 light chain variable region (VL)

<400>109

DIQMTQSPSSLSASVGDRVTITCKASQSVINDVAWYQQKPGQPPKLLIYYASNRNTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDYSPPFTFGQGTKLEIK

<210>110

<211>351

<212>DNA

<213> Humanization

<220>Humanized antibody clone 12 heavy chain variable region (VH) nucleotide sequence

<400>110

CAAGTGCAGCTTGTGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCCTGCGGCTGTCTTGTGTTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGGTTGCCGAAATTAGAAGCAAAGCTAATAATCATGCACCATACTACTGAGTCTGTGAAAGGGAGGT TCACCATCTCACGAGATGACTCCAAAAACCCCTCTACCTGCAAATGAACAGCTTAAAGACCGAAGACACTGCCGTGTATTACTGTGCCAGGGATAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

<210>111

<211>117

<212>PRT

<213> Humanization

<220> humanized antibody clone 12 heavy chain variable region (VH) amino acid sequence

<400>111

QVQLVESGGGLVQPGGSLRLSCVASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHAPYYTESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDSTATHWGQGTLVTVSS

<210>112

<211>321

<212>DNA

<213> Humanization

<220> Humanized Antibody Clone 12 Light Chain Variable Region (VL) Nucleotide Sequence

<400>112

GACATTCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGGTGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGGCCCCTAGGCTGCTGATATACTATGCATCCAATCGCAACACTGGAGTTCCTGATCGCTTCAGCGGCAGTGGATCCGGGACGGATTTCACTCTC AAAATCAGCCGGGTGGAGGCTGAAGACGTGGGGGTTTATTACTGTCAGCAGGATTATAGTCCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAA

<210>113

<211>107

<212>PRT

<213> Humanization

<220> Humanized Antibody Clone 12 Light Chain Variable Region (VL) Amino Acid Sequence

<400>113

DIQMTQSPSSLSASVGDRVTITCKASQSVINDVAWYQQKPGQAPRLLIYYASNRNTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQDYSPPFTFGQGTKLEIK

TCCAGCTGTGGAAGCACGACCTGGAATACTTCAAGTCGCTGCCCCAG

GCTGACCAGGACAATATCATCGGCCGCCGCCTGAGCGACAACGAAAA

GCTCGGCGATGCCCCCGAGTCCGCGCACGTCAAGCGCACTGCCCAGG

AAAGCTTTGAACCCGAAGCGTTCATGGTCCGTCGCTCGGTAGCCTGG

GCCGACCAGCGCGGCGCCGGCCTGGCCTTCGTCGCCTTGGGCAAGAG

TTTCGATGCATTCGGAGTGCAATTGCGGCGCATGAGTGGCCTGGAAGA

CGGCATCATCGACGGATTGTACCGCTTTAGCCGCCCGCTGACGGGTGG

CTACTACTGGTGCCCGCCGATGGGCGAGACGGGGGTTGATCTGAGCT

CCTTGCTGCGGGCCTGA

<210>4

<211>287

<212> amino acid

Amino acid sequence of <213> type B dye-depigmenting peroxidase mutant

<220>

<223>

<400>4

MPFQQGLLATPVPAHARHLFFTLQSPEALPAALDALLPQVDGEQLL

LGIGAPLVKALGREVPGLRAFPLLLDTAVENPSTQHALWLWLRGDERGDLLLRAQALEQALAPALQLADSVDGFLRRGGYDLTGYEDGTENPVDEDVV

QAAIAADGSSFAAFQLWKHDLEYFKSLPQADQDNIIGRRLSDNEKLGDA

PESAHVKRTAQESFEPEAFMVRRSVAWADQRGAGLAFVALGKSFDAFGV

QLRRMSGLEDGIIDGLYRFSRPLTGGYYWCPPMGETGVDLSSLLRA

Claims (9)

1. The monoclonal antibody of anti-human CD147, the aminoacid sequence of the heavy chain variable region and the light chain variable region of this antibody is: Antibody WBP247.hAb12: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:111, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:113. 2. the monoclonal antibody of anti-human CD147 as claimed in claim 1, is characterized in that, the nucleotide sequence of the heavy chain variable region of WBP247.hAb12 antibody comprises specific complementarity determining region CDR1, CDR2 and CDR3 The sequences are respectively SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and the sequences of CDR1, CDR2 and CDR3 of the specific complementarity determining region contained in the nucleotide sequence of the light chain variable region are respectively SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15. 3. the gene of coding anti-human CD147 monoclonal antibody described in claim 1 is characterized in that, the nucleotide sequence of the heavy chain variable region and the light chain variable region of coding antihuman CD147 monoclonal antibody is: WBP247.hAb12 antibody: the nucleotide sequence encoding the heavy chain variable region is the sequence shown in SEQ ID NO:110; the nucleotide sequence encoding the light chain variable region is the sequence shown in SEQ ID NO:112. 4. the cell strain expressing the monoclonal antibody described in claim 1, the denomination and deposit number of the cell strain are: Cell line expressing WBP247.hAb12 antibody: named: 247C-B4Z2-01-C-005, deposit number: CCTCCNO. C2019147. 5. The antibody expressed by the cell line of claim 4. 6. The expression vector of the monoclonal antibody of claim 1. 7. The method for preparing the antibody according to claim 1, characterized in that the method comprises: a) obtaining the DNA molecular sequence of the antibody described in claim 1; b) constructing an expression vector, which contains the DNA molecule described in step a) and the regulatory sequence for expressing the DNA molecule; c) transfecting host cells with the expression vector described in step b); culturing under suitable culture conditions for the host cells; d) Obtaining the monoclonal antibody of claim 1 through separation and purification steps. 8. The application of the antibody conjugated drug coupled to the antibody described in claim 1 to prepare a drug for treating liver cancer or lung cancer, the antibody conjugated drug is the maytansine derivative DM1 coupled to the antibody described in claim 1, the coupling right The tubulin polymerase inhibitor MMAE of the antibody of claim 1 or the radionuclide iodine conjugated to the antibody of claim 1. 9. The use of the antibody of claim 1 in the preparation of a medicament for diagnosing CD147-expression-positive diseases, wherein the CD147-expression-positive diseases are liver cancer or lung cancer.