阅读说明：本技术 抗人cd147的单克隆抗体、表达载体、细胞株及其应用 (Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof ) 是由 陈志南 朱平 边惠洁 杨向民 张征 张阳 于 2019-08-27 设计创作，主要内容包括：本发明公开了抗人CD147的单克隆抗体、细胞株、表达载体及其应用。本发明抗体可用于制备抗体偶联药物,也可用于制备诊断和治疗CD147表达阳性疾病药物及其生物技术产品。(The invention discloses an anti-human CD147 monoclonal antibody, a cell strain, an expression vector and application thereof. The antibody of the invention can be used for preparing antibody conjugate drugs, and can also be used for preparing drugs for diagnosing and treating CD147 expression positive diseases and biotechnological products thereof.)

1. The monoclonal antibody of anti-human CD147 has the amino acid sequences of the heavy chain variable region and the light chain variable region:

wbp247.hab12 antibody: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 111 or the sequence with more than 90 percent of sequence homology with the sequence shown as SEQ ID NO. 111, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 113 or the sequence with more than 90 percent of sequence homology with the sequence shown as SEQ ID NO. 113;

wbp247.hab4 antibody: the amino acid sequence of the heavy chain variable region is the sequence shown by SEQ ID NO. 103 or the sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 103, and the amino acid sequence of the light chain variable region is the sequence shown by SEQ ID NO. 105 or the sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 105;

or the one or more of the following components,

wbp247.hab6 antibody: the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO. 107 or the sequence with homology of more than 90% 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 the sequence with homology of more than 90% with the sequence shown in SEQ ID NO. 109.

2. The anti-human CD147 monoclonal antibody of claim 1 wherein the amino acid sequence of the heavy chain variable region of wbp247. haba 12 antibody comprises the sequences of the specific antigen complementarity determining regions CDR1, CDR2 and CDR3 of seq id NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18, the amino acid sequence of the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences of the specified antigen complementarity determining region of SEQ ID NOs: 13. SEQ ID NO: 14 and SEQ ID NO: 15.

3. the monoclonal antibody of anti-human CD147 is characterized in that the nucleotide sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are as follows:

wbp247.hab12 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown by SEQ ID NO. 110 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 110; the nucleotide sequence of the light chain variable region is a sequence shown by SEQ ID NO. 112 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 112;

wbp247.hab4 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown by SEQ ID NO. 102 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 102; the nucleotide sequence of the light chain variable region is a sequence shown by SEQ ID NO. 104 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 104;

or the one or more of the following components,

wbp247.hab6 antibody: the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 106 or a sequence with homology of more than 90 percent with the sequence shown as SEQ ID NO. 106; the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO. 108 or the sequence with homology of more than 90 percent with the sequence shown in SEQ ID NO. 108.

4. A cell line expressing the monoclonal antibody of claim 1,2 or 3, the cell line having the designation and deposit number:

cell line expressing wbp247.hab12 antibody: is named as: 247C-B4Z2-01-C-005, accession number: CCTCC NO. C2019147;

cell line expressing wbp247.hab4 antibody: is named 247A-B9Z4-02-C-T9 and has the preservation number: CCTCC NO. C2019148;

or the one or more of the following components,

cell line expressing wbp247.hab6 antibody: is named 247B-B9Z4-01-C-T9 and has the preservation number: CCTCC NO. C2019149.

5. An antibody expressed by the cell line of claim 4.

6. An expression vector for the monoclonal antibody of claim 1,2 or 3.

7. A method of producing the antibody of claim 1,2 or 3, comprising:

a) obtaining the DNA molecule sequence of the antibody of claim 1,2 or 3;

b) constructing an expression vector, wherein the expression vector comprises the DNA molecule in the step a) and a regulatory sequence for expressing the DNA molecule;

c) transfecting a host cell, in particular a mammalian cell, preferably a CHO cell, with the expression vector of step b); culturing under culture conditions suitable for the host cell;

d) obtaining the monoclonal antibody of claim 1,2 or 3 through separation and purification steps.

8. Use of an antibody according to claim 1,2, 3 or 5 for the preparation of an antibody conjugated drug which is a maytansine derivative DM1, a tubulin polymerase inhibitor MMAE or radionuclide iodine.

9. A maytansine derivative DM1 conjugated to an antibody according to claim 1,2, 3 or 5.

10. A tubulin polymerase inhibitor MMAE conjugated to an antibody of claim 1,2, 3 or 5.

11. Radionuclide iodine conjugated to the antibody of claim 1,2, 3 or 5.

12. Use of the antibody of claim 1,2, 3 or 5 for the manufacture of a medicament for the diagnosis or treatment of a disease positive for CD147 expression.

Technical Field

The invention relates to the technical field of biology, and particularly provides three humanized antibody antihuman antibodies, related cell strains, expression vectors and application of CD147 molecules.

Background

CD147 has been named in many different ways, including TCSF/EMMPRIN, M6, Basigin, Neurothelin, with high homology to antigens from different species such as mouse Basing/gp42, rat OX-47/CE-9 and chicken HT7/5A11, which were finally identified as Basigin by the HUGO (human gene nomenclature Committee); the human leukocyte differentiation antigen cooperative group was assigned to the endothelial cell group by unifying different nomenclature from each laboratory as CD 147. The molecule is a highly glycosylated transmembrane glycoprotein with the molecular weight of 50-60 kD, and belongs to a member of an immunoglobulin superfamily (IgSF). In humans, CD147 has a total of 269 amino acids, and is divided into extracellular, transmembrane and intracellular regions. The first 21 residues after the initial translation of the N terminal are signal peptides, 22-205 constitute extracellular regions, 206-229 are transmembrane regions and have typical leucine zipper structures, and the C terminal 230-269 is an 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 the CD147 molecule is a functional membrane protein important in the tumor development process and is involved in various cancer-related phenomena, and various retrospective studies have also shown that there is a close correlation between the expression intensity of the CD147 molecule in tumor tissues and the prognosis of tumor patients. In non-small cell lung cancer patients, elevated levels of CD147 expression are closely correlated with the prognosis of the patient. Thus, the CD147 molecule has become a new target for tumor therapy, in which the antibody drug "iodine" [ solution ]¹³¹I]The successful development of the rituximab injection-linagliptin proves that the target point patent medicineSafety and effectiveness of.

The monoclonal antibody (McAb) has the advantages of high specificity, high affinity, small toxic and side effects, low immunogenicity, long in-vivo action time, capability of exerting curative effect by an in-vivo autoimmune system and the like, is widely applied to diagnosis and treatment of a plurality of diseases, and becomes an effective way for developing novel medicines. However, repeated injections of murine McAb into humans can cause the patient to induce human anti-mouse antibody (HAMA) responses, develop systemic anaphylactoxic responses and block the development of antibody efficacy.

Disclosure of Invention

Based on the needs, deficiencies and drawbacks of the prior art, the present invention provides anti-human CD147 monoclonal antibodies, expression vectors, cell lines and uses thereof.

The invention provides an anti-human CD147 monoclonal antibody, wherein the amino acid sequences of a heavy chain variable region and a light chain variable region of the antibody are as follows:

wbp247.hab12 antibody: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 111 or the sequence with more than 90 percent of sequence homology with the sequence shown as SEQ ID NO. 111, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 113 or the sequence with more than 90 percent of sequence homology with the sequence shown as SEQ ID NO. 113;

wbp247.hab4 antibody: the amino acid sequence of the heavy chain variable region is the sequence shown by SEQ ID NO. 103 or the sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 103, and the amino acid sequence of the light chain variable region is the sequence shown by SEQ ID NO. 105 or the sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 105;

or the one or more of the following components,

wbp247.hab6 antibody: the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO. 107 or the sequence with homology of more than 90% 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 the sequence with homology of more than 90% with the sequence shown in SEQ ID NO. 109.

Further, the amino acid sequence of the heavy chain variable region of the wbp247. habb 12 antibody of the present invention comprises the sequences of the specific antigen complementarity determining regions CDR1, CDR2, and CDR3 of SEQ ID NOs: 16. SEQ ID NO: 17 and SEQ ID NO: 18, the amino acid sequence of the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences of the specified antigen complementarity determining region of SEQ ID NOs: 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 are as follows:

wbp247.hab12 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown by SEQ ID NO. 110 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 110; the nucleotide sequence of the light chain variable region is a sequence shown by SEQ ID NO. 112 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 112;

wbp247.hab4 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown by SEQ ID NO. 102 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 102; the nucleotide sequence of the light chain variable region is a sequence shown by SEQ ID NO. 104 or a sequence with homology of more than 90 percent with the sequence shown by SEQ ID NO. 104;

or the one or more of the following components,

wbp247.hab6 antibody: the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 106 or a sequence with homology of more than 90 percent with the sequence shown as SEQ ID NO. 106; the nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID NO. 108 or the sequence with homology of more than 90 percent with the sequence shown in SEQ ID NO. 108.

In another aspect, the present invention provides a cell line expressing the monoclonal antibody, wherein the cell line has the following designation and deposit numbers:

cell line expressing wbp247.hab12 antibody: the cell strain is preserved in China center for type culture Collection in 2019, 7, 16 and the preservation address is as follows: wuhan university in Wuhan, China, zip code: 430072, named: the recombinant Chinese hamster ovary cells 247C-B4Z2-01-C-005 have the preservation number: CCTCC NO. C2019147;

cell line expressing wbp247.hab4 antibody: the cell strain is preserved in China center for type culture Collection in 2019, 7, 16 and the preservation address is as follows: wuhan university in Wuhan, China, zip code: 430072, named: recombinant Chinese hamster ovary cells 247A-B9Z4-02-C-T9 with the deposit number: CCTCC NO. C2019148;

or the one or more of the following components,

cell line expressing wbp247.hab6 antibody: the cell strain is preserved in China center for type culture Collection in 2019, 7, 16 and the preservation address is as follows: wuhan university in Wuhan, China, zip code: 430072, named: recombinant Chinese hamster ovary cells 247B-B9Z4-01-C-T9 with the deposit number: CCTCC NO. C2019149.

The invention also provides an expression vector of the monoclonal antibody.

Further, the present invention provides a method for producing the monoclonal antibody. The provided method comprises the following steps:

a) obtaining the DNA molecule sequence of the antibody of claim 1,2 or 3;

b) constructing an expression vector, wherein the expression vector comprises the DNA molecule in the step a) and a regulatory sequence for expressing the DNA molecule;

c) transfecting a host cell, in particular a mammalian cell, preferably a CHO cell, with the expression vector of step b); culturing under culture conditions suitable for the host cell;

d) the monoclonal antibody is obtained through separation and purification steps.

The antibody can reduce the human anti-mouse antibody (HAMA) reaction caused by repeated injection of the murine McAb into a human body, and avoid systemic anaphylactic toxicity reaction. The affinity of the obtained antibody was analyzed by ELISA analysis, and the humanized antibody of the present invention had an EC50 value of about half that of the chimeric antibody wbp247. cabb 1, and an affinity about twice higher than that of the chimeric antibody.

Preferably, the humanized antibody WBP247.hAb12 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, and the EC50 of the antibody prepared by expression is 0.002614ug/ml, which is about one time higher than the EC50(0.0382nM) of the parent chimeric antibody WBP247.cAb1, and the antibody can be specifically combined with solid tumors such as lung cancer, liver cancer and the like.

In addition, humanized antibodies WBP247.hAb4 and WBP247.hAb6 are stably screened, and the EC50 of the two antibodies which are prepared by expression is 0.002864ug/ml and 0.003022ug/ml respectively through detection, and the antibodies can be specifically combined with solid tumors such as liver cancer.

In the above example antibody construction, where the light chain constant region is kappa and the heavy chain constant region consists of IgG1, the invention also protects other antibody isotypes, such as IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, IgE. Antigen binding fragments of antibodies, including Fab, Fv, ScFv and single chain antibodies, are also protected by the invention.

The antibody of the invention can be applied to the preparation of medicines for diagnosing and treating CD147 expression positive diseases, biotechnological products, detection reagents, imaging, inspection and diagnosis and the like.

The invention also provides application of the antibody in preparing antibody conjugate drugs. The drug is maytansine derivative DM1, tubulin polymerase inhibitor MMAE or radionuclide iodine.

Drawings

FIG. 1 SDS-PAGE analysis of humanized antibody clones 1-4; wherein: the left panel according to the figure is a non-reduced electrophoretic sample of purified protein, while the right panel is a reduced electrophoretic sample of purified protein. From left to right in sequence: m is a 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. 2SPR dissociation spectra for humanized antibody affinity determination, wherein the abscissa represents the response time of the sample and the ordinate represents the signal value after binding, and each line in the figures is a detection curve of different concentration dilutions of the same sample, indicating a specific change in signal with dose variation;

FIG. 3 SDS-PAGE non-reducing electrophoretic analysis of humanized antibody clones 5-12; wherein Lane1 clone 5 non-reduced; 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;

FIG. 4 SDS-PAGE reduction electrophoretic 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;

FIG. 5 SDS-PAGE reduction and non-reduction electrophoretic 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;

FIG. 6 shows gradient dilution curves of the binding of 3 selected antibodies to antigen;

FIG. 7 full length humanized antibody immunohistochemical screening profile; wbp247. haba 5 (liver cancer), wbp247. haba 12(32270) liver cancer and wbp247. haba 12(32270) lung cancer were stained sequentially from left to right in the drawing, wherein brown regions were indicated as antibody staining positive regions;

FIG. 8 plasmid map of expression vector pWX2.1-LC-B-247B 4;

FIG. 9 plasmid map of expression vector pWX2.1-LC-B-247B 12;

FIG. 10 plasmid map of expression vector pWX1.1-HC-Z-247B 4;

FIG. 11 plasmid map of expression vector pWX1.1-HC-Z-247B 6;

FIG. 12 immunohistochemical staining profiles of CD147 humanized antibodies on different tissues; the brown (dark) area in the figure is the antibody staining positive area, and the white Bar is 100 μm;

FIG. 13 in vitro killing experiment of humanized antibody against tumor cells.

Detailed Description

First, term interpretation:

immunoglobulin: refers to 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), all four chains being linked to each other by disulfide bonds. 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 regions of hypervariability (or in the form of hypervariable regions and/or structurally defined loops in sequence), also known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, 1987). Typically, the numbering of the amino acid residues in this segment is by Kabat's rule. Sequencesof proteins of immunologicalcalest, 5th ed, public health service, national institutes of health, Bethesda, MD (1991) (methods described herein for both heavy chain variable domains and light chain variable domains using this numbering system).

Humanized antibody: as used herein, refers to an antibody derived from a non-human antibody, typically a murine, which has been engineered to retain or substantially retain the antigen binding properties of a parent antibody (parent antibody), but which has reduced immunogenicity in humans. Since the antibodies of the invention are defined by structural and functional features, "humanized antibodies" can be used interchangeably with "antibodies".

Complementarity Determining Region (CDR): refers to a characteristic sequence of an antibody comprising a plurality of amino acids that together define the binding affinity and specificity of the variable fragment (Fv) region of the immunoglobulin binding site for the target antigen CD 147.

Framework Region (FR): refers to the amino acid sequence inserted between the CDRs. These portions of the antibody serve 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 comprise a Framework Region (FR) 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 of the invention are all derived from human immunoglobulins. The heavy chain constant region can be selected from five isotypes: α, δ, ε, γ or μ. Further, heavy chains of various subclasses (e.g., IgG subclasses of heavy chains) can elicit different effector functions, and thus, by selecting a desired heavy chain constant region, antibodies with a desired effector function can be produced. Preferred heavy chain constant regions are γ 1(IgG1), γ 2(IgG2), γ 3(IgG3) and γ 4(IgG4), more preferably γ 2(IgG 2). The light chain constant region may be of the kappa or lambda type, preferably of the kappa type.

The chimeric Fab is obtained by recombining the light chain variable region gene and the heavy chain variable region gene of a functional antibody with the kappa chain and the heavy chain CH1 constant region gene of a human antibody respectively, cloning the genes into an expression vector to construct a mouse-human chimeric Fab gene expression vector, and then transferring the mouse-human chimeric Fab gene expression vector into a host cell for expression. The first human-murine chimeric Fab antibody, ReoPro (which is an antiplatelet receptor gIIb/IIIa), was approved by the FDA in 1994 for antithrombotic therapy.

CDR grafted antibody is prepared by cloning all six CDRs on murine monoclonal antibody to corresponding Frame Region (FR) of human antibody by PCR and other method. Compared with the chimeric antibody, CDR transplantation further reduces the content of heterologous sequences in the antibody and reduces the heterogeneity of the antibody.

Affinity of antibody (avidity): refers to the overall strength of the interaction between two molecules, e.g., between an antibody and an antigen. Avidity characterizes the strength of binding between a molecular pair (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 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 ligand concentration is required to occupy the site.

The variable or constant regions of the immunoglobulin heavy or light chains may be joined as described by using standard recombinant DNA techniques to create a polynucleotide that can be expressed in a suitable host to produce the immunoglobulin chain(s), or the joined variable and constant regions may be synthesized by using peptide chemistry.

The humanized antibody of the invention preserves the important part of the binding property of the parent murine antibody, namely the murine antibody HAb18 called anti-human CD147 molecule, and the establishment of the cell strain is described in monoclonal antibody communication 1989; 33-36, Chenzhinan, Liuyan, Yangquan, etc. (known patent: 1. ANTI-HUMAN liver cancer MONOCLONAL antibody HAb18 light AND HEAVY chain variable region GENE AND its application, patent No. ZL02114471.0, publication No. CN1381461A, AND 2 VARIABLELEREGION GENE OF HEAVY/LIGHT CHAIN OF ANTI-HUMAN HEPATOMA MONOCLONAL ANTIBODYHAb18 AND USE THEOFF, US patent No. US 7638619). Particularly, the humanized modification is carried out on the basis of the anti-human CD147 murine parent antibody, and the capacity of specifically binding the parent antibody to recognize the antigen is reserved. Through optimization and screening, the obtained humanized antibody exhibits the same or substantially the same antibody binding affinity (affinity) or avidity (avidity) as the parent antibody.

"humanized antibodies" or "antibodies", as used herein, include intact molecules as well as fragments thereof which are capable of binding to an epitope determinant, e.g., Fab, F (ab') 2, and Fv. These antibody fragments retain the ability to selectively bind to human CD147, examples of which include, but are not limited to, the following:

(1) fab: defined as a fragment containing a monovalent antigen-binding fragment of an antibody molecule, whole antibody (whole) is degraded by the enzyme papain to produce an intact light chain and a portion of one heavy chain;

(2) fab': a fragment defined as a monovalent antigen-binding fragment containing an antibody molecule, the whole antibody being treated with pepsin followed by reduction, thereby generating intact light and heavy chain portions; two Fab' fragments are available per antibody molecule;

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

(4) fv: defined as a genetically engineered fragment containing the variable light and variable heavy chains regions represented as two chains;

(4) fv: homology (homology) is a central concept in comparative biology and refers generally to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution studies. Homology is usually checked by sequencing, but DNA-DNA or DNA-RNA hybridization provides valuable judgment (5) Coupling, oxidative Coupling, which is a process of obtaining one organic molecule by some kind of chemical reaction between two organic chemical units (moieities). Depending on the type, cross-coupling and self-coupling reactions can be distinguished. The antibody conjugation herein refers to cross-linking a specific group of chemical reaction of a drug or a small molecule drug to an antibody molecule by a specific reagent reaction, thereby improving the killing and therapeutic effects of the antibody or the small molecule drug.

The invention has clarified the CDR and FR region genes of the light and heavy chain variable regions of the murine antibody HAb18 (ZL02114471.0) of the anti-human CD147 molecule through bioinformatics analysis, carries out humanized design by using a computer-aided antibody structure, adopts molecular biology means such as phage display antibody library technology and the like to carry out humanized transformation on the framework region in the variable region, obtains the humanized antibody variable region gene sequence of the anti-human CD147 molecule, constructs a eukaryotic expression system containing the full-length antibody molecule gene, and further expresses in CHO host cells to prepare the anti-human CD147 humanized CDR-grafted antibody. Immunohistochemistry and ELISA results show that the screened humanized antibody of the anti-human CD147 molecule keeps the affinity equivalent to that of the murine parent antibody, and maintains the specificity of the parent antibody for recognizing antigen.

The present invention will be described in further detail with reference to examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the present invention. The experimental procedures used in the following examples are all conventional molecular biological procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are available from commercial reagents companies unless otherwise specified.

Second, construction of phage display vector containing light and heavy chain variable regions of mouse antibody HAb18 of anti-human CD147 molecule

1. Material

Hybridoma cell line HAb18, the establishment of which is described in monoclonal antibody communication, 1989; 2:33-36, Chenzhinan, Liuyan, Yangjie, etc. (patent: CN 1381461A).

PCR amplification primers for murine VH and VL of HAb 18G: wherein, VH gene amplification primer:

an upstream primer: HAb18-F, shown as SEQ ID NO 1 in the sequence list;

a downstream primer 1: Linker-HAb18-R1, shown as SEQ ID NO:2 in the sequence list.

A downstream primer 2: HAb18-linker-R2, shown as SEQ ID NO 3 in the sequence list.

Wherein, the VL gene amplification primer:

an upstream primer: HAb18-Linker-F1, shown as SEQ ID NO 4 in the sequence list;

a downstream primer: HAb18-R shown in SEQ ID NO. 5 of the sequence list.

2. Method and results

2.1 Total RNA extraction: total RNA from hybridoma HAb18GC2 was extracted with reference to a Total RNA extraction kit (OMEGA Total RNA R6834), and the integrity of the Total RNA was checked by agarose gel electrophoresis.

2.2 reverse transcription of cDNA: take 1. mu.g of the total RNA obtained in 2.1, referenceFirst strand cDNA was synthesized using RT reagent Kit DRR037A reverse transcription Kit instructions and frozen at-20 ℃ for use.

2.3 PCR amplification of VH and VL genes:

using cDNA prepared from 2.2 as template, using VH gene amplification primer and VL gene amplification primer to respectively amplify to obtain mouse-derived VH gene fragment and VL gene fragment, and using amplification system according to the formula

High-Fidelity DNA Polymerase (NEB) protocol configuration;

and (3) PCR reaction conditions: 94 ℃ for 5 min; 94 ℃, 15s, 54 ℃, 30s, 72 ℃, 1min, 35 cycles; 72 ℃ for 10 min. The size of the amplified fragment was observed by electrophoresis on a 1% agarose gel.

The VH gene is amplified by an upstream primer HAb18-F and a downstream primer 1Linker-HAb18-R1, and after a product is purified, the VH gene is amplified by selecting HAb18-F and a downstream primer 2HAb18-Linker-R2 again to obtain the VH gene. The VL gene is obtained by directly amplifying an upstream primer HAb18-Linker-F1 and a downstream primer HAb 18-R.

2.4 amplification of ScFv Gene:

the VH gene fragment and the VL gene fragment obtained in 2.3 were mixed in the same molar number, and ScFv gene was amplified by Overlap-PCR in the following amplification system:

and (3) PCR reaction conditions: 95 ℃ for 5 min; 35 cycles of 95 ℃, 15s, 56 ℃, 30s, 72 ℃,1 min; 72 ℃ for 10 min. The band of interest was visualized by 1% agarose gel electrophoresis and recovered.

2.5ScFv gene enzyme digestion and connection with vector and transformation: the amplified ScFv gene fragment and the vector plasmid pGEM-T vector (Promega) prepared above are digested by enzyme digestion with Nco I (NEB: C ^ CATGG) and Not I (NEB: GC ^ GGCCGC), respectively. The recovered fragment product of 2.4 and pGEM-T vector were taken as 600ng and 3. mu.g, respectively, and 1. mu.L each of restriction enzyme (NEB) and 5. mu.L of 10 XCutSmart buffer were added to the mixture, and 50. mu.L of water was added thereto, and the mixture was digested at 37 ℃ for 1.5 hours.

The enzyme digestion system is as follows:

after digestion in a 37 ℃ water bath, 5ul of 10 Xloading buffer was added to stop the reaction. The band of the restriction enzyme was recovered by 1% agarose gel electrophoresis and quantified by UV.

The T4 phage DNA ligase is used for ligation reaction, and the reaction system comprises the following components:

the reaction was carried out at 16 ℃ overnight. The ligation product was used to transform TG1 competent cells. Spread on LB agar plates and incubated overnight at 37 ℃. The next day, 10 single clones were picked up at random, and the colony of the positive clones was identified with the vector universal primers, recorded as pGEM-ScFv, and verified by sequencing, and the correctly sequenced clones were stored at-40 ℃ for future use.

Third, anti-CD 147 parent monoclonal antibody HAb18 CDR and FR sequence calibration

The light and heavy chain variable region nucleotide sequences of the murine antibody HAb18 encoding anti-CD 147, also known as HAb18G monoclonal antibody (CN021144710), were translated into their encoded amino acid sequences using www.expasy.org on-line software, and these signature sequences were identified according to Kabat database principles and were retained in further humanization.

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

Selection of framework sequence FR for antibody humanization

In order to select a suitable human antibody framework sequence to which mouse CDRs are grafted, the present inventors constructed a human antibody sequence information database with the aid of antibody sequences derived from the Kabat protein database; the molecular structure of the antibody before and after humanization is analyzed and replaced by using discovery studio (VIOVIA, version 3.5) through homologous modeling and mechanical optimization technology, so that the replaced amino acid can not change the whole framework structures of VH and VL, especially can not damage the secondary structure of beta-strand, and the original affinity of the antibody can be maintained. The FR shuffling method was originally proposed by Dall' Acqua, and humanization was achieved by random substitution of the FR region of a human antibody sequence having a high homology with the FR region of a murine antibody sequence with a murine antibody sequence, which reduced theoretical library capacity and irrelevant FR recombination.

The FR and CDR regions of the antibody variable regions were determined according to the Kabat database. In the antibody database, homology alignments were performed using the variable region sequences of the murine antibody HAb 18. For the modification of FR1, FR2 and FR3 of VH, 12 germlines of FR1, 4 germlines of FR2 and 10 germlines of FR3 are respectively referred to in the invention, and the sequence of VH-FR4 is consistent with the sequence of IGHJ1 & lt 01 & gt of human and is kept unchanged; for the modification of FR1, FR2, FR3 of VL, reference is made to 6 germlines, 9 germlines and 7 germlines; the FR4 regions of VH and VL were confirmed by JH and JK, respectively. Through homologous modeling, the molecular structures of the antibody before and after humanization are analyzed, and the substituted amino acid can not change the overall framework structures of VH and VL, particularly can not damage a beta-strand secondary structure, so that the original affinity of the antibody can be maintained. The design scheme is shown in Table 1 and Table 2, and the theoretical library capacity of the humanized antibody library finally designed is 1.8 x 10^ 5.

TABLE 1V_HSelectively substituted amino acids in framework

Table 2 amino acids selected for replacement in VL frame

Fifthly, construction and screening of phage display CD147 humanized antibody library

The FR sequence variable region sequence of the human antibody obtained according to the above (three) and the alternative amino acid sequence thereof, the combination of which retains the CDR sequence (II) of the parent mAb HAb18, i.e., the light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO:8, characteristic sequence; the heavy chain variable region sequence comprises SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11 a characteristic sequence; in the FR framework region, designing corresponding PCR primers according to the candidate sites shown in the attached figures 1 and 2, and obtaining the designed full-length gene containing the mutation sites by an overlapping PCR method; meanwhile, codons commonly used in mammalian cells are selected for partial replacement, and the obtained full-length gene is totally cloned into a phage vector pComb3Xss for construction and screening of a humanized antibody library. Finally, 16 effective humanized sequence combination clones for light chain and heavy chain pairing of anti-CD 147 are obtained.

Primer design for CD147 humanized antibody library

According to the selection and analysis results of the framework sequence FR of the antibody humanized modification, 70 antibody library primers are designed, all the sequences are 247-headed, such as 247-A1, 247-A2 and the like, and are respectively shown as SEQ ID NO: 12 to SEQ ID NO: 81.

2. gene amplification protocol

Following the Overlapping-PCR procedure, a specific humanized antibody library amplification protocol was as follows:

primer selection template length

First step of

Second step of

247-A1,B1,F1,G1,A12 VH 404bp

247-A13,A24 VL 394bp

The third step

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

Amplifying a target fragment; the reaction conditions are 95 ℃ and 3 min; 95 ℃ for 30 sec; 55 ℃ for 30 sec; 72 ℃, 40 sec; 30 cycles, final extension at 72 ℃ for 10 min. After the PCR reaction was completed, the PCR product was purified and recovered by 1% agarose gel electrophoresis.

3 phage display library construction

3.1 cloning of the phage vector

The recovered PCR-amplified humanized antibody library gene fragment was digested with Nco I (NEB: C ^ CATGG) and Not I (NEB: GC ^ GGCCGC) in a double digestion under the same conditions as (I) 2.5, separated by 1% agarose electrophoresis, and then purified with Gel Extraction Kit (Omega bio-tek). Then, the purified digested fragment was ligated with NcoI/Not I double-digested phage vector pComb3Xss using T4DNA ligase (TaKaRa), and after deionization, TG1 competent cells were transformed, and inoculated on an LB culture plate for colony screening. Randomly picking 48 monoclones, and carrying out colony identification on positive clones by using M13-48 and M13-47 primers; counting the library capacity, and storing the library at-80 ℃ for later use.

3.2 preparation and titration of helper phage

XL-1Blue single colony is picked to inoculate 5m 1SB-T⁺(20ug/ml) and cultured overnight at 37 ℃ with shaking. Diluting according to the ratio of 1:500 and inoculating 10ml of SB-T⁺(20ug/ml) the solution was shaken at 37 ℃ for one hour. Picking single bacteriophage M13K07 virus plaque, inoculating into the above 10ml bacterial solution, culturing at 37 deg.C under shaking for 2 hr, adding 200M1SB-T⁺(20ug/ml)K⁺(70 ug/ml) and cultured overnight at 37 ℃ with shaking. Centrifuging at 4000rpm at 4 deg.C for 15 min, collecting supernatant to determine phage titer, aseptically packaging, and storing at 4 deg.C.

3.3 phage rescue experiment

Scraping 3.1 mL of electrotransformation bacteria, diluting to 400mL of SOB-GAT solution, culturing at 30 deg.C until A600 is about 0.5, adding M13K 0737 deg.C, standing for superinfection for 1 hr (preferably multiple infection value is 5: 1, adding M13K07 pfu amount: 5 × 10)⁸bacteria/A600 units). times.multiple infection value (5). times.A 600 value (0.5). times.final volume of bacteria (mL), centrifugation at 4 ℃ at 3500RPM for 10min, resuspension of the pellet in 2YT-AKT bulk, shake culture at 30 ℃ at medium speed overnight. The rescue culture solution is centrifuged at 4000rpm at 4 ℃ for 20min, the supernatant is taken and added with 4 percent PEG8000 and 3 percent N aC1 for ice bath precipitation of the phage for 1h, the supernatant is centrifuged at 15000g at 4 ℃ for 20min, 1-2mL of sterilized PBS (containing 1 percent BSA, 0.02mol/L and PH7.4) is used for heavy suspension precipitation, the supernatant is the rescued original phage antibody library (split charging and 4 ℃ preservation for later use) after short-time low-speed centrifugation.

3.4 panning of phage antibody libraries

The antibody phage library obtained in 3.1 was subjected to antigen-specific panning by solid phase panning. The antigen coating concentration of each round of panning was decreased in turn and positive/negative controls were set. The specific operation is as follows: resuscitated 3.1 stored antibody phage libraries were grown in 60ml 2YT medium at 37 ℃ in a shaker to OD600 ═ 0.3-0.4. Adding M13KO7 helper phage (Invitrogen); incubated at 37 ℃ for 30 minutes with standing and 60 minutes with shaking. Centrifuging at 1500 rpm for 10min, discarding the supernatant, resuspending the cells in 60ml of 50ug/ml kanamycin (no glucose) medium, and incubating overnight in a shaker at 30 ℃; the phage library was pelleted by centrifugation at 12000 rpm for 10 minutes and the supernatant was transferred to a centrifuge tube, 30 ml/tube. 7.5ml of PEG/NaCl was added to each tube, mixed well and placed on ice for 1 h. Centrifuging, rotating at 12000 r for 5min, and discarding the supernatant; the phage were resuspended in 2.2ml of PBS-5% BSA, centrifuged, 12000 rpm, 5 minutes, and cell debris removed. Thereafter, the library of affinity sieves was applied to the expressed CD147 molecule coated plate, and the antigen coating concentration was decreased in the order of 5 rounds of panning (adsorption-elution-amplification) (1ug/ml, 0.1ug/ml, 0.01ug/ml, 0.001ug/ml, 0.0001 ug/ml). Calculating the input/output ratio (recovery rate) of the phage screened in each round as an index of the enrichment of the specific phage antibody, wherein the calculation formula is as follows: the recovery (% yield) — (eluent volume × eluent titer × 100)/(input antibody pool volume × antibody pool titer), and when panning was performed until the recovery was less than 10, the panning experiment was terminated, 768 clones obtained were selected, expression was induced, and a gene product containing the humanized gene antibody heavy chain VH and light chain VL, i.e., ScFv antibody, was obtained for further Elisa detection.

Sixth, ELISA and sequencing analysis

With coating buffer (200mM Na)₂CO₃/NaHCO₃pH 9.2) CD147 was diluted to 1. mu.g/ml, 50. mu.l was added to each reaction well and coated overnight at 4 ℃; discarding the solution in the reaction well, washing with 1XPBS buffer solution for 3 times, adding 200. mu.l blocking buffer solution (2% BSA/1XPBS buffer) and blocking at room temperature for 1 h; wash 3 times with 200. mu.l 1XPBS buffer; adding (IV) 3.4 cell culture supernatant 768 samples (8 pieces of 96-well micro-perforated plates) containing ScFv antibody for induced expression and a negative control (50 mu.l/well), and incubating for 2h at room temperature; wash 3 times with 200 μ l 1X PBS buffer; anti- -c-MycAb (HRP) (Abcam Cat # ab19312, 50. mu.l/well), incubation at room temperature for 1 h; wash 6 times with 200 μ l 1XPBS buffer; adding TMB substrate solution (50 μ l/well) into each reaction well for reaction for 10 min; stop solution (2M HCl, 50. mu.l/well) was added to stop the reaction; reading the absorbance of A450nm with an ELISA plate reader;

based on the ELISA results, 50 clone samples with A450>3.0 were selected for sequencing (sequencing work was done by Shanghai Boshang Biotech Co., Ltd.). DNA sequence analysis: the humanization degree of the sequences was evaluated with reference to the germline database of human antibodies and http:// www.bioinf.org.uk/abs/shab/while affinity ranking the molecules with better humanization degree.

Seventhly, SPR measurement of the affinity of ScFv antibody

7.1 SPR binding assay

Affinity determination of the ELISA antibody strongly positive antibody in step (five) was performed with a ProteOn XPR36(Bio-Rad, XPR36) instrument. GLC chips (Bio-Rad, 1765011) were 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 30ul/min to couple the antigen to the activated chip via the amino group. Finally, inactivating the chip by using 1M ethanomine-HCl (Bio-Rad); the chip was rotated 90 degrees and washed with buffer (PBS/0.005% Tween 20) until baseline plateaus. Cell culture supernatants containing ScFv antibody induced by (IV) 3.4 were injected on 6 horizontal channels, respectively. The injection rate was 30. mu.l/min. The sample binding time is 60s, and the dissociation time is 900 s; data analysis was performed using Langmuir kinetics (Kinetic-Langmuir) model; the fully humanized antibody was constructed by selecting a clone with high affinity.

7.2 SPR determination of ScFv antibody affinity ranking

Binding of coated CD147 to the (tetra) 3.4 inducible expression of ScFv antibody-containing cell culture supernatant was monitored in real time using SPR by determining the dissociation rate constant (K)_off) Reflecting the magnitude of affinity of CD147 for the humanized ScFv antibody. The results are shown in Table 3, and according to the change of the affinity, a plurality of molecules with better affinity are selected for the construction of the complete humanized antibody.

TABLE 3 degree of humanization of HAb18Sequence analysis and K_offSorting

Eight, full-length humanized antibody construction and detection

8.1 materials

Based on the results of the aforementioned affinity ranking, first, 4 clones (i.e., clones 26601,26602,27028 and 27044) of the first 2SPR measurement fragment antibody ScFv antibodies were picked up for the first round of full-length antibody construction and expression.

Namely, 4 clones selected firstly are inoculated and cultured overnight, plasmids are respectively extracted, and sequencing is carried out to confirm a template sequence.

PCR primers: humanized antibody VH gene amplification primers: an upstream primer: 247-VH _ F1, as shown in SEQ ID NO: 84; a downstream primer: 247-VH _ R1 as set forth in SEQ ID NO: 85, the size of the target product is 392 bp; humanized antibody VL gene amplification primers: upstream of the first set: 247-33_ F1, downstream primer: 247-33_ R1, which are respectively shown as SEQ ID NO: 82 and SEQ ID NO: 83; according to the sequencing results, the second set of upstream primers: 247-VL-2_ F2; a downstream primer: 247-vl _ R1, corresponding to SEQ ID NOs: 87 and SEQ ID NO: 86.

8.2 PCR amplification Process

Respectively extracting plasmids, after sequencing to confirm that the sequence of the template is correct, respectively carrying out PCR amplification on target fragments by using the primers and the template, wherein the amplification schemes are respectively as follows:

length of target fragment of primer template

8.3 preparation of transient expression vectors

And (3) adding the light chain variable region gene or the heavy chain variable region gene amplified in the step (7.2) into II type restriction enzymes NgoMIV and SnaBI respectively for double enzyme digestion, purifying by a DNA purification kit after enzyme digestion, and connecting with a mammalian cell expression vector pCI-vector containing hIgG1/k digested by the same restriction enzymes NgoMIV/SnaBI. The ligation product was transformed into TOP10 E.coli, spread on LB agar medium containing 100. mu.g/ml ampicillin, and the obtained positive clone was cultured in LB liquid medium containing 100. mu.g/ml ampicillin, and plasmid extraction and sequencing were performed to obtain full-length eukaryotic expression vector clones 1-VH 1-4 containing humanized antibody VH gene and full-length eukaryotic expression vector clones 1-VL 1-4 containing humanized antibody VL gene, respectively. Through sequence determination, the nucleotide sequence corresponding to the heavy chain variable region of clone 4 is shown as SEQ ID NO. 102; the light chain variable region has the corresponding nucleotide sequence as shown in SEQ ID No. 104.

Jiu, transient transfection of cell and antibody expression purification

9.1 transient transfection of cells with antibodies

Respectively pairwise pairing VH gene full-length eukaryotic expression vector clones 1-VH 1-4 obtained in step (VII) 7.3 and full-length eukaryotic expression vector clones 1-VL 1-4 containing humanized antibody VL genes by using Freestyle Max Reagent transfection Reagent of Invitrogen company for co-transfection into HEK293 cells (1.0 × 10)⁶Pieces/ml), the transfected cells were placed in a shaker at 37 ℃ in CO₂Shake culturing in an incubator with 5% concentration, and rotating the shaking table at 120 turns. After 7 days of transfection, the cell supernatant after transfection was collected by centrifugation, the target full-length humanized antibody was separated and purified from the cell culture supernatant using a ProteinA affinity column, and the protein concentration of the antibody thus purified was measured and used for further measurement of affinity SPR of the purified antibody (the antibody was referred to as clones 1 to 4, respectively).

9.2 SDS-PAGE analysis of antibodies

The method adopts the conventional SDS-PAGE analysis method in a laboratory, namely, NuPAGE LDS sample buffer and NuPAGE Reducing Agent are respectively mixed with each sample, water bath is carried out for 10 minutes at 75 ℃, and centrifugation is carried out. The loading was 2. mu.g/well and the run was carried out at 200V for 35 minutes. After completion of electrophoresis, the gel was rinsed three times for 5 minutes each. Adding a dyeing solution SimplyBlue safestain for one hour, discarding the dyeing solution after finishing, and adding deionized water for decolorization until the gel background is completely decolorized; the observation results are shown in figure 1.

Wherein, the left picture is a non-reduction electrophoresis sample of the purified protein, and the right picture is a reduction 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 (prepared in different batches); and M is a protein Marker.

Ten, humanized antibody affinity assay

SPR determination of humanized antibody affinity: the affinity of the purified first 4 humanized antibodies was determined by SPR as in (VI) 6.1, and the results showed that the affinity of the purified antibodies clone 1, clone 2, clone 3 and clone 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), and the results are shown in FIG. 2, wherein the binding dissociation pattern of the antibodies is shown in FIG. 2.

TABLE 4 humanized antibody SPR kinetic data

Eleventh, screening and identification of humanized antibody specificity by immunohistochemical staining method

Although the affinity of the humanized antibody was measured by the SPR method, since the antigen used was a purified antigen prepared by in vitro expression, the antibody was further immunohistochemically stained with a tumor tissue specimen held in a laboratory in order to further observe the binding specificity of the humanized antibody obtained.

And detecting the specific binding capacity of the expressed series of humanized antibody clones 1-4 and tumor tissues, and inspecting the immune tissue cross reaction of the antibody.

The specific operation is as follows:dewaxing by conventional xylene, dehydrating by gradient alcohol, and hydrating the tissue chip; 3% H₂O₂Blocking and inactivating endogenous peroxidase; sealing the normal sheep serum working solution; the antibody clone 1-4 is used as a primary antibody, a biotin-labeled rabbit anti-human Fc antibody is used as a secondary antibody, a horseradish peroxidase-labeled streptomycin ovalbumin working solution is used as a tertiary antibody, DAB color development, hematoxylin counterstaining, dehydration and transparency are carried out, and then mounting and microscopic examination are carried out. The organized screening result shows that only antibody clone 4, namely WBP247.hAb4 can be specifically stained in malignant tumor tissues of lung cancer, liver cancer and the like, and the staining degree is "+" or "+ +"; but with little binding to normal tissue. While the other three antibodies were negative. The results show that only 1 available candidate humanized antibody WBP247.hAb4 is screened by the expression of the round, the specificity of the antibody for recognizing the antigen is similar to that of the murine parent antibody, the humanized antibody is successfully constructed, but the available number is very small, the quantity requirement cannot be met, and therefore, multiple rounds of expression and screening are needed.

Eleven, repeated rounds of full-length humanized antibody construction and immunohistochemical screening

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

According to the results provided by immunohistochemistry, since there is a possibility that the specificity of the antibody is changed after humanization, the strategy of the previous stage was adjusted by first constructing and expressing a full-length antibody for all 16 clones ranked first in the order of affinity of the SPR measurement fragment antibody ScFv antibody (the sixth) 2; the method is the same as the method of 7.1, and the carrier construction and the expression of the full-length antibody are carried out on the selected clone. Wherein, the nucleotide sequence corresponding to clone 6, namely WBP247.hAb6, is shown as SEQ ID NO 106; the light chain variable region has the corresponding nucleotide sequence as shown in SEQ ID No. 108. Clone 12, WBP247.hAb12, the nucleotide sequence of its corresponding heavy chain variable region is set forth in SEQ ID NO: 110; the corresponding nucleotide sequence of the light chain variable region is set forth in SEQ ID NO: 112. The obtained antibodies were then purified according to (eight) 8.1 and transient cell transfection and antibody expression purification (clones 5 to 16 for each antibody), and analyzed by SDS-PAGE according to the method described above for 8.2, and the results are shown in FIGS. 3, 4 and 5.

Twelve, ELISA analysis of humanized antibody affinity

Coating 200ng of recombinant CD147 protein into an enzyme label plate, and standing overnight at 4 ℃; blocking with 1 XPBS/2% BSA at room temperature for 1 hour. And (5) diluting the purified antibody product clones 1-6 prepared in the step (eight) and the subsequent steps from 1ug/ml by using a blocking solution 1:3.16 in a gradient manner, adding 100 mu l of the blocking solution into each hole, and standing for 1 hour at room temperature. Mu.l of a Goat anti-human secondary antibody, Goat anti-human IgG Fc HRP (Bethy Cat # A80-304P) diluted 1:4000 was added and allowed to stand at room temperature for 1 hour. Color development with addition of TMB and 2M H₂SO₄The reaction was terminated. The reading was done with a microplate reader at 450 nm. As can be seen from the ELISA results, screening clone 1-clone 16 totaled 16 humanized antibodies with EC50 values about half that of the chimeric antibody wbp247. cabb 1 and an affinity about twice higher than that of the chimeric antibody. (see Table 5, FIG. 6).

TABLE 5 antibody binding to antigen CD147 EC₅₀Measurement of

Sample:	EC50(ug/ml)	EC50(nM)
			Chimeric cAb1	0.005733	0.0382
Clone 1	0.002725	0.0182
			Clone 2	0.002944	0.0196
Clone 3	0.003076	0.0205
			Clone 4	0.002864	0.0191
Clone 5	0.002811	0.0187
			Clone 6	0.003022	0.0201
Clone 7	0.002507	0.0167
			Clone 8	0.001701	0.0113
Clone 9	0.002099	0.0140
			Clone 10	0.002061	0.0137
Clone 11	0.002362	0.0157
			Clone 12	0.002614	0.0174
Clone 13	0.002527	0.0168
			Clone 14	0.002509	0.0167
Clone 15	0.002432	0.0162
			Clone 16	0.00325	0.0217

Thirteen, full length humanized antibody immunohistochemical screening

In order to further detect the specific binding capacity of the expressed serial humanized antibody clones 5-16 and tumor tissues, the immune tissue cross reaction of the antibody is examined, and the specificity of the humanized antibody is further screened by an immunohistochemical staining method. The specific operation is as the tenth step: dewaxing by conventional xylene, dehydrating by gradient alcohol, and hydrating the tissue chip; 3% H₂O₂Blocking and inactivating endogenous peroxidase; sealing the normal sheep serum working solution; and (3) taking antibody clone 5-16 as a primary antibody, a biotin-labeled rabbit anti-human Fc antibody as a secondary antibody, a horseradish peroxidase-labeled streptomycin avidin working solution as a tertiary antibody, performing DAB color development, performing hematoxylin counterstaining, dehydrating and transparentizing, and performing mounting and microscopic examination. And (4) positive judgment: positive for brown staining of cell membrane. Through the expression screening of the round, the expression screening is obtained againTwo candidate humanized antibodies, wbp247.hab6 and clone 12, were available, wherein clone 12 bound not only to liver cancer specimens, but also to lung cancer specimens. The specific information is shown in table 6 below:

TABLE 6 selection of humanized antibody specificity by immunohistochemical staining

Through multiple histochemical stains and the binding specificity of the obtained humanized antibody, three humanized monoclonal antibodies, namely clone 4(WBP247.hAb4), clone 6(WBP247.hAb6) and clone 12(WBP247.hAb12), are primarily screened to carry out screening of downstream stable cell strains.

Fourteen, humanized antibody high-efficiency expression vector construction and stable expression cell strain screening

Based on the results of ELISA and immunization, the gene sequences of three clones with best affinity and specificity, namely clone 4, clone 6 and clone 12, namely 'WBP247. hAb4, WBP247.hAb6 and WBP247.hAb 12', are selected for constructing high-efficiency expression vectors and screening stable cell strains.

TABLE 7 WBP247.hAb4, WBP247.hAb6 and WBP247.hAb12 sequence combination information

(wherein, clone 12 light chain variable region amino acid sequence is shown as SEQ ID NO:113, the corresponding light chain variable region (VL) nucleotide sequence is shown as SEQ ID NO:112, the heavy chain variable region amino acid sequence is shown as SEQ ID NO:111, and the corresponding heavy chain variable region (VH) nucleotide sequence is shown as SEQ ID NO: 110).

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

The gene DNA (27989-VL) fragment was amplified in vitro, using this DNA as a template, the variable region fragment WBP247B4-VL was amplified with 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), and an EcoRI restriction site was introduced into the 5 'end of WBP247B4-VL, a BsiWI restriction site was introduced into the 3' end, this fragment and the expression vector pWX2.1 containing the light chain constant region gene were subjected to double digestion with EcoRI and BsiWI, and a 5119bp DNA fragment was recovered from the gel after 1% agarose gel electrophoresis. The DNA fragment after gel cutting purification and the plasmid pWX2.1 fragment are subjected to ligation reaction, T4 ligase is subjected to 20 mul system and is reacted for 20min at 16 ℃, and 10 mul of ligation solution is taken to transform the escherichia coli TOP10 competent cells. After colony PCR identification, enzyme digestion identification and sequencing, a correct single clone is picked up and cultured in 200ml LB culture medium at 37 ℃ and 220rpm overnight with shaking, a large amount of plasmids are extracted, and finally the obtained plasmid is named as pWX2.1-LC-B-247B4(5521bp) (as shown in figure 8).

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

The amplification of a gene DNA (32270-VL) fragment was carried out in vitro, using the 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 site was introduced into the 5 'end of WBP247B12-VL, a BsiWI restriction site was introduced into the 3' end, the fragment and an expression vector pWX2.1 containing a light chain constant region gene were subjected to double digestion with EcoRI and BsiWI, and a 5119bp DNA fragment was recovered from a 1% agarose gel electrophoresis and a gel. The DNA fragment after gel cutting purification and the fragment from the plasmid pWX2.1 are subjected to ligation reaction, and the escherichia coli TOP10 competent cells are transformed. After colony PCR identification, enzyme digestion identification and sequencing, a correct single clone is picked up and cultured in 200ml LB culture medium at 37 ℃ and 220rpm overnight with shaking, a large amount of plasmids are extracted, and finally the obtained plasmid is named as pWX2.1-LC-B-247B12(5521bp) (as shown in figure 9).

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

Carrying out in vitro gene DNA (27989-VH) fragment amplification, using the DNA as a template, amplifying variable region fragments WBP247B4-VH by 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 a sequence table), introducing EcoRI restriction enzyme cutting sites at the 5 'end of WBP247B4-VH, introducing NheI restriction enzyme cutting sites at the 3' end, carrying out EcoRI and NheI double enzyme cutting on the fragments and an expression vector pWX1.1 containing light chain constant region genes, carrying out 5202bp DNA fragment recovery by cutting gel after 1% agarose gel electrophoresis. The DNA fragment after gel cutting purification and the fragment from the plasmid pWX1.1 are subjected to ligation reaction, and the escherichia coli TOP10 competent cells are transformed. After colony PCR identification, enzyme digestion identification and sequencing, a correct single clone is picked up and cultured in 200ml LB culture medium at 37 ℃ and 220rpm overnight with shaking, a large amount of plasmids are extracted, and finally the obtained plasmid is named as pWX1.1-HC-Z-247B4(5625bp) (shown in figure 10).

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

Carrying out in vitro gene DNA (32338-VH) fragment amplification, using the DNA as a template, amplifying variable region fragments WBP247B6-VH by 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 a sequence table), introducing an EcoRI restriction enzyme site at the 5 'end of WBP247B6-VH, introducing a NheI restriction enzyme site at the 3' end, carrying out EcoRI and NheI double digestion on the fragment and an expression vector pWX1.1 containing a light chain constant region gene, carrying out 5201% agarose gel electrophoresis, and then cutting gel to recover a DNA fragment of 2 bp. The DNA fragment after gel cutting purification and the fragment from the plasmid pWX1.1 are subjected to ligation reaction, and the escherichia coli TOP10 competent cells are transformed. After colony PCR identification, enzyme digestion identification and sequencing, a correct single clone is picked up and cultured in 200ml LB culture medium at 37 ℃ and 220rpm overnight with shaking, a large amount of plasmids are extracted, and finally the obtained plasmid is named as pWX1.1-HC-Z-247B6(5625bp) (shown in figure 11).

14.5 construction and screening of CHO stably expressing cell line

After the vector construction, the humanized antibody light chain expression vector and the humanized antibody heavy chain expression vector were assigned to Mingkudan as described above according to (1) pWX2.1-LC-B-247B4 and pWX1.1-HC-Z-247B4, respectively; (2) pWX2.1-LC-B-247B6, pWX1.1-HC-Z-247B 4; (3) WX2.1-LC-B-247B12 and pWX1.1-HC-Z-247B4 were co-transfected into CHO/DHFR cells, medium of appropriate concentration was added for selection passage, and single clones were selected to obtain stable expression CHO cell lines.

Thereafter, Minipool batch feeding experiments were performed to measure titers. After four-round batch feeding optimization, screening to obtain a cell strain 247C-B4Z2-01-C-005(CCTCC NO. C2019147) which stably expresses WBP247.hAb12 antibody (named: mehozumab monoclonal antibody), a cell strain 247A-B9Z4-02-C-T9(CCTCC NO. C2019148) which expresses WBP247.hAb4 and a cell strain 247B-B9Z4-01-C-T9(CCTCC NO. C2019149) which expresses WBP247.hAb6 respectively, wherein the cell strains are preserved in the Wuhan China center for type culture collection at present and the preservation date is 16 months and 7 months in 2019.

Fifteen, tumor cell in vitro killing experiment of humanized anti-CD 147 antibody

The method comprises the following steps: taking human lung cancer cell NCI-H520 in logarithmic growth phase, performing pancreatin digestion to obtain single cell suspension, adjusting concentration to 1 × 10⁴One per ml. 2000 cells (200. mu.l cell suspension) were seeded into 96-well plates with 5% CO₂Incubate overnight at 37 ℃ waiting for the cells to adhere and recover to a state of logarithmic proliferation.

According to the conventional ADC drug laboratory preparation method, three humanized antibodies are subjected to DM1 labeling, and WBP247.hAb12-DM1, WBP247.hAb4-DM1 and WBP247.hAb6-DM1 are obtained. The following gradients were diluted with serum-free RPMI1640 medium, respectively: 1000. 100, 10, 1, 0.1, 0.01, 0.001, 0.0001. mu.g/mL; 20. mu.l of each concentration was added to the corresponding well, and 6 duplicate wells were designed for each concentration. Negative antibody controls (human IgG) and background (cell-free medium only) were designed simultaneously. 5% CO₂Incubating at 37 ℃ for 48h, and adding 10 mu L of CCK-8 developing solution into each well. Incubating for 2h at 37 ℃, and reading the light absorption value at 450nm by a full-wavelength microplate reader. The results are shown in fig. 13, and the screened humanized antibodies all have better killing effect on tumor cells (fig. 13), which indicates that the prepared humanized antibodies all have good tumor specificity and targeting property. Based on the effect, those skilled in the art can deduce that the antibody of the present invention can be used for preparing (drugs for and biotechnological products thereof, detection reagents, imaging reagents, and reagents for testing diagnosis) and treating CD147 expression positive diseases.

Nucleotide sequence list electronic file

<110> the fourth military medical university of the Chinese people liberation army

<120> anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof

<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

TACCACCACCACCAGAACCGCCACCACCTGAAGAGACAGTGACCAGAGTC

<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

GGATCCTCCAGGTTGCACCAAGCCTCCTCCAGACTCCTCAAG

<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

CCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAAGC

<210>35

<211>48

<212>DNA

<213>

<220>247-A24

<400>35

ATCCTCTTCTGAGATGAGTTTTTGTTCTGCGGCCGCTTTGATTTCCAG

<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

GGATCCTCCAGGTTGCACCAAGCCTCCTCCAGACTCCACAAG

<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

GGTGCATGATTATTAGCTTTGCTTCTAATTTCAGAAACCCACTCAAGTCC

<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

GGATCCTCCAGGCTTCACCAAGCCTCCTCCAGACTCCACAAG

<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

GGATCCTCCAGGTTKCACCAAGCCTCCTCCAGACTCCAGAAG

<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

GGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG

<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> humanized antibody WBP247.hAb4 heavy chain variable region (VH) nucleotide sequence

<400>102

CAAGTGCAGCTTGTGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCCTGCGGCTGTCTTGTGTTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGGTTGCCGAAATTAGAAGCAAAGCTAATAATCATGCACCATACTATACTGAGTCTGTGAAAGGGAGGTTCACCATCTCACGAGATGACTCCAAAAACACCCTCTACCTGCAAATGAACAGCTTAAAGACCGAAGACACTGCCGTGTATTACTGTGCCAGGGATAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

<210>103

<211>117

<212>PRT

<213> humanization

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

<400>103

QVQLVESGGGLVQPGGSLRLSCVASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHAPYYTESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDSTATHWGQGTLVTVSS

<210>104

<211>321

<212>DNA

<213> humanization

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

<400>104

GACATTCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGCCCCCTAAACTGCTGATATACTATGCATCCAATCGCAACACTGGAGTTCCTAGCCGCTTCAGCGGCAGTGGAAGCGGGACGGATTTCACTTTCACCATCAGCAGCCTCCAGCCCGAAGACATCGCAACCTATTACTGTCAGCAGGATTATAGTCCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAA

<210>105

<211>107

<212>PRT

<213> humanization

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

<400>105

DIQMTQSPSSLSASVGDRVTITCKASQSVINDVAWYQQKPGQPPKLLIYYASNRNTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDYSPPFTFGQGTKLEIK

<210>106

<211>351

<212>DNA

<213> humanization

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

<400>106

GAAGTGCAGCTTGTGGAGTCTGGAGGAGGCTTGGTGAAGCCTGGAGGATCCCTGAGGCTGTCTTGTGCCGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGGTTGCTGAAATTAGAAGCAAAGCTAATAATCATGCACCATACTATACTGAGTCTGTGAAAGGGAGGTTCACCATCTCACGAGATGATTCCAAAAGTATTACCTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGCCGTGTATTACTGTGCCAGGGATAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

<210>107

<211>117

<212>PRT

<213> humanization

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

<400>107

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHAPYYTESVKGRFTISRDDSKSITYLQMNSLRAEDTAVYYCARDSTATHWGQGTLVTVSS

<210>108

<211>321

<212>DNA

<213> humanization

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

<400>108

GACATTCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGCCCCCTAAACTGCTGATATACTATGCATCCAATCGCAACACTGGAGTTCCTAGCCGCTTCAGCGGCAGTGGAAGCGGGACGGATTTCACTTTCACCATCAGCAGCCTCCAGCCCGAAGACATCGCAACCTATTACTGTCAGCAGGATTATAGTCCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAA

<210>109

<211>107

<212>PRT

<213> humanization

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

<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

CAAGTGCAGCTTGTGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCCTGCGGCTGTCTTGTGTTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGGCCCCAGGCAAGGGACTTGAGTGGGTTGCCGAAATTAGAAGCAAAGCTAATAATCATGCACCATACTATACTGAGTCTGTGAAAGGGAGGTTCACCATCTCACGAGATGACTCCAAAAACACCCTCTACCTGCAAATGAACAGCTTAAAGACCGAAGACACTGCCGTGTATTACTGTGCCAGGGATAGCACGGCTACCCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

<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

GACATTCAGATGACCCAGTCCCCCTCCTCCCTGAGCGCCTCAGTCGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGATTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGGCCCCTAGGCTGCTGATATACTATGCATCCAATCGCAACACTGGAGTTCCTGATCGCTTCAGCGGCAGTGGATCCGGGACGGATTTCACTCTCAAAATCAGCCGGGTGGAGGCTGAAGACGTGGGGGTTTATTACTGTCAGCAGGATTATAGTCCTCCATTCACGTTCGGCCAGGGGACAAAGCTGGAAATCAAA

<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

<213> amino acid sequence of type B dye-decolorized peroxidase mutant

<220>

<223>

<400>4

MPFQQGLLATPVPAHARHLFFTLQSPEALPAALDALLPQVDGEQLL

LGIGAPLVKALGREVPGLRAFPLLDTAVENPSTQHALWLWLRGDERGDLLLRAQALEQALAPALQLADSVDGFLRRGGYDLTGYEDGTENPVDEDVV

QAAIAADGSSFAAFQLWKHDLEYFKSLPQADQDNIIGRRLSDNEKLGDA

PESAHVKRTAQESFEPEAFMVRRSVAWADQRGAGLAFVALGKSFDAFGV

QLRRMSGLEDGIIDGLYRFSRPLTGGYYWCPPMGETGVDLSSLLRA