CN117586397A - Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof - Google Patents

Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof Download PDF

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CN117586397A
CN117586397A CN202310558186.1A CN202310558186A CN117586397A CN 117586397 A CN117586397 A CN 117586397A CN 202310558186 A CN202310558186 A CN 202310558186A CN 117586397 A CN117586397 A CN 117586397A
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antibody
seq
variable region
chain variable
sequence
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陈志南
朱平
边惠洁
杨向民
张征
张阳
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Fourth Military Medical University FMMU
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Fourth Military Medical University FMMU
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
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Abstract

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 coupling medicines, medicines for diagnosing and treating CD147 expression positive diseases and biotechnological products thereof.

Description

Anti-human CD147 monoclonal antibody, expression vector, cell strain and application thereof
The present application is application number CN201910796766.8, application date 2019, 08 and 27, and is mainly the divisional application of monoclonal antibody, expression vector, cell strain and application of the invention for resisting human CD 147.
Technical Field
The invention relates to the field of biotechnology, and in particular provides three humanized antibodies of anti-CD 147 molecules, anti-human cells, related cell lines, expression vectors and application thereof.
Background
CD147 has been named differently and includes TCSF/EMMPRIN, M6, baskin, neurochelin, which has higher homology with the different species-derived antigens of mouse basin/gp 42, rat OX-47/CE-9 and chicken HT7/5A11, which are ultimately identified as Baskin by the human Committee for Gene nomenclature HUGO; the human leukocyte differentiation antigen-cooperating group was designated as CD147 from each laboratory, and was classified as endothelial cell group. The molecule is a highly glycosylated transmembrane glycoprotein with the molecular weight of 50-60 kD, and belongs to immunoglobulin superfamily (IgSF) members. In humans, CD147 is composed of 269 amino acids and is divided into extracellular, transmembrane and intracellular domains. The first 21 residues after N-terminal initial translation are signal peptides, 22-205 form extracellular regions, 206-229 are transmembrane regions, a typical leucine zipper structure is provided, and the C-terminal 230-269 is an intracellular region. CD147 has been demonstrated to be overexpressed in many types of human solid tumors, such as lung, liver, cervical, colon, breast, ovarian, esophageal, or gastric cancers.
Previous studies have shown that CD147 molecules are important functional membrane proteins in the tumor development process, are involved in a variety of cancer-related phenomena, and that there is a close correlation between the expression intensity of CD147 molecules in tumor tissues and prognosis of tumor patients. In non-small cell lung cancer patients, increased levels of CD147 expression are closely related to the prognosis of the patient. Thus, CD147 molecules have become new targets for tumor therapy, where the antibody drug "iodine 131 I]The successful development of the rituximab injection-Li Kating' proves the safety and effectiveness of the target patent medicine.
Monoclonal antibodies (McAb) have the advantages of high specificity, high affinity, small toxic and side effects, low immunogenicity, long in-vivo action time, capability of exerting curative effects by using an in-vivo autoimmune system and the like, are widely applied to diagnosis and treatment of a plurality of diseases, and become an effective way for developing novel medicines. However, repeated injection of murine mcabs into humans can cause patients to elicit a response to human anti-murine antibodies (human anti mouse antibody, HAMA), develop systemic allergic toxic responses and block the development of antibody efficacy.
Disclosure of Invention
Based on the needs, deficiencies and shortcomings of the prior art, the present invention provides anti-human CD147 monoclonal antibodies, expression vectors, cell lines and uses thereof.
The invention provides a monoclonal antibody of anti-human CD147, which has the amino acid sequences of a heavy chain variable region and a light chain variable region:
wbp247.hab12 antibody: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 111 or the sequence with the homology of more than 90 percent with the sequence shown in SEQ ID NO. 111, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 113 or the sequence with the homology of more than 90 percent with the sequence shown in SEQ ID NO. 113;
wbp247.hab4 antibody: the amino acid sequence of the heavy chain variable region is a sequence shown as SEQ ID NO. 103 or a sequence with homology of more than 90% with the sequence shown as SEQ ID NO. 103, and the amino acid sequence of the light chain variable region is a sequence shown as SEQ ID NO. 105 or a sequence with homology of more than 90% with the sequence shown as SEQ ID NO. 105;
or alternatively, the method can be used for preparing the composite,
wbp247.hab6 antibody: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 107 or the sequence with the 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 shown in SEQ ID NO. 109 or the sequence with the homology of more than 90% 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 comprises the sequences of the specific complementarity determining regions CDR1, CDR2 and CDR3 of SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO:18, the CDR1, CDR2 and CDR3 sequences of the specific complementarity determining regions comprised by the nucleotide sequence of the light chain variable region are 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 of the present invention are:
wbp247.hab12 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown as SEQ ID NO. 110 or a sequence with the homology of more than 90% with the sequence shown as SEQ ID NO. 110; the nucleotide sequence of the light chain variable region is a sequence shown as SEQ ID NO. 112 or a sequence with the homology of more than 90% with the sequence shown as SEQ ID NO. 112;
wbp247.hab4 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown as SEQ ID NO. 102 or a sequence with the homology of more than 90% with the sequence shown as SEQ ID NO. 102; the nucleotide sequence of the light chain variable region is a sequence shown as SEQ ID NO. 104 or a sequence with the homology of more than 90% with the sequence shown as SEQ ID NO. 104;
or alternatively, the method can be used for preparing the composite,
wbp247.hab6 antibody: the nucleotide sequence of the heavy chain variable region is a sequence shown as SEQ ID NO. 106 or a sequence with the homology of more than 90% with the sequence shown as SEQ ID NO. 106; the nucleotide sequence of the light chain variable region is the sequence shown as SEQ ID NO. 108 or the sequence with the homology of more than 90 percent with the sequence shown as SEQ ID NO. 108.
In another aspect, the invention provides a cell line expressing the monoclonal antibody, wherein the cell line is named and has the accession number:
Cell line expressing wbp247.hab12 antibody: the name is: 247C-B4Z2-01-C-005, accession number: cctccc No. c2019147;
cell line expressing wbp247.hab4 antibody: designated 247A-B9Z4-02-C-T9, accession number: cctccc No. c2019148;
or alternatively, the method can be used for preparing the composite,
cell line expressing wbp247.hab6 antibody: designated 247B-B9Z4-01-C-T9, accession number: cctccc No. c2019149.
The invention also provides an expression vector of the monoclonal antibody.
Further, the invention provides a preparation method of 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 containing the DNA molecule described in 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 described in 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 reaction of the human anti-mouse antibody (human anti mouse antibody, HAMA) caused by repeatedly injecting the murine McAb into human bodies, and avoid systemic anaphylactic toxicity reaction. The obtained antibodies were analyzed for affinity by ELISA assay, and the humanized antibodies of the present invention had EC50 values of about half that of the chimeric antibody wbp247.Cab1, with affinities about one time higher than those 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, the light chain variable region amino acid sequence shown in SEQ ID NO. 113, the EC50 of the antibody prepared by expression is 0.002614ug/ml, which is improved by about one time compared with the EC50 (0.0382 nM) 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 prepared by detection and expression are 0.002864ug/ml and 0.003022ug/ml respectively, so that the antibodies can be specifically combined with solid tumors such as liver cancer.
In the construction of the above example antibodies, the light chain constant region is kappa and the heavy chain constant region is of the IgG1 composition, and the 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 invention can be applied to the preparation of medicines for diagnosing and treating CD147 expression positive diseases, and biotechnology products, detection reagents, imaging, inspection and diagnosis and the like thereof.
The invention also provides application of the antibody in preparation of an antibody coupling drug. The medicine is maytansine derivative DM1, tubulin polymerase inhibitor MMAE or radionuclide iodine.
Drawings
SDS-PAGE analysis of humanized antibody clones 1-4 of FIG. 1; wherein: the left panel according to the drawing shows a non-reducing electrophoretic sample of purified protein, and the right panel shows a reducing electrophoretic sample of purified protein. The method comprises the following steps in sequence from left to right: 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. 2 is a graph of SPR dissociation for determining affinity of humanized antibodies, wherein the abscissa represents response time of a sample and the ordinate represents signal values after binding, each line in the graph being a detection curve of different concentration dilutions of the same sample, indicating a specific change in signal with dose;
FIG. 3 SDS-PAGE non-reducing electrophoretic analysis of humanized antibody clones 5-12; wherein Lane1 is cloned 5non-reduced; lane2 WBP247.hAb6 non-reduced; lane3 clone 7non-reduced; lane4 clone 8non-reduced; lane5 clone 9non-reduced; lane6 clone 10non-reduced; lane7 clone 11non-reduced; lane8 clone 12non-reduced;
FIG. 4 SDS-PAGE reduction electrophoresis analysis of humanized antibody clones 5-12; lane1 clone 5reduced; lane2 WBP247.HAb 6reduced; lane3 clone 7reduced; lane4 clone 8reduced; lane5 clone 9reduced; lane6 clone 10reduced; lane7 clone 11reduced; lane8 clone 12reduced;
FIG. 5 SDS-PAGE reduction and non-reduction electrophoretic analysis of humanized antibody clones 13-16; lane1 clone 13reduced; lane2 clone 14reduced; lane3 clone 15reduced; lane4 clone 16reduced; lane5 clone 13non-reduced; lane6 clone 14non-reduced; lane7 clone 15non-reduced; lane8 clone 16non-reduced;
FIG. 6 shows a gradient dilution of 3 antibodies screened against antigen;
FIG. 7 is a diagram of a full-length humanized antibody immunohistochemical screen; wbp247.hab5 (liver cancer), wbp247.hab12 (32270) liver cancer and wbp247.hab12 (32270) lung cancer are stained in this order from left to right, wherein the brown area is indicated as antibody staining positive area;
FIG. 8 shows a plasmid map of the expression vector pWX2.1-LC-B-247B 4;
FIG. 9 shows a plasmid map of the expression vector pWX2.1-LC-B-247B 12;
FIG. 10 shows a map of the vector pWX1.1-HC-Z-247B 4;
FIG. 11 shows a plasmid map of the expression vector pWX1.1-HC-Z-247B 6;
FIG. 12 immunohistochemical staining of different tissues with CD147 humanized antibodies; the brown (dark) areas in the figure are antibody staining positive areas, and the white Bar is 100 μm;
FIG. 13 in vitro killing experiments of humanized antibodies against tumor cells.
Detailed Description
1. 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 interconnected 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 regions of hypervariability in sequence and/or in the form of structurally defined loops), also known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL is typically composed 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 by the Kabat rule. SequencesofProteinsofImmunologicalInterest,5thEd,PublicHealthService,NationalInstitutesofHealth,Bethesda,MD (1991) (this numbering system describes the procedure used herein for either the heavy chain variable domain or the light chain variable domain).
Humanized antibodies: as used herein, refers to antibodies derived from non-human antibodies, typically murine, that retain or substantially retain the antigen binding properties of the parent antibody (parent antibody) after being engineered, but have reduced immunogenicity in humans. Since the antibodies of the present invention are defined by structural and functional features, "humanized antibodies" may be used interchangeably with "antibodies".
Complementarity Determining Regions (CDRs): refers to a characteristic sequence of antibodies containing a plurality of amino acids which 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 parts of the antibody are used to hold the CDRs in place (allowing the CDRs to bind antigen). The light chain variable region and the heavy chain variable region each comprise a Framework Region (FR) and typically three CDRs.
Constant Region (CR): refers to the portion of the antibody molecule that confers effector function. The constant regions of the humanized antibodies of the invention are derived from human immunoglobulins. The heavy chain constant region may be selected from five isoforms: alpha, delta, epsilon, gamma or mu. Further, the heavy chains of the various subclasses (e.g., the IgG subclasses of heavy chains) can cause different effector functions, and thus, by selecting the desired heavy chain constant regions, antibodies with the desired effector functions can be produced. Preferred heavy chain constant regions are γ1 (IgG 1), γ2 (IgG 2), γ3 (IgG 3) and γ4 (IgG 4), more preferably γ2 (IgG 2). The light chain constant region may be kappa or lambda type, preferably kappa type.
The chimeric Fab is prepared by recombining the light and heavy chain variable region genes of a functional antibody with the kappa chain and heavy chain CH1 constant region genes of a human antibody respectively, cloning the recombinant into an expression vector to construct a mouse-human chimeric Fab gene expression vector, and transferring the recombinant into a host cell for expression. The first human-murine chimeric Fab antibody, reoPro (anti-platelet receptor gIIb/IIIa), was approved by the FDA in 1994 for antithrombotic treatment.
The CDR grafted antibody is a new antibody constructed by cloning all six CDRs of murine monoclonal antibody onto the corresponding Framework Regions (FRs) of human antibody by PCR and other methods. CDR grafting further reduces the content of heterologous sequences in the antibody and reduces antibody heterology compared to chimeric antibodies.
Affinity (avidity) of antibodies: refers to the total strength of interaction between two molecules, e.g., between an antibody and an antigen. The affinity characterizes the strength of binding between a pair of molecules (e.g., 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 half of the binding sites of the X molecules present in the solution. A smaller Kd indicates stronger or higher affinity interactions and lower ligand concentrations are 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 a polynucleotide that can be expressed in a suitable host (thereby producing the immunoglobulin chain (s)), or can be joined by chemical synthesis using peptides.
The humanized antibody of the present invention retains an important part of the binding properties of the parent murine antibody, namely murine antibody HAb18, known as anti-human CD147 molecule, established as a cell line in monoclonal antibody communication, 1989;2:33-36, chen Zhina, liu Yanfang, yang Jizhen, etc. (prior patent: 1. ANTI-human liver cancer monoclonal antibody HAb18 light AND heavy chain variable region gene AND USE THEREOF, patent No. ZL02114471.0, publication No. CN1381461A, AND 2VARIABLE REGION GENE OF HEAVY/LIGHT CHAIN OF ANTI-HUMAN HEPATOMA MONOCLONAL ANTIBODY HAb AND USE THEREOF, (U.S. patent No. US 7 638619). In particular, the humanized modification of the invention based on the parent antibody of anti-human CD147 murine origin retains the ability to specifically bind antigen recognition by the parent antibody. The humanized antibodies obtained exhibit the same or substantially the same antibody binding affinity (avidity) or avidity as the parent antibody by optimization and screening.
"humanized antibodies" or "antibodies", as used herein, include intact molecules as well as fragments thereof capable of binding epitope determinants, such as 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: a fragment defined as a monovalent antigen binding fragment containing an antibody molecule, which is degraded by the enzyme papain to form a complete light chain and a portion of a heavy chain;
(2) Fab': a fragment defined as a monovalent antigen binding fragment containing an antibody molecule, treating the whole antibody with pepsin followed by reduction to produce a portion of the intact light and heavy chains; two Fab' fragments are available per antibody molecule;
(3)(Fab’) 2 : defined as the treatment with the enzyme pepsin without subsequent reduction to obtain antibody fragments;
(Fab) 2 is a dimer of two Fab' fragments bound 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 represented as two chains;
(4) Fv: homology (homology) is a central concept in comparative biology, meaning that in molecular evolution studies, homology generally refers to the degree of similarity between nucleotide sequences of two nucleic acid molecules or between amino acid sequences of two protein molecules. Typically, homology is checked by sequencing, but DNA-DNA or DNA-RNA hybridization provides valuable judgment (5) of Coupling, also called Coupling, oxidative Coupling, which is the process of obtaining an organic molecule from two organic chemical units (moities) by some chemical reaction. Depending on the type, it can be classified into cross-coupling and self-coupling reactions. The antibody coupling refers to the cross-linking of specific drug chemical reaction groups or small molecular drugs to antibody molecules through specific reagent reactions, so as to improve the killing and therapeutic effects of the antibodies or the small molecular drugs.
The invention determines the CDR and FR region genes of the mouse antibody HAb18 (ZL 02114471.0) light and heavy chain variable region of the anti-human CD147 molecule through bioinformatics analysis, uses a computer-aided antibody structure to carry out humanized design, adopts molecular biological 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 and prepares the anti-human CD147 humanized CDR grafted antibody in CHO host cells. Immunohistochemistry and ELISA results showed that the humanized antibodies against human CD147 molecules screened maintained affinity comparable to the murine parent antibody and maintained the specificity of the parent antibody for antigen recognition.
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 methods used in the examples below are all conventional molecular biology methods unless otherwise indicated.
Materials, reagents, and the like used in the examples described below are available from commercial reagent company unless otherwise specified.
2. Construction of phage display vector containing the light and heavy chain variable region of murine antibody HAb18 against human CD147 molecule 1. Material
Hybridoma cell strain HAb18, the establishment of which is shown in monoclonal antibody communication, 1989;2:33-36, chen Zhina, liu Yanfang, yang Jizhen, etc. (patent: CN 1381461A).
PCR amplification primers for murine VH and VL of HAb 18G: wherein, the VH gene amplification primer:
an upstream primer: HAb18-F, SEQ ID NO 1 in the sequence Listing;
downstream primer 1: linker-HAb18-R1 is shown in SEQ ID NO 2 of the sequence Listing.
Downstream primer 2: HAb18-linker-R2, see SEQ ID NO 3 of the sequence Listing.
Wherein, VL gene amplification primers:
an upstream primer: HAb18-Linker-F1, SEQ ID NO 4 in the sequence Listing;
a downstream primer: HAb18-R, SEQ ID NO. 5 of the sequence Listing.
2. Method and results
2.1 total RNA extraction: total RNA in the hybridoma cells HAb18GC2 was extracted with reference to the 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: 1 μg of total RNA obtained in 2.1 was taken and referenced to TaKaRaRT reagent Kit DRR037A reverse transcription kit instruction manual synthesizes the first strand of cDNA, and the cDNA is frozen at-20 ℃ for standby.
2.3PCR amplification of VH and VL genes:
Using cDNA prepared in 2.2 as template, using VH gene amplification primer and VL gene amplification primer to respectively amplify to obtain VH gene fragment and VL gene fragment of murine origin, the amplification system is according to the following stepsHigh-Fidelity DNA Polymerase (NEB) instruction configuration;
PCR reaction conditions: 94 ℃ for 5min;94 ℃,15s,54 ℃,30s,72 ℃,1min,35 cycles; 72℃for 10min. The size of the amplified fragment was observed by 1% agarose gel electrophoresis.
Wherein, the VH gene is amplified by an upstream primer HAb18-F and a downstream primer 1Linker-HAb18-R1, and after the product is purified, the VH gene is obtained by amplifying the HAb18-F and the downstream primer 2HAb18-Linker-R2 again. 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 VL gene fragment obtained in 2.3 were mixed in the same molar number, and ScFv genes were amplified by overlay-PCR using the following amplification system:
PCR reaction conditions: 95 ℃ for 5min;95 ℃,15s,56 ℃,30s,72 ℃,1min,35 cycles; 72℃for 10min. The target band was observed and recovered by 1% agarose gel electrophoresis.
2.5ScFv Gene cleavage, ligation with vector and transformation: the amplified ScFv gene fragment and the vector plasmid pGEM-T vector (Promega) prepared above were 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 at 600ng and 3. Mu.g, respectively, 1. Mu.L of restriction enzyme (NEB) each, 5. Mu.L of 10 XCutSmart buffer was added, and water was added to 50. Mu.L, and digested at 37℃for 1.5 hours.
The enzyme digestion system is as follows:
after digestion in a 37℃water bath, the reaction was stopped by adding 5ul of 10 Xloading buffer, respectively. The enzyme-cut strips were recovered by 1% agarose gel electrophoresis and quantified by ultraviolet.
The ligation reaction was performed using T4 phage DNA ligase, and the reaction system consisted of:
the reaction was carried out at 16℃overnight. The ligation product was used to transform TG1 competent cells. Plated on LB agar plates and incubated overnight at 37 ℃. The next day, 10 single clones were randomly picked, positive clone colony identification was performed with vector universal primers, recorded as pGEM-ScFv, sequencing verified, and clones with correct sequencing were stored at-40℃for use.
3. CDR and FR sequence calibration of anti-CD 147 parent monoclonal antibody HAb18
The nucleotide sequences encoding the light and heavy chain variable region of the murine antibody HAb18, also known as the HAb18G monoclonal antibody (CN 021144710) against CD147 were translated into their encoded amino acid sequences using www.expasy.org on-line software, calibrated according to the Kabat database principle, and these marker sequences were retained for further humanization.
Amino acid sequences of CDR1, CDR2 and CDR3 of the complementarity determining regions marked in the sequence of the light chain variable region of monoclonal antibody HAb18 are shown in SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: shown at 8. Amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 marked in the variable region sequence of the monoclonal antibody HAb18 heavy chain are shown in SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO: 11.
4. Selection of framework sequence FR for antibody humanization engineering
In order to select a suitable human antibody framework sequence onto which the mouse CDRs are grafted, the inventors constructed a database of human antibody sequence information by means of antibody sequences derived from the Kabat protein database; the antibody molecular structure 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 is ensured not to change the integral framework structure of VH and VL, especially not to damage the beta-strand secondary structure, thereby maintaining the original affinity of the antibody. The FR-stuffer method was first proposed by Dall' Acqua, and the humanized antibody was achieved by randomly substituting the murine antibody with the FR region of a human antibody sequence having higher homology with the FR region of the murine antibody sequence, which reduces theoretical reservoir capacity and irrelevant FR recombination.
The FR and CDR regions of the antibody variable region were determined according to the Kabat database. In the antibody database, homology alignment was performed using the variable region sequence of the murine antibody HAb 18. For the transformation of FR1, FR2 and FR3 of VH, the invention refers to the sequences of 12 FR1 germlines, 4 FR2 germlines and 10 FR3 germlines respectively, and the sequences of VH-FR4 are consistent with the sequence IGHJ1 of human and are kept unchanged; for engineering FR1, FR2, FR3 of VL, reference is made to 6, 9 and 7; the FR4 region of VH and VL are confirmed by JH and JK, respectively. By homologous modeling, the molecular structure of the antibody before and after humanization is analyzed, so that the replaced amino acid can not change the overall framework structure of VH and VL, especially can not damage the beta-strand secondary structure, thereby maintaining the original affinity of the antibody. The design schemes are shown in tables 1 and 2, and the theoretical storage capacity of the finally designed humanized antibody library is 1.8X10-5.
TABLE 1V H Amino acids selected for replacement in frame works
TABLE 2 selection of alternative amino acids in VL frame works
5. Construction and screening of phage display CD147 humanized antibody library
The human antibody FR sequence variable region sequence obtained according to (iii) above, and its replaceable amino acid sequence, in combination, retains CDR sequence (two) of parent mab HAb18, i.e., the light chain variable region sequence comprises SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 a feature sequence; the heavy chain variable region sequence comprises SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11 a signature sequence; in the FR frame region, according to the candidate sites of tables 1 and 2, corresponding PCR primers are designed, and the full-length gene containing the mutation site is obtained by a method of overlapping PCR; and simultaneously selecting codons commonly used in mammalian cells for partial replacement, and cloning all obtained full-length genes into a phage vector pComb3Xss for constructing and screening humanized antibody libraries. Eventually 16 effective humanized sequence combination clones of the light and heavy chain pairing of the anti-CD 147 are obtained.
Primer design for CD147 humanized antibody library
According to the selection analysis result of the framework sequence FR of the humanized transformation of the antibody, 70 antibody library primers are designed, and all sequences are 247, such as 247-A1, 247-A2 and the like, and are respectively SEQ ID NO in sequence table: 12 to SEQ ID NO:81.
2. Gene amplification protocol
According to the procedure of overlay-PCR, a specific humanized antibody library amplification protocol is as follows:
template length for primer selection
First step
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Second step
247-A1,B1,F1,G1,A12 VH 404bp
247-A13,A24 VL 394bp
Third step
247-A1,B1,F1,G1,A24 VH+VL 774bp
Amplifying the target fragment; the reaction condition is 95 ℃ for 3min;95 ℃ for 30sec;55 ℃,30sec;72 ℃,40sec;30 cycles, last 72℃extension, 10min. After the completion of 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 vectors
PCR amplified humanized antibody library gene fragments recovered by double digestion with NcoI (NEB: C≡CATGG) and Not I (NEB: GC fact GGCCGC) were selected, and the specific conditions were the same as those (one) 2.5, and after separation by 1% agarose electrophoresis, the digested fragments were purified by Gel Extraction Kit (Omega bio-tek). Then, the purified fragment was ligated with the Nco I/Not I double-digested phage vector pComb3Xss using T4DNA ligase (TaKaRa), and the resulting fragment was deionized, electrically transformed into TG1 competent cells, and inoculated onto LB plates for clone selection. Randomly picking 48 monoclonals, and carrying out positive clone colony identification by using M13-48 and M13-47 primers; counting the storage capacity, and storing the storage at-80 ℃ for standby.
3.2 preparation and titration of helper phages
XL-1Blue single colony was picked and inoculated with 5m 1SB-T + (20 ug/ml) solution, cultured overnight at 37℃with shaking. 10ml SB-T was inoculated at a dilution of 1:500 + (20 ug/ml) solution, shaking at 37℃for one hour. Picking single phage M13K07 virus plaque, inoculating into the 10ml bacterial liquid, shake culturing at 37deg.C for 2 hr, adding 200M1SB-T + (20ug/ml)K + (70 ug/ml) solution, cultured overnight at 37℃with shaking. Centrifugation was performed at 4000rpm for 15 minutes at 4℃and the supernatant was taken for phage titer determination, aseptically dispensed and stored at 4 ℃.
3.3 phage rescue experiments
Scraping 6mL of electrotransfer bacteria of 3.1, diluting to 400mL of SOB-GAT liquid, culturing at 30 ℃ until A600=0.5, adding M13K07 ℃ for standing and super-infecting for 1 hour (preferably, the multiple infection value reaches 5:1, and the pfu number of adding M13K07 is 5 multiplied by 10) 8 bacteria/A600 units) x multiple infection values (5) x A600 values (0.5) x final volume of bacteria (mL), centrifuged at 3500RPM at 4℃for 10min, and the pellet resuspended in 2YT-AKT stock volume and shake-cultured at 30℃at medium speed overnight. Centrifuging the rescue culture solution at 4000rpm at 4deg.C for 20min, adding 4% PEG8000 and 3% N aC1 ice-bath to supernatant to precipitate phage for 1h, centrifuging at 15000g at 4deg.C for 20min, and sterilizing 1-2mL PBS (containing1% BSA,0.02mol/L, pH 7.4), and performing short-term low-speed centrifugation to obtain supernatant, wherein the supernatant is the original phage antibody library after rescue (sub-packaging, and storing at 4 ℃ for later use).
3.4 panning of phage antibody libraries
Antigen-specific panning was performed on the antibody phage library obtained in 3.1 using a solid phase panning method. The antigen coating concentration was decreased in each round of panning, and positive/negative controls were set. The specific operation is as follows: resuscitates 3.1 the stored antibody phage library was incubated in 60ml of 2YT medium in a shaker at 37 ℃ until the od600=0.3-0.4. M13KO7 helper phage (Invitrogen) was added; incubate at 37℃for 30 min and shake for 60 min. The cells were resuspended in 60ml of 50ug/ml kanamycin (no glucose) medium and incubated overnight in a 30℃shaker after centrifugation at 1500 rpm for 10 min, the supernatant was discarded; phage library was pelleted by centrifugation at 12000 rpm for 10 min, and the supernatant was transferred to a centrifuge tube at 30 ml/tube. 7.5ml PEG/NaCl was added to each of the tubes, mixed well and placed on ice for 1h. Centrifuging for 12000 r/s for 5 min, and discarding supernatant; phage were resuspended in 2.2ml of solution containing PBS-5% BSA, centrifuged, 12000 rpm, and cell debris removed for 5 minutes. Thereafter, the affinity screening library was performed with the expressed CD147 molecular encapsulation plate, and the antigen coating concentration was successively decreased (1 ug/ml, 0.1ug/ml, 0.01ug/ml, 0.001ug/ml, 0.0001 ug/ml) for each round of panning through 5 rounds of panning (adsorption-elution-amplification). The input/output ratio (recovery rate) of each round of screening phage is calculated as an index of enrichment of the specific phage antibody, and the calculation formula is as follows: recovery (% yield) = (eluent volume×eluent droplet size×100)/(input antibody library volume×antibody library titer), panning was performed until recovery was less than 10, the panning experiment was terminated, 768 clones obtained were picked up, expression was induced, and ScFv antibody, which was a gene product containing humanized gene antibody heavy chain VH and light chain VL, was obtained for further Elisa detection.
6. ELISA and sequencing assays
With coating buffer (200 mM Na 2 CO 3 /NaHCO 3 pH 9.2) CD147 was diluted to 1. Mu.g/ml, 50. Mu.l per well was added and coated overnight at 4 ℃; the solution in the reaction wells was discarded and buffered with 1XPBSWashing 3 times, adding 200 μl of blocking buffer (2% BSA/1XPBS buffer) and blocking for 1h at room temperature; wash 3 times with 200 μl 1XPBS buffer; adding 768 samples of cell culture supernatant containing ScFv antibody (8 96-well microplates) and negative control (50 μl/well) which are induced to express by 3.4, and incubating for 2h at room temperature; wash 3 times with 200 μl 1XPBS buffer; anti-c-Myc Ab (HRP) (Abcam Cat#ab19312, 50 μl/well) diluted with blocking buffer (1:2500) was added and incubated for 1h at room temperature; wash 6 times with 200 μl with 1XPBS buffer; TMB substrate solution (50 μl/well) was added to each reaction well for reaction for 10min; stop solution (2M HCl, 50. Mu.l/well) was added to stop the reaction; reading the absorbance at A450nm by ELISA plate reader;
based on ELISA results, 50 clone samples with A450>3.0 were selected for sequencing (sequencing work was done by Shanghai Boshang Biotechnology Co., ltd.). DNA sequence analysis: the degree of humanization of the sequences was evaluated with reference to the human antibody's germline database and http:// www.bioinf.org.uk/abs/shab, while affinity ordering was performed for molecules with better degrees of humanization.
7. SPR determination of the affinity of ScFv antibodies
7.1SPR binding assay
Affinity determination of ELISA antibodies strong positive in step (five) was performed with a Proteon XPR36 (Bio-Rad, XPR 36) 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 amino groups. Finally, inactivating the chip by using 1M ethane-HCl (Bio-Rad); after 90 degrees rotation the chip was rinsed with buffer (PBS/0.005% Tween 20) to baseline plateau. Cell culture supernatants containing (IV) 3.4-induced expression of ScFv-containing antibodies were injected separately on 6 horizontal channels. The sample injection rate was 30. Mu.l/min. Sample binding time is 60s, and dissociation time is 900s; data analysis was performed using the Langmuir Kinetic (Kinetic-Langmuir) model; clones with high affinity were selected for the construction of fully humanized antibodies.
7.2SPR determination of affinity ranking of ScFv antibodies
Real-time monitoring of coated CD147 and (IV) 3.4 mutagenesis using SPRCell culture supernatant binding of the expressed ScFv-containing antibodies was measured by determining dissociation rate constants (K off ) Reflecting the size of the affinity of CD147 for humanized ScFv antibodies. The results are shown in Table 3, and a plurality of molecules with better affinity are selected for the construction of the fully humanized antibodies according to the change of the affinity.
TABLE 3 sequence analysis of the degree of humanization of HAb18 and K off Ordering of
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8. Construction and detection of full-Length humanized antibody 8.1 Material
Based on the results of the affinity ranking described above, the first 4 clones (i.e., clones 26601,26602,27028 and 27044) of the (sixth) 2, i.e., SPR assay fragment antibody ScFv antibody, with the top affinity ranking, were first picked for the first round of full-length antibody construction and expression.
That is, the first 4 clones were inoculated overnight for culture, plasmids were extracted separately, and the template sequence was confirmed by sequencing.
PCR primer: 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 shown in SEQ ID NO:85, the size of the target product is 392bp; humanized antibody VL gene amplification primers: upstream of the first set: 247-33_f1, downstream primer: 247-33_r1 are respectively shown as SEQ ID NOs in the sequence table: 82 and SEQ ID NO:83, a step of detecting the position of the base; based on sequencing results, the second set of upstream primers: 247-VL-2_f2; a downstream primer: 247-vl_r1, respectively corresponding to SEQ ID NO:87 and SEQ ID NO:86.
8.2PCR amplification procedure
Respectively extracting plasmids, sequencing to confirm that the template sequences are correct, and respectively carrying out PCR (polymerase chain reaction) amplification on target fragments by using the primers and the templates, wherein the amplification schemes are as follows:
Primer template target fragment Length
8.3 preparation of transient expression vectors
Adding the light chain variable region gene or heavy chain variable region gene amplified in the step 7.2 into type II restriction enzymes NgoMIV and SnaBI respectively for double 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, followed by plasmid extraction and sequencing to obtain full-length eukaryotic expression vector clones 1-VH1 to 4 containing the humanized antibody VH gene and full-length eukaryotic expression vector clones 1-VL1 to 4 containing the humanized antibody VL gene, respectively. The nucleotide sequence corresponding to the heavy chain variable region of clone 4 is shown as SEQ ID NO. 102; the nucleotide sequence corresponding to the light chain variable region is shown as SEQ ID NO. 104.
9. Cell transient transfection and antibody expression purification
9.1 cell transient transfection of antibodies
The full-length eukaryotic expression vector clone 1-VH 1-4 of the VH gene obtained in (seventh) 7.3 and the full-length eukaryotic expression vector clone 1-VL 1-4 containing the VL gene of the humanized antibody were co-transfected into HEK293 cells (1.0X10 6 Each ml), the transfected cells were placed in a shaker at 37℃and placed in CO 2 Shake culturing in an incubator with concentration of 5%, and shaking table rotation speed of 120 rotation. Centrifuging and collecting transfected cell supernatant 7 days after transfection, separating and purifying the target full-length humanized antibody from the cell culture supernatant by using a ProteinA affinity chromatography column, and measuring the protein concentration of the expressed and purified antibodyAnd used for further purification of antibodies (antibodies were designated clone 1-4, respectively) for determination of affinity SPR.
9.2 SDS-PAGE analysis of antibodies
The samples were centrifuged by mixing NuPAGE LDS sample buffer, nuPAGE Reducing Agent with each sample, respectively, using a laboratory conventional SDS-PAGE analysis, in a 75℃water bath for 10 minutes. The loading was 2. Mu.g/well and the 200V voltage was run for 35 minutes. After electrophoresis was completed, the gel was taken and rinsed three times for 5 minutes each. Adding the dyeing liquid SimplyBlue Safestain for dyeing for one hour, and adding deionized water to decolor after finishing the dyeing, until the decoloring of the rubber background is complete; the observation results are shown in figure 1.
Wherein the left panel is a non-reducing electrophoretic sample of purified protein and the right panel is a reducing electrophoretic sample of purified protein. The six lanes in the left and right figures are, in order 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); m is a protein Marker.
10. Humanized antibody affinity assay
SPR assay of humanized antibody affinity: the first 4 purified humanized antibodies were assayed for affinity by SPR as described in (six) 6.1, which shows that the purified antibodies clone 1, clone 2, clone 3 and clone 4 have slightly lower affinities (1.9E-9,2.63E-9,1.61E-9 and 1.83E-9) than the parent chimeric antibody WBP247.CAb1 (3.94E-10), as shown in FIG. 2.
TABLE 4 humanized antibody SPR kinetic data
11. Specific screening and identification of humanized antibodies by immunohistochemical staining method
Although affinity measurement was performed on humanized antibodies by SPR method, since the antigen used was a purified antigen prepared by in vitro expression, the above antibodies were subjected to further immunohistochemical staining by tumor tissue specimens maintained in the laboratory in order to further observe the binding specificity of the obtained humanized antibodies.
And detecting the specific binding capacity of the expressed serial humanized antibody clones 1-4 and tumor tissues, and examining the immune tissue cross reaction of the antibody.
The specific operation is as follows: conventional xylene dewaxing, gradient alcohol dehydration and hydration tissue chip; 3%H 2 O 2 Blocking inactivation of endogenous peroxidases; sealing the normal sheep serum working solution; antibody clone 1-4 is used as a primary antibody, a biotin-marked rabbit anti-human Fc antibody is used as a secondary antibody, a horseradish peroxidase-marked streptomycin ovalbumin working solution is used as a tertiary antibody, DAB color development, hematoxylin counterstain, dehydration and transparency are carried out, and then the piece is sealed and subjected to microscopic examination. The result of the screening by the organization shows that only the antibody clone 4, namely WBP247.hAb4, can be specifically colored in malignant tumor tissues such as lung cancer, liver cancer and the like, and the degree of the coloring is "++" or "++ +"; while rarely binding to normal tissue. While the other three antibodies were negative. The above results show that only 1 available candidate humanized antibody WBP247.hAb4, which is similar to the murine parent antibody in terms of antigen recognition specificity, was screened by this round of expression, and the humanized antibody was successfully constructed, but the available number was very small and did not meet the quantitative requirements, thus requiring multiple rounds of expression and screening.
11. Repeated 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, the specificity of the humanized antibodies possibly changes, so that the strategy of the earlier stage is adjusted, and firstly, the 16 clones with the earlier affinity sequence of the SPR measurement fragment antibody ScFv antibody (the sixth) 2 are constructed and expressed by the full-length antibodies; the method is the same as the previous 7.1, and the vector construction and 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 nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 108. Clone 12, WBP247.HAb12, has the corresponding heavy chain variable region nucleotide sequence shown in SEQ ID NO. 110; the corresponding nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 112. The obtained antibodies were then purified according to the above-described (eight) 8.1 and cell transient transfection and antibody expression purification (antibodies were designated as clones 5 to 16, respectively), and SDS-PAGE analysis was performed according to the method of the above-described 8.2, and the results are shown in FIG. 3, FIG. 4 and FIG. 5.
12. ELISA analysis of humanized antibody affinity
200ng of recombinant CD147 protein is coated in an ELISA plate, and the ELISA plate is kept stand at 4 ℃ overnight; blocking was performed with 1XPBS/2% BSA for 1 hour at room temperature. Clones 1 to 6 of the purified antibody product of step (eight) and subsequent preparations were diluted in a gradient from 1ug/ml with a blocking solution of 1:3.16, 100. Mu.l each well was added, and allowed to stand at room temperature for 1 hour. 100 μl of 1:4000 dilution of horseradish peroxidase-labeled Goat anti-human IgG Fc HRP (Bethy Cat#A80-304P) was added, and the mixture was allowed to stand at room temperature for 1 hour. TMB was added for color development and the mixture was incubated with 2M H 2 SO 4 The reaction was terminated. Read with a microplate reader at 450 nm. ELISA results showed that the EC50 value of 16 humanized antibodies of screening clone 1-clone 16 total was about half that of chimeric antibody WBP247.CAb1, with approximately one-fold higher affinity than chimeric antibody. (see Table 5, FIG. 6).
TABLE 5 binding of antibodies to antigen CD147 EC 50 Measurement
Sample: EC 50 (ug/ml) EC 50 (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
13. 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 follows: conventional xylene dewaxing, gradient alcohol dehydration and hydration tissue chip; 3%H 2 O 2 Blocking inactivation of endogenous peroxidases; sealing the normal sheep serum working solution; antibody clone 5-16 is used as primary antibody,the biotin-marked rabbit anti-human Fc antibody is a secondary antibody, the horseradish peroxidase-marked streptomycin ovalbumin working solution is a tertiary antibody, DAB is developed, hematoxylin is counterstained, and the transparent rabbit anti-human Fc antibody is subjected to dehydration, sealing and microscopic examination. Positive judgment: positive with brown staining of cell membrane. Through this round of expression screening, two available candidate humanized antibodies WBP247.hAb6 and clone 12 were obtained again, wherein clone 12 was able to bind not only to liver cancer specimens, but also to lung cancer specimens. Specific information is shown in table 6 below:
TABLE 6 specific screening of humanized antibodies by immunohistochemical staining method
Three humanized monoclonal antibodies, namely clone 4 (WBP 247.hAb4), clone 6 (WBP 247.hAb6) and clone 12 (WBP 247.hAb12) were initially screened for downstream stable cell line selection by multiple histochemical staining.
14. Construction of humanized antibody high-efficiency expression vector and screening of stable expression cell strain
According to the results of the ELISA and the immunity, three clones with the best affinity and specificity, namely clone 4, clone 6 and clone 12, namely the gene sequences of WBP247.hAb4, WBP247.hAb6 and WBP247.hAb12 are selected to construct a high-efficiency expression vector and screen stable cell strains.
TABLE 7WBP247.hAb4, WBP247.hAb6 and WBP247.hAb12 sequence combination information
(wherein the clone 12 light chain variable region has the amino acid sequence shown as SEQ ID NO:113, the corresponding light chain variable region (VL) nucleotide sequence shown as SEQ ID NO:112, the heavy chain variable region has the amino acid sequence shown as SEQ ID NO:111, and the corresponding heavy chain variable region (VH) nucleotide sequence shown as SEQ ID NO: 110).
14.1 construction of the light chain expression vector pWX2.1-LC-B-247B4
In vitro gene DNA (27989-VL) fragment amplification is carried out, the DNA is used as a template, 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) are used for amplifying variable region fragments WBP247B4-VL, ecoRI restriction enzyme sites are introduced at the 5 'end of WBP247B4-VL, bsiWI restriction enzyme sites are introduced at the 3' end, ecoRI and BsiWI double enzyme digestion are carried out on the fragments and an expression vector pWX2.1 containing a light chain constant region gene, and 5119bp DNA fragments are recovered by gel cutting after 1% agarose gel electrophoresis. The DNA fragment after gel cutting and purification was subjected to ligation reaction with the fragment from plasmid pWX2.1, and the reaction was carried out at 16℃for 20min with 20. Mu.l of T4 ligase system, and 10. Mu.l of ligation solution was used to transform E.coli TOP10 competent cells. After colony PCR identification, restriction enzyme identification and sequencing, a correct single clone was selected and cultured in 200ml LB medium at 37℃overnight with shaking at 220rpm, and the plasmid was extracted in large quantities, and the resulting plasmid was designated pWX2.1-LC-B-247B4 (5521 bp) (see FIG. 8).
14.2 construction of the light chain expression vector pWX2.1-LC-B-247B12
In vitro gene DNA (32270-VL) fragment amplification is carried out, the DNA is used as a template, 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) are used for amplifying variable region fragments WBP247B12-VL, ecoRI restriction enzyme cutting sites are introduced at the 5 'end of WBP247B12-VL, bsiWI restriction enzyme cutting sites are introduced at the 3' end, ecoRI and BsiWI double enzyme cutting is carried out on the fragments and an expression vector pWX2.1 containing light chain constant region genes, and 5119bp DNA fragments are recovered by gel cutting after 1% agarose gel electrophoresis. The DNA fragment after gel cutting purification was subjected to ligation reaction with the fragment derived from plasmid pWX2.1 to transform E.coli TOP10 competent cells. After colony PCR identification, restriction enzyme identification and sequencing, a correct single clone was selected and cultured in 200ml LB medium at 37℃overnight with shaking at 220rpm, and the plasmid was extracted in large quantities, and the resulting plasmid was designated pWX2.1-LC-B-247B12 (5521 bp) (see FIG. 9).
14.3 construction of the heavy chain expression vector pWX1.1-HC-Z-247B4
In vitro gene DNA (27989-VH) fragment amplification is carried out, the DNA is taken as a template, 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) are used for amplifying variable region fragment WBP247B4-VH, ecoRI restriction enzyme sites are introduced at the 5 'end of WBP247B4-VH, nheI restriction enzyme sites are introduced at the 3' end, ecoRI and NheI double enzyme digestion are carried out on the fragment and an expression vector pWX1.1 containing heavy chain constant region genes, and a 5202bp DNA fragment is recovered after agarose gel electrophoresis. The DNA fragment after gel cutting purification was subjected to ligation reaction with the fragment derived from plasmid pWX1.1 to transform E.coli TOP10 competent cells. After colony PCR identification, restriction enzyme identification and sequencing, a correct single clone was selected and cultured in 200ml LB medium at 37℃overnight with shaking at 220rpm, and the plasmid was extracted in large quantities, and the resulting plasmid was designated pWX1.1-HC-Z-247B4 (5625 bp) (see FIG. 10).
Construction of the 4-fold chain expression vector pWX1.1-HC-Z-247B6
In vitro gene DNA (32338-VH) fragment amplification is carried out, the DNA is taken as a template, 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) are used for amplifying variable region fragments WBP247B6-VH, ecoRI restriction enzyme sites are introduced at the 5 'end of WBP247B6-VH, nheI restriction enzyme sites are introduced at the 3' end, ecoRI and NheI double enzyme digestion are carried out on the fragments and an expression vector pWX1.1 containing heavy chain constant region genes, and 5202bp DNA fragments are recovered after agarose gel electrophoresis. The DNA fragment after gel cutting purification was subjected to ligation reaction with the fragment derived from plasmid pWX1.1 to transform E.coli TOP10 competent cells. After colony PCR identification, restriction enzyme identification and sequencing, a correct single clone was selected and cultured in 200ml LB medium at 37℃overnight with shaking at 220rpm, and the plasmid was extracted in large quantities, and the resulting plasmid was designated pWX1.1-HC-Z-247B6 (5625 bp) (see FIG. 11).
14.5 construction and screening of CHO Stable expression cell lines
After vector construction, the humanized antibody light chain expression vector and the humanized antibody heavy chain expression vector are carried out by the delegated drug Ming Kangde 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-247B4; (3) WX2.1-LC-B-247B12 and pWX1.1-HC-Z-247B4 are co-transfected into CHO/DHFR cells, medium with proper concentration is added for screening and passage, and monoclonal is selected to obtain CHO stable expression cell strain.
Thereafter, a miniport batch feed experiment was performed to measure titers. The four-wheel batch feed optimization is carried out, and cell strains 247C-B4Z2-01-C-005 (CCTCC NO. C2019147), 247A-B9Z4-02-C-T9 (CCTCC NO. C2019148) and 247B-B9Z4-01-C-T9 (CCTCC NO. C2019149) which respectively express the WBP247.hAb12 antibody stably (named as mehozumab monoclonal antibody) are obtained through screening, and the cell strains are preserved in the China center for type culture collection of Wuhan at present, and the preservation date is 2019, 7 months and 16 days.
15. In vitro killing experiment of tumor cells of humanized anti-CD 147 antibody
The method comprises the following steps: taking human lung cancer cell NCI-H520 in logarithmic phase, digesting with pancreatin to obtain single cell suspension, and adjusting concentration to 1×10 4 And each ml. 2000 cells (200. Mu.l cell suspension) were seeded into 96-well plates, 5% CO 2 Incubate overnight at 37 ℃, wait for cells to adhere and resume log-proliferated state.
Marking three humanized antibodies with DM1 according to the conventional ADC pharmaceutical laboratory preparation method to obtain WBP247.hAb12-DM1, WBP247.hAb4-DM1 and WBP247.hAb6-DM1. The following gradients were diluted with serum-free RPMI1640 medium, respectively: 1000. 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 μg/mL; mu.l of each concentration was added to the corresponding well, and 6 multiplex wells were designed for each concentration. Negative antibody control (human IgG) and background (cell-free medium only) were designed at the same time. 5% CO 2 Incubation was carried out at 37℃for 48h, and 10. Mu.L of CCK-8 chromogenic solution was added to each well. Incubate at 37℃for 2h, and read absorbance at 450nm with a full wavelength microplate reader. The results are shown in FIG. 13, and the humanized antibodies have good killing effect on tumor cells (FIG. 13), indicating that the prepared humanized antibodies have good killing effectGood tumor specificity and targeting. From this effect, one skilled in the art can infer that the antibody of the present invention can be used for preparing a medicine for diagnosing and treating (a medicine for diagnosing CD147 expression positive diseases and biotechnological products, detection reagents, imaging and examination diagnostic reagents thereof).

Claims (8)

1. A monoclonal antibody against human CD147, the heavy chain variable region and the light chain variable region of which have the amino acid sequences:
wbp247.hab6 antibody: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 107, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 109.
2. A gene encoding the anti-human CD147 monoclonal antibody of claim 1, wherein the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the anti-human CD147 monoclonal antibody are:
wbp247.hab6 antibody: the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 106; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 108.
3. A cell line expressing the monoclonal antibody of claim 1, the cell line being designated and deposited with the accession number:
cell line expressing wbp247.hab6 antibody: designated 247B-B9Z4-01-C-T9, accession number: cctcno. c2019149.
4. An antibody expressed by the cell line of claim 3.
5. An expression vector for the monoclonal antibody of claim 1.
6. The method of producing an antibody of claim 1, comprising:
a) Obtaining the DNA molecule sequence of the antibody of claim 1;
b) Constructing an expression vector containing the DNA molecule described in step a) and a regulatory sequence for expressing the DNA molecule;
c) Transfecting a host cell with the expression vector described in step b); culturing under culture conditions suitable for the host cell;
d) The monoclonal antibody of claim 1 is obtained through a separation and purification step.
7. Use of an antibody conjugate of the antibody of claim 1 coupled to maytansine derivative DM1 of the antibody of claim 1, a tubulin polymerase inhibitor MMAE of the antibody of claim 1 or a radionuclide iodine of the antibody of claim 1 for the preparation of a medicament for treating liver cancer or lung cancer.
8. Use of the antibody of claim 1 for the preparation of a medicament for diagnosing a CD147 expression positive disease, said CD147 expression positive disease being liver cancer or lung cancer.
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