CN113549152B - anti-BASIGIN humanized antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-BASIGN humanized antibody and application thereof, wherein the antibody comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, and the immunoglobulin light chain variable region comprises a CDR1: as shown in SEQ ID NO. 13 of the sequence table; CDR2: as shown in SEQ ID NO. 14 of the sequence table; CDR3: as shown in SEQ ID NO. 15 of the sequence table; the immunoglobulin heavy chain variable region comprises CDR1: as shown in SEQ ID NO. 16 of the sequence table; CDR2: as shown in SEQ ID NO. 17 of the sequence table; CDR3: as shown in SEQ ID NO. 18 of the sequence Listing. The application of the anti-BASIGIN humanized antibody in preparing medicaments for treating diseases related to CD147 expression.

Description

anti-BASIGIN humanized antibody and application thereof

Technical Field

The invention relates to the field of biotechnology, and in particular provides an anti-BASIGN humanized antibody and application thereof.

Background

BASIGN (EMMPRIN, neurothelin, M6 antigen, BASIGIN) is a highly glycosylated, transmembrane glycoprotein with a molecular weight of 50-60 kD, a member of the immunoglobulin superfamily (IgSF). In humans, BASIGIN has a total 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. BASIGIN has been shown 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 the basign molecule is an important functional membrane protein in the tumor development process, and is involved in various cancer-related phenomena, such as:

(1) BASIGIN mediates tumor cell adhesion and movement: BASIGAN expressed on the surface of tumor cells interacts with vinclin to promote the formation, spreading and adhesion of tumor cell pseudopodia; interacting with annexin II, promoting secretion of tumor cell MMP-2, and enhancing movement ability and invasion potential of tumor cell; by stimulating the tumor cells and surrounding fibroblasts, various matrix metalloproteinases (extracellular matrix metalloproteinase, MMP) including MMP-1, MMP-2, MMP-3, MMP-11, MT1-MMP, MT2-MMP, etc. are secreted, so that the degradation of the matrix around the tumor is accelerated, and the metastasis of the tumor is promoted.

(2) BASIGIN is involved in anaerobic metabolism of tumor cells: BASIGN is an important chaperone for the monocarboxylic acid transporter (monocarboxylate transporter) MCT-1 and MCT-4. BASIGN can bind to MCT-1 and MCT-4, assist in their proper localization on cell membranes, and continue to regulate their transport function for lactate metabolites. Since tumor cells rely primarily on anaerobic metabolism to produce energy, BASIGIN can regulate tumor cell energy metabolism indirectly by affecting MCT expression and function.

(3) BASIGIN promotes tumor cell resistance: the BASIGN molecule influences the transcription of MDR gene through a coexpression regulation mechanism and promotes the expression of P-g, so that the multi-drug resistance of tumors can be induced. Kanekura et al demonstrate that BASIGN leads to MDR via P-g, so down-regulating BASIGN expression may be a target for efficient resistance to MDR. In addition, BASIGAN promotes the nuclear localization of p-TFII-I, up-regulates the expression of Bip, a key factor of Unfolded Protein Response (UPR), and causes the stress and UPR of the endoplasmic reticulum of tumor cells, so that apoptosis and insensitivity to drugs are inhibited, and BASIGAN molecules are inhibited to promote apoptosis of liver cancer cells and enhance the sensitivity of tumor cells to the existing antitumor drugs.

(4) The BASIGN can up regulate VEGF expression, promote tumor angiogenesis, inhibit BASIGN expression, and remarkably inhibit VEGF secretion and tumor angiogenesis.

(5) BASIGIN binds to a variety of molecules involved in interactions: the existence of a highly evolutionarily conserved negative charge of glutamate in the transmembrane segment of the BASIGIN protein is a structural basis for interaction with a variety of cell membrane proteins, and also suggests that BASIGIN can influence a variety of physiological activities of cells through interaction with a variety of proteins. BASIGIN can affect tumor cell growth and metastasis processes from multiple aspects of energy metabolism, cell-matrix interactions, signaling, etc. through interactions with various proteins such as CD98, integrin, caveolin-1, cyclophilins (CyP), etc.

Furthermore, several retrospective studies have shown that there is a close correlation between the expression intensity of basignin molecules in tumor tissue and prognosis in tumor patients. In non-small cell lung cancer patients, elevated levels of BASIGN expression are closely related to the prognosis of the patient.

Therefore, BASIGN molecule has become a new target for tumor treatment, wherein the development of antibody drug Li Kating proves the safety and effectiveness of the target patent medicine.

In addition, the application of the CD147 specific monoclonal antibody 5A12 can inhibit the proliferation level of Tm cells, and can simultaneously down regulate the expression of the early activation marker CD69 molecules and the late activation marker CD25 molecules on the surfaces of the Tm cells; at the same time, the secretion of cytokine IL-2 is reduced, which suggests that CD147 molecules play an important role in the activation of Tm cells. The research basis of the CD147-Tm/Osteoclast-RA chain is likely to provide a new theoretical basis for the treatment of RA by the humanized 5A12 monoclonal antibody in the future.

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 utilizing an in-vivo autoimmune system to exert curative effects 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. Therefore, humanized antibodies and humanized antibodies are becoming a mainstream trend of antibody drug development with their unique advantages.

Based on the research background, the invention screens novel anti-BASIGIN humanized antibody with high affinity through phage antibody library, and the biological activity of the novel anti-BASIGIN humanized antibody is verified through experiments.

Disclosure of Invention

In view of the shortcomings and drawbacks of the prior art, an object of the present invention is to provide an anti-BASIGN humanized antibody and uses thereof.

An anti-human BASIGIN humanized antibody comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region;

the immunoglobulin light chain variable region comprises CDR1: as shown in SEQ ID NO. 13 of the sequence table; CDR2: as shown in SEQ ID NO. 14 of the sequence table; CDR3: as shown in SEQ ID NO. 15 of the sequence table;

the immunoglobulin heavy chain variable region comprises CDR1: as shown in SEQ ID NO. 16 of the sequence table; CDR2: as shown in SEQ ID NO. 17 of the sequence table; CDR3: as shown in SEQ ID NO. 18 of the sequence Listing.

An anti-human BASIGN humanized antibody has the amino acid sequence of a light chain variable region shown as SEQ ID NO. 1 and the amino acid sequence of a heavy chain variable region shown as SEQ ID NO. 3.

An anti-human BASIGN humanized antibody has the amino acid sequence of a light chain variable region shown in SEQ ID NO. 5 and the amino acid sequence of a heavy chain variable region shown in SEQ ID NO. 7.

An anti-human BASIGN humanized antibody has the amino acid sequence of a light chain variable region shown in SEQ ID NO. 9 and the amino acid sequence of a heavy chain variable region shown in SEQ ID NO. 11.

The humanized antibody of the anti-human BASIGIN has a light chain constant region of kappa or lambda type and a heavy chain constant region of IgG2, igG1, igG3, igG4, igM, igA1, igA2, igD or IgE.

An antigen binding fragment comprising an anti-human basign humanized antibody of the invention and Fab, fv, scFv or single chain antibody.

The application of the humanized antibody of the anti-human BASIGIN in preparing the medicine for treating the CD147 expression related diseases.

Alternatively, the CD147 expression-related disease is selected from lung cancer, liver cancer, colon cancer and rheumatoid arthritis.

The anti-human BASIGIN humanized antibody provided by the invention recognizes and binds to CD147 molecules, and the experimental result of in-vitro inhibition of inflammation by the anti-human BASIGIN humanized antibody suggests the application of the antibody in the aspect of treating inflammation.

A gene expression vector selected from pcdna3.3-LC-104new-M and ploftivec-HC-104 new-M; the carrier is embedded with the nucleotide sequence of any anti-human BASIGIN humanized antibody.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a schematic illustration of selected replacement amino acids in VH frame work;

FIG. 2 is a view of selected replacement amino acids in VL frame work;

FIG. 3 is an SDS-PAGE analysis of humanized and chimeric antibodies;

FIG. 4 is an ELISA analysis of chimeric and humanized antibodies;

FIG. 5 is a schematic diagram of the structure of the light chain gene expression vector pcDNA3.3-LC-N-203;

FIG. 6 is a schematic diagram of the structure of the heavy chain gene expression vector pOptivec-HC-D-203;

FIG. 7 shows the results of immunofluorescent staining of humanized antibody cells;

FIG. 8 is an in vitro inhibition of CD4 by humanized antibodies ⁺ CD45RO ⁺ T cell proliferation activation assay results.

Detailed Description

The invention is described in detail below with reference to the drawings and the detailed description.

1. Term interpretation:

immunoglobulins refer 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, numbering of amino acid residues in this region can be performed by the methods described in Kabat et al SequencesofProteinsofImmunologicalInterest,5thEd,PublicHealthService,NationalInstitutesofHealth,Bethesda,MD (1991) (phrase variable domain residue numbering, as in or according to Kabat, herein referring to such numbering system for heavy chain variable domains or light chain variable domains).

"humanized antibody", as used herein, refers to an antibody derived from a non-human antibody, typically a murine antibody, which retains or substantially retains the antigen binding properties of the parent antibody (parent antibody) but which is less immune 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), as used herein, refer to a variety of amino acid sequences that collectively define the binding affinity and specificity of the variable fragment (Fv) region of an immunoglobulin binding site.

Framework Regions (FR), as used herein, refer to amino acid sequences inserted between 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), as used herein, refers to the portion of an antibody molecule that confers effector function. The constant regions of the subject humanized antibodies 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 skilled artisan will appreciate that the variable or constant regions of an immunoglobulin heavy or light chain can be linked 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 linked by chemical synthesis using peptides.

The humanized antibody of the present invention retains an important part of the binding property of a parent antibody, namely, monoclonal antibody 5A12, which is a murine antibody called an anti-human BASIGIN molecule, produced by hybridoma cell line HAb18Gedomab1, which is deposited with China center for type culture collection (CCTCC-C200408, see patent number ZL200410078858.6 for details) at 12/13 of 2006. In particular, the anti-human BASIGIN humanized antibodies of the present invention retain the ability to specifically bind to the antigen recognized by the parent antibody used to produce such humanized antibodies. Preferably, the humanized antibody exhibits the same or substantially the same antibody binding affinity (avidity) and avidity (avidity) as the parent antibody. Ideally, the affinity (KD) of the antibody will be no more than 10 times the affinity of the parent antibody.

The skilled artisan will appreciate that "avidity" refers to the total strength of interaction between two molecules, e.g., between an antibody and an antigen. The affinity depends on the affinity and valence of the interaction. Also, "affinity" refers to the strength of binding between a single binding site of a molecule (e.g., an antibody) and a ligand (e.g., an 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.

"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 some ability to selectively bind to human antigens, examples of which include, but are not limited to, the following:

(1) Fab, fragments comprising monovalent antigen binding fragments of an antibody molecule which can be degraded by the enzyme papain to form complete light chains and a portion of one heavy chain;

(2) Fab', can be obtained by treating whole antibodies with pepsin followed by reduction to generate intact light and heavy chain portions; two Fab' fragments are available per antibody molecule;

(3) (Fab') 2, obtainable by 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;

2. the specific scheme of the invention is as follows:

in a first aspect, the present invention provides three anti-human BASIGIN humanized antibodies, designated monoclonal antibody HP5A12-1, monoclonal antibody HP5A12-2, and monoclonal antibody HP5A12-3, respectively, each comprising an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region;

(1) The immunoglobulin light chain variable region comprises CDR1: as shown in SEQ ID NO. 13 of the sequence table; CDR2: as shown in SEQ ID NO. 14 of the sequence table; CDR3: as shown in SEQ ID NO. 15 of the sequence table;

(2) The immunoglobulin heavy chain variable region comprises CDR1: as shown in SEQ ID NO. 16 of the sequence table; CDR2: as shown in SEQ ID NO. 17 of the sequence table; CDR3: as shown in SEQ ID NO. 18 of the sequence table;

In a preferred embodiment, the monoclonal antibody HP5A12-1 comprises the light chain variable region amino acid sequence shown as SEQ ID NO. 1 and the heavy chain amino acid sequence shown as SEQ ID NO. 3; affinity kd=5.96×10 of the antibody ^-10 M。

In a preferred embodiment, the monoclonal antibody HP5A12-2 comprises the light chain variable region amino acid sequence shown as SEQ ID NO. 5 and the heavy chain amino acid sequence shown as SEQ ID NO. 7; affinity kd=7.19×10 of the antibody ^-10 M。

In a preferred embodiment, monoclonal antibody HP5A12-3 comprises the light chain variable region amino acid sequence shown as SEQ ID NO. 9 and the heavy chain amino acid sequence shown as SEQ ID NO. 11; affinity kd=6.74×10 of the antibody ^-10 M。

It is another object of the present invention to provide a DNA molecule encoding the above recombinant humanized antibody.

In a preferred embodiment, the monoclonal antibody HP5A12-1 has the nucleotide sequence of the light chain variable region shown in SEQ ID NO. 2, and the nucleotide sequence of the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO. 4;

in a preferred embodiment, the monoclonal antibody HP5A12-2 has the nucleotide sequence of the light chain variable region shown in SEQ ID NO. 6, and the nucleotide sequence of the heavy chain variable region shown in SEQ ID NO. 8;

in a preferred embodiment, the monoclonal antibody HP5A12-3 has the nucleotide sequence of the light chain variable region shown in SEQ ID NO. 10, and the nucleotide sequence of the heavy chain variable region shown in SEQ ID NO. 12.

In a preferred embodiment, the light chain constant region of the above antibody is kappa and the heavy chain constant region is IgG2, however the invention also protects the isotypes of other antibodies, such as IgG1, igG3, igG4, igM, igA1, igA2, igD, igE. The invention also protects antigen binding fragments of antibodies, including Fab, fv, scFv, and single chain antibodies.

It is still another object of the present invention to provide a method for preparing the anti-BASIGIN monoclonal antibody, which comprises:

a) Providing a DNA molecule of said anti-BASIGIN monoclonal antibody;

b) Providing an expression vector comprising the DNA molecule described in step a) and regulatory sequences for expressing the DNA molecule;

c) Transforming a host cell, in particular a mammalian cell, preferably a CHO cell, with the expression vector described in step a);

d) Culturing the host cell obtained in step c) under culture conditions suitable for said monoclonal antibody and isolating and purifying said monoclonal antibody expressed in step c).

Another object of the present invention is the use of humanized anti-human BASIGN antibodies in the manufacture of a medicament for the treatment of diseases associated with BASIGN expression, including lung cancer, liver cancer, colon cancer and rheumatoid arthritis.

In order to overcome the defect that repeated injection of a murine McAb into a human body can cause a patient to induce a human anti-mouse antibody (human anti mouse antibody, HAMA) to react, systemic anaphylactic toxicity reaction occurs and the exertion of antibody efficacy is blocked, the invention adopts phage display antibody library technology, computer-aided antibody structure design and other technologies, takes hybridoma cell strain HAb18Gedomab1 (preservation number CCTCC-C200408, detailed description in patent number ZL 200410078858.6) of a murine antibody 5A12 secreting anti-human BASIGAN molecules as a basis, defines the CDR and FR regions of a light-heavy chain variable region of the antibody through bioinformatics, designs a framework region of a humanized antibody by utilizing a computer-aided antibody structure design means, and adopts a molecular biological means to carry out gene construction of full molecules, thereby eukaryotic expressing the anti-human BASIGAN humanized antibody. The antibody affinity and immunohistochemical analysis result shows that the anti-human BASIGIN humanized antibody has better affinity than the murine parent antibody, and the specificity of the parent antibody for recognizing antigen is maintained. The results of in vitro inhibition of inflammation experiments with anti-human BASIGIN humanized antibodies suggest the use of the antibodies in the treatment of inflammation.

The present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are set forth merely for purposes of illustration and are not intended to limit the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

1. Construction of phage display vector containing murine antibody 5A12 light and heavy chain variable region of anti-human BASIGIN molecule

PCR amplified VH gene. The VH fragment of the 5A12 antibody synthesized by the genes is used as a template, and the VH gene fragment 5A12VH1 is obtained by amplification of a VH gene primer.

The amplification system is as follows: 1pmol of the VH fragment of the genetically synthesized 5A12 antibody, 100pmol of pFL-5A12-F1 (SEQ ID NO: 19) 100pmol,Linker R1 (SEQ ID NO: 20) and 10. Mu.L of 10 XPCR buffer. 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.

PCR amplified VL gene. And (3) using the VL fragment of the genetically synthesized 5A12 antibody as a template, and respectively amplifying by using VL gene primers to obtain a VL gene fragment 5A12VL1.

The amplification system is as follows: gene 5A12 antibody VL 1pmol, linker F1 (SEQ ID NO: 21) 100pmol, pFL-5A12-R2 (SEQ ID NO: 22) 100pmol,10 XPCR buffer 10. Mu.L. 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.

Amplification of scFv Gene: the VH gene fragment 5A12VH1 and the VL gene fragment 5A12VL1 obtained in the steps 1 and 2 were mixed in the same molar number, and the scFv gene was amplified by overlap-PCR.

The amplification system is as follows: VH gene fragment 5A12VH 1pmol, VL gene fragment 5A12VL1 1pmol,pFL-5A12-F1 (SEQ ID NO: 21) 100pmol, pFL-5A12-R2 (SEQ ID NO: 22) 100pmol, 10. Mu.L of 10 XPCR buffer. PCR reaction conditions: 95 ℃ for 5min;95 ℃,15s,56 ℃,30s,72 ℃,1min,35 cycles; 72℃for 10min. The size of the amplified fragment was observed by 1% agarose gel electrophoresis. The target band was excised, and scFv gene fragments containing cleavage sites Nco I and Not I were recovered using a DNA fragment purification kit (Omega bio-tek).

scFv gene cleavage and ligation to vector and ligation product conversion: the scFv gene fragment containing the cleavage sites Nco I and Not I prepared above and the vector plasmid pGEM-T vector (Promega) were subjected to cleavage after measuring the nucleic acid concentration with an ultraviolet spectrophotometer. The enzyme digestion method comprises the following steps: 3. Mu.g of scFv recovered product or pGEM-T vector was taken, 1. Mu.L each of Nco I-HF and Not I-HF restriction enzyme (NEB) was added, 5. Mu.L of 10 XCutSmart buffer was added, water was added to 50. Mu.L, and the mixture was digested at 37℃for 1 hour. The target band was cut out by 1% agarose gel electrophoresis, and the gel was recovered.

The digested scFv was taken and 0.7. Mu.g of the recovered product, 1.4. Mu.g of the digested vector pGEM-T, 40U of T4 ligase, 40. Mu.L of 5 Xligation buffer, and ligated overnight at 16 ℃. The ligation product was used to transform TG1 competent cells. Plated on LB agar plates and incubated overnight at 37 ℃. The next day, 10 monoclonals are randomly picked, and positive clone colony identification is carried out by utilizing a vector universal primer; 5 positive clones are selected, sequencing verification is carried out, and clones with correct sequencing are stored at the temperature of minus 40 ℃ for standby, so that the scFv gene expression vector pFL-5A12 containing the complete 5A12 light and heavy chain variable region genes is obtained.

2. Defining CDR and framework residues of murine anti-BASIGIN monoclonal antibody 5A12

The nucleotide sequence encoding the light and heavy chain variable region of the murine antibody 5A12 (patent number ZL 200410078858.6) against BASIGIN was translated into the amino acid sequence encoded by the same using www.expasy.org on-line software.

Determining the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 in the light chain variable region sequence according to the Kabat database as shown in SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO: 15. The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 in the heavy chain variable region sequence are shown in SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO: shown at 18.

3. Selection of appropriate human antibody framework sequences

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. And splicing the human antibody variable region framework in the human antibody sequence information database with the CDR, and screening a reasonable antibody variable region.

The result is a VH frame work as shown in fig. 1 and a vlframe work as shown in fig. 2.

4. Construction and screening of phage display BASIGIN humanized antibody library

The antibody sequence obtained according to the third step and the antibody amino acid sequence obtained by computer simulation comprise the light chain variable region amino acid sequences SEQ ID NO. 1, SEQ ID NO. 5 and SEQ ID NO. 9, and the heavy chain variable region amino acid sequences SEQ ID NO. 3, SEQ ID NO. 7 and SEQ ID NO. 11. The codons commonly used in mammalian cells are selected for reverse translation to obtain the corresponding antibody gene nucleotide sequences. Designing corresponding primers by using a gene total synthesis PCR technology, and obtaining a full-length gene by an overlapping PCR method; cloning into cloning vector and sequencing.

1. Material

Humanized VH gene amplification primers:

an upstream primer: 5A12-L1_F1, see SEQ ID NO 23 in the sequence Listing; 5A12-L1_F2, see SEQ ID NO 25 in the sequence Listing; 5A12-L1_F3, see SEQ ID NO 27 in the sequence Listing;

a downstream primer: 5A12-L1_R1, see SEQ ID NO 24 in the sequence Listing; 5A12-L1_R3, see SEQ ID NO 26 in the sequence Listing; 5A12-L1_R4, which is shown in SEQ ID NO 28 in the sequence table; 5A12-L1_R2, see SEQ ID NO 33 in the sequence Listing; 5A12-L1_R5, see SEQ ID NO 34 in the sequence Listing;

Humanized VL gene amplification primers:

an upstream primer: 5A12-L1_F4, see SEQ ID NO 29 in the sequence Listing; 5A12-L1_F6, see SEQ ID NO. 31 in the sequence Listing; 5A12-L1_F5, see SEQ ID NO 36 in the sequence Listing;

a downstream primer: 5A12-L1_R6, see SEQ ID NO 30 in the sequence Listing; 5A12-L1_R8, see SEQ ID NO. 32 in the sequence Listing; 5A12-L1_R7, see SEQ ID NO 35 in the sequence Listing;

2. PCR amplification of the target fragment was performed using the above primers:

the amplification protocol was as follows:

2.1, pFL-5A12 is used as a template, and 5A12-L1_F1+5A12-L1_R1, 5A12-L1_F2+5A12-L1_R3, 5A12-L1_F3+5A12-L1_R4, 5A12-L1_F4+5A12-L1_R6 and 5A12-L1_F6+5A12-L1_R8 are respectively used as primer pairs to amplify target genes; the reaction condition is 95 ℃ for 3min;95 ℃ for 30sec;55 ℃,30sec;72 ℃,40sec;40 cycles, final 72℃extension, 10min. After the completion of the PCR reaction, the PCR product was purified and recovered by 1% agarose gel electrophoresis and ligated into pMD18-T vector (TaKaRa). Fragments with correct sequencing were designated as 5A12-L1-1 fragment, 5A12-L1-2 fragment, 5A12-L1-3 fragment, 5A12-L1-4 fragment and 5A12-L1-5 fragment, respectively.

2.2 amplification of the target gene with the 5A12-L1-1 fragment, 5A12-L1-2 fragment, 5A12-L1-3 fragment, 5A12-L1-4 fragment and 5A12-L1-5 fragment obtained in 2.1 as templates and with 5A12-L1_F1+5A12-L1_R2, 5A12-L1_F2+5A12-L1_R5, 5A12-L1_F4+5A12-L1_R7, 5A12-L1_F5+5A12-L1_R8 as primers, respectively; the reaction conditions were the same as 2.1. After the PCR reaction is finished, the PCR product is purified and recovered by using 1% agarose gel electrophoresis and is connected with a pMD18-T vector (TaKaRa), positive clone sequencing is screened and verified, and fragments with correct sequencing are respectively named as 5A12-L1-6 fragments, 5A12-L1-9 fragments, 5A12-L1-7 fragments and 5A12-L1-8 fragments.

2.3. Amplifying target genes by using the 5A12-L1-6 fragment and the 5A12-L1-9 fragment obtained in 2.2 as templates and using 5A12-L1_F1+5A12-L1_R5 as primers, wherein the reaction conditions are the same as 2.1, and the sequencing result is named as 5A12-L1-10 fragment; amplifying target genes by using 5A12-L1-7 fragments and 5A12-L1-8 fragments as templates and 5A12-L1_F4+5A12-L1_R8 as primers under the same reaction conditions as 2.1, wherein the sequencing result is named as 5A12-L1-11 fragments; the reaction conditions were the same as 2.1.

2.4. Amplifying target genes by using the 5A12-L1-10 fragment and the 5A12-L1-11 fragment obtained in 2.3 as templates and using 5A12-L1_F1+5A12-L1_R8 as primers, wherein the sequencing result is named as 5A12-L1-12 fragment; the reaction conditions were the same as 2.1.

2.5. The PCR-amplified 5A12-L1-12 fragment was digested with NcoI-HF and NotI-HF (NEB), separated by 1% agarose electrophoresis, and the digested fragments were purified by Gel Extraction Kit (Omega bio-tek). Then, the purified fragment was ligated with the co-digested phage vector pComb3Xss using T4DNA ligase (TaKaRa), and the TG1 competent cells were electrically transformed after deionization. Cloning and screening are carried out by inoculating on LB culture plates. Counting the library capacity of the 5A12-L1 antibody phage library, and preserving the library at-80 ℃ for standby.

2.6 Panning of a 5A12-L1 phage display antibody library

Antigen-specific panning was performed on the 5A12-L1 antibody phage library using a solid phase panning method. The specific operation is as follows:

resuscitating the 5a12-L1 antibody phage library in 60mL of 2YT medium and incubating in a shaker at 37 ℃ until od600=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 50. Mu.g/mL kanamycin (no glucose) medium after centrifugation at 1500 rpm for 10 min, and incubated overnight in a 30℃shaker; 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 turns for 25 minutes, and discarding the supernatant; phage were resuspended in 2.2ml of solution containing PBS-5% BSA, centrifuged, 12000 rpm, and cell debris removed for 5 minutes.

The affinity screening library was performed with expressed BASIGN molecular plates, and the antigen coating concentration was successively decreased (1. Mu.g/mL, 0.1. Mu.g/mL, 0.01. Mu.g/mL, 0.001. Mu.g/mL, 0.0001. Mu.g/mL) for each round of panning through 4 rounds of panning (adsorption-elution-amplification). Panning was performed until S/N was less than 10 (S/N expressed as signal to noise ratio), the panning experiment was terminated, 864 clones were picked, expression was induced, and human scFv antibodies were obtained for Elisa detection.

5. ELISA analysis, humanized sequence analysis and affinity analysis

With coating buffer (200 mM Na ₂ CO ₃ /NaHCO ₃ pH 9.2) BASIGN was diluted to 1. Mu.g/ml, 50. Mu.L was added to each reaction well and coated overnight at 4 ℃; the solution in the reaction well is discarded, washed 3 times with 1XPBS buffer, and blocked for 1h at room temperature by adding 200. Mu.L of blocking buffer (2% BSA/1XPBS buffer); wash 3 times with 200 μl 1XPBS buffer; cell culture supernatants containing scFV antibodies (8 96-well microplates) and negative controls (50. Mu.L/well) were added and incubated for 2h at room temperature; wash 3 times with 200 μl 1X PBS buffer; anti-c-Myc Ab (HRP) (Abcam Cat#ab19312, 50. Mu.L/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 mu L/hole) is added into each reaction hole for reaction for 10min; stop solution (2M HCl, 50. Mu.L/well) was added to stop the reaction; reading the light absorption value of 450nm by using an ELISA plate reader;

258 clones with A450>2.0 were selected for sequencing analysis based on ELISA results, and sequence information was determined (sequencing work was done by Shanghai Boshang Biotechnology Co., ltd.).

DNA sequence analysis: the degree of humanization of the sequences was assessed with reference to the human antibody's germline database and http:// www.bioinf.org.uk/abs/shab. And antibody affinity was determined using the SPR method.

Affinity assay: the affinity between the human scFv antibody and the antigen CD147 molecule was determined using the SPR method. GLM chips were first activated with activator for 60s. The activator was mixed with 0.01M sulfo-NHS (Bio-Rad) using 0.04M EDC. Protein A/G was diluted with 10mM NaAc (pH 4.0) and injected onto the chip at a rate of 30ul/min to couple protein A/G to the activated chip via amino groups. The 6 parallel channels were simultaneously coupled to protein A/G. Finally, the chip was inactivated with 1Methanolamine-HCl (Bio-Rad).

Humanized ScFv antibodies were diluted to 4. Mu.g/mL with buffer (PBS/0.01% Tween 20) and injected into two channels on the chip at 30. Mu.L/min. After 90 degrees of rotation, the chip was rinsed with buffer until the baseline was stable. Analyte CD147 was diluted to 8.0,4.0,2.0,1.0 and 0.5nM with the same buffer. Five concentrations of sample and buffer were injected on 6 horizontal channels, respectively. The sample injection speed is 100ul/min. The sample binding time was 240s and the dissociation time was 1800s. After each sample test, 0.3% H was used ₃ PO ₄ Regenerating the chip. Data analysis was performed using Langmuir kinetics (Kinetic-Langmuir) model.

The best 14 scFV molecules of the degree of humanization were selected for affinity ranking, and the results are shown in table 1, with 10596 selected based on the degree of humanization analysis and affinity assay results; 10750;10752;10742; five clones 10765 were subsequently humanized antibody constructed.

Table 1 sequence analysis and affinity ordering of antibodies

6. Fully humanized antibody construction

1. Material

1.1 templates: selecting 10596 based on the results of the affinity ranking described above; 10750;10752;10742; five clones 10765 were inoculated overnight for culture, plasmids were extracted separately, and sequencing confirmed that the template sequence was correct.

1.2 primers:

fully humanized antibody VL gene amplification primers:

an upstream primer: 5A12-L2_F1, see SEQ ID NO 37 in the sequence Listing; 5A12-L2_F4, see SEQ ID NO 39 in the sequence Listing;

a downstream primer: pCI-5A12_R8 is shown in SEQ ID NO 41 of the sequence list;

fully humanized antibody VH gene amplification primers:

an upstream primer: 5A12-L2_F3, see SEQ ID NO 38 in the sequence Listing; 5A12-L2_F2, see SEQ ID NO. 40 in the sequence Listing;

a downstream primer: new-pCI-5A12_R3 is shown in SEQ ID NO. 42 in the sequence table;

2 PCR amplification of the target fragment using the above primers and templates, respectively:

the amplification protocol was as follows:

2.1 amplifying the complete humanized antibodies WBP203-201-VL and VH by using the 10596 as a template and 5A12-L1_F1+pCI-5A12_R8 and 5A12-L1_F3+new-pCI-5A12_R3 as primer pairs respectively;

2.2 amplification of fully humanized antibodies WBP203-202-VL and VH with the 10750 described above as template, and with 5a12-l2_f4+pci-5a12_r8 and 5a12-l1_f3+new-pCI-5a12_r3 as primer pairs, respectively;

2.3 amplification of the fully humanized antibodies WBP203-203-VL and VH with the 10752 template described above, primer pairs 5a12-l2_f4+pci-5a12_r8 and 5a12-l1_f3+new-pCI-5a12_r3, respectively;

2.4 amplification of fully humanized antibodies WBP203-204-VL and VH with the 10742 described above as template, and with 5a12-l1_f1+pci-5a12_r8 and 5a12-l1_f3+new-pCI-5a12_r3 as primer pairs, respectively;

2.5 amplification of fully humanized antibodies WBP203-205-VL and VH with the 10678 described above as template, and 5a12-l2_f4+pci-5a12_r8 and 5a12-l1_f2+new-pCI-5a12_r3 as primer pairs, respectively; after PCR amplification, the PCR product was recovered and purified by agarose gel electrophoresis.

2.6 restriction enzymes NgoMIV and SnaBI are added to the light chain variable region genes for cleavage, restriction enzymes NgoMIV and SnaBI are added to the heavy chain variable region genes for cleavage, and the cleavage is followed by purification by a DNA purification kit and ligation to the mammalian cell expression vector pCI-vector containing hIgG2/k digested with the same restriction enzymes. The ligation product was transformed into TOP10 E.coli and plated on LB agar medium containing 100. Mu.g/ml ampicillin. The obtained positive clones were cultured in LB liquid medium containing 100. Mu.g/ml ampicillin, and after sequencing verification by Invitrogen company, positive clone plasmids were extracted with a plasmid large sample kit, which were designated WBP203-201, WBP203-202, WBP203-203, WBP203-204 and WBP203-205, respectively.

8. Cell transient transfection and antibody purification

Plasmids containing light and heavy chain genes of WBP203-201 to 205 were co-transfected into 293F cells (1.0X10-s) using Invitrogen company Freestyle Max Reagent transfection reagent, respectively ⁶ Individual/ml), the transfected cells were placed in a shaker at 37℃with 5% CO ₂ Shake culturing in incubator, shaking table rotational speed is 120 revolutions. The transfected cell supernatant was collected by centrifugation 7 days after transfection, and the antibody of interest was isolated and purified from the cell culture supernatant using a protein A affinity column. The 3 humanized antibody WBP203-201 with the highest expression level is selected to contain the light chain variable region amino acid sequence shown as SEQ ID NO. 1 and the heavy chain amino acid sequence shown as SEQ ID NO. 3, and is named HP5A12-1; WBP203-202 contains the light chain variable region amino acid sequence shown as SEQ ID NO. 5 and the heavy chain amino acid sequence shown as SEQ ID NO. 7 and is named HP5A12-2; WBP203-203 contains the light chain variable region amino acid sequence shown as SEQ ID NO. 9 and the heavy chain amino acid sequence shown as SEQ ID NO. 11 and is designated HP5A12-3.

SDS-PAGE analysis of the highest 3 humanized antibodies 203-201, 203-202, 203-203 and chimeric antibody 5A12 was performed, as shown in FIG. 3, lanes 1-4 were non-reduced bands of HP5A12-1, HP5A12-2, HP5A12-3 and chimeric antibody, lanes 5-8 were reduced bands of HP5A12-1, HP5A12-2, HP5A12-3 and chimeric antibody, respectively, and two bands with molecular weights of about 50kD and 25kD were clearly seen from the reduced bands of lanes 5-8, and no bands, indicating that the humanized antibody and chimeric antibody were substantially identical in molecular weight.

9. Humanized antibody affinity assay

ELISA determination of humanized antibody affinity

CD147 was diluted to 1. Mu.g/ml with coating buffer (200 mM Na2CO3/NaHCO3, pH 9.2), and coated overnight at 4℃with 50. Mu.L of each reaction well; the solution in the reaction well was discarded, washed 3 times with 1XPBS buffer, blocked 1h at room temperature with 200. Mu.L of blocking buffer (2% BSA/1XPBS buffer), and washed 3 times with 200. Mu.L of 1XPBS buffer; adding scFV supernatant (8 96-well microwell plates) and negative control (50. Mu.L/well), and incubating at room temperature for 2h; wash 3 times with 200 μl 1X PBS buffer; anti-c-Myc Ab (HRP) (Abcam Cat#ab19312, 50. Mu.L/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; adding TMB substrate solution (50 mu L/hole) into each reaction hole for reaction for 10min; stop solution (2M HCl, 50. Mu.L/well) was added to stop the reaction; the absorbance at 450nm was read using an ELISA plate reader. Read with a microplate reader at 450 nm.

The results are shown in FIG. 4, and under the experimental conditions, the 3 antibodies and chimeric antibodies obtained by screening have obvious affinity to BASIGIN, and the affinity is superior to that of the control murine parent antibody (FIG. 4).

SPR determination of humanized antibody affinity

GLM chips were first activated with activator for 60s. The activator was mixed with 0.01M sulfo-NHS (Bio-Rad) using 0.04M EDC. Protein A/G was diluted with 10mM NaAc (pH 4.0) and injected onto the chip at a rate of 30ul/min to couple protein A/G to the activated chip via amino groups. The 6 parallel channels were simultaneously coupled to protein A/G. Finally, the chip was inactivated with 1M ethane-HCl (Bio-Rad).

Murine 5A12 and HP5A12 were diluted to 4. Mu.g/mL with buffer (PBS/0.01% Tween 20) and injected into both channels on the chip at 30. Mu.L/min. After 90 degrees of rotation, the chip was rinsed with buffer until the baseline was stable. Analyte CD147 was diluted to 8.0,4.0,2.0,1.0 and 0.5nM with the same buffer. At 6Five concentrations of sample and buffer were injected separately on each horizontal channel. The sample injection speed is 100ul/min. The sample binding time was 240s and the dissociation time was 1800s. The above steps were repeated three times, with 0.3% H after each sample test ₃ PO ₄ Regenerating the chip. Data analysis was performed using Langmuir kinetics (Kinetic-Langmuir) model.

As a result, as shown in Table 2, the binding rate constant (ka) of the HP5A12-1 was 8.35E+05, the dissociation rate constant (KD) was 5.08E-04, and the affinity constant (KD) was 5.96E-10; the binding rate constant (ka) of HP5A12-2 was 7.38E+05, the dissociation rate constant (KD) was 5.30E-04, and the affinity constant (KD) was 7.19E-10; the binding rate constant (ka) of HP5A12-3 was 7.04E+05, the dissociation rate constant (KD) was 4.75E-04, and the affinity constant (KD) was 6.74E-10;

Table 2 SPR assay of humanized antibody affinity

Sample of	Ka(1/Ms)	Kd(1/S)	KD(M)	Rmax(RU)	Chi 2 (RU)
						Mouse 5A12	5.92E+05	7.59E-04	1.28E-09	91.73	3.66
Chimeric 5A12	5.17E+05	7.78E-04	1.50E-09	50.47	3.32
						HP5A12-1	8.35E+05	5.08E-04	5.96E-10	62.94	3.46
HP5A12-2	7.38E+05	5.30E-04	7.19E-10	55.34	4.03
						HP5A12-3	7.04E+05	4.75E-04	6.74E-10	61.88	3.50

The above results suggest that the affinity of the humanized antibody is substantially identical to that of the murine parent antibody 5a12, indicating that the humanized engineering does not alter the affinity of the antibody.

10. Efficient humanized antibody expression vector construction

1. Construction of light chain gene expression vector

VL fragment of gene synthesis 203, 5'-3' was introduced with restriction enzymes XbaI and BsiWI, respectively, and the fragment and pcDNA3.3-LC-104new-M containing the light chain constant region gene were subjected to double digestion with XbaI and BsiWI, and 1% agarose gel electrophoresis followed by gel cutting and recovery. The DNA fragment (416 bp fragment from the gene synthesis fragment, 5712bp fragment from the plasmid pcDNA3.3-LC-104 new-M) after gel cutting purification was subjected to ligation reaction, and the ligation reaction was performed at 16℃for 20min with 20. Mu.L of T4 ligase, 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℃and 220rpm overnight with shaking, and the plasmid was extracted in large quantities, and the resulting plasmid was designated pcDNA3.3-LC-N-203 (shown in FIG. 5).

2. Construction of heavy chain gene expression vector

The VH fragment of gene synthesis 203, 5'-3' was introduced with restriction enzymes XbaI and NheI, respectively, and the fragment and pOptivec-HC-104new-M containing the heavy chain constant region gene were subjected to double digestion with XbaI and NheI, and after electrophoresis on 1% agarose gel, the rubber cut was recovered. The DNA fragment (434 bp fragment from the gene synthesis fragment, 5376bp fragment from plasmid pOptivec-HC-104 new-M) after the purification of the rubber cutting was subjected to ligation reaction, and the T4 ligase was reacted at 16℃for 20min in 20. Mu.L system, and 10. Mu.L 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 to obtain a large plasmid, and the final plasmid was designated pOptivec-HC-D-203 (shown in FIG. 6).

11. Determination of the specificity of humanized antibodies by cellular immunofluorescence

The specific binding capacity of the antibody and tumor cells is detected by using a cell immunofluorescence method, and the immunological cross reaction of the antibody is examined. The specific operation is as follows: will be 1X 10 ⁴ Inoculating the individual cells into a 35mm glass bottom culture dish, discarding the cell culture medium after the cells are attached, adding 1mL of tissue fixative (Boschia) to fix the cells, and fixing the cells at room temperature for 15 minutes; the fixative was discarded, then washed once with 1 XPBS, 1mL of 0.2% X-triton 100 was added and the wells were punched at room temperature for 2 minA clock; discard 0.2% x-triton 100, then wash once with 1 x PBS, add 1mL goat serum working solution (bi yun tian) and block for half an hour at room temperature; goat serum was discarded, after which 1 XPBS was used for washing once, 1mL of 1. Mu.g/mL HP5A12 antibody was added as primary antibody and incubated for 3 hours at room temperature; after three washes with 1 XPBS, 1mL Alexa Fluor 488 labeled donkey anti-human antibody (Abcam, dilution concentration 1:200) was added, DAPI stained nuclei, followed by imaging using a laser scanning confocal microscope.

As shown in FIG. 7, antibodies HP5A12-1, HP5A12-2 and HP5A12-3 all showed specific membrane staining in malignant cells such as lung cancer, liver cancer, colon cancer; whereas no membrane staining was seen on the corresponding CD147 knockout cells. Three antibodies were shown to specifically bind CD147.

12. Flow cytometry determination of humanized antibodies inhibiting memory T cell activation

CD4 by flow cytometry ⁺ CD45 ^RO+ T cell (memory T cell) proliferation inhibition experiments examined the ability of the antibodies to inhibit inflammation. The specific operation is as follows:

1) PBMC sorting: diluting the freshly collected 50mL of peripheral blood with 50mL of 1640 medium; respectively adding 100mL of human lymphocyte separating liquid and 100mL of diluted blood in sequence; 300g of the mixture is centrifuged for 20 minutes at normal temperature; after centrifugation, the tube was removed and visibly divided into three distinct layers: the middle white membrane layer is the PBMC layer; carefully aspirate the middle PBMC layer into a new centrifuge tube, centrifuge at 2000rpm for 5 minutes and discard the supernatant; 10mL of erythrocyte lysate is added, and after being gently blown and beaten uniformly by a pipetting gun, the erythrocyte lysate is kept stand for 15 minutes at normal temperature; adding 10mL of lymphocyte washing liquid, gently blowing with a pipetting gun uniformly, centrifuging at 2000rpm for 5 minutes, and discarding the supernatant; the cells were washed once with 10mL of serum-free 1640, centrifuged at 2000rpm, and the supernatant was discarded, and 10mL of 1640 was diluted and then subjected to cell counting to prepare PBMC cells for use.

2)CD4 ⁺ CD45 ^RO+ T cell sorting: using CD4 ⁺ CD45 ^RO+ T cell sorting kit (negative selection) (Miltenyi Biotec, germany) for separating CD4 from the PBMC cells ⁺ CD45 ^RO+ T cell and its operation steps are shown in the specification. Separating CD4 ⁺ CD45 ^RO+ T cells were adjusted to a cell concentration of 1X 10 in 1640 medium containing 10% FBS ⁶ Cell suspension per mL;

3)CD4 ⁺ CD45 ^RO+ t cell activation: 100. Mu.L of FSE solution (50. Mu.M) was added to 1mL of the cell suspension and CFSE was added to a final concentration of 5. Mu.M, and the mixture was labeled at 37℃for 10min in the dark. Washing twice with serum-free medium, centrifuging, and re-suspending with fresh medium to adjust cell concentration to 1×10 ⁶ Cell suspension per mL; 1mL of the cell suspension was added with 1mL of CD3/28 coated immunomagnetic beads (BD Co.).

4) CD4 ⁺ CD45 ^RO+ T CD3/28 activated cell suspensions were added to 24 well plates at 500 μl/well, and the experiment was divided into 11 groups, blank groups: add 500. Mu.l PBS; isotype control group: human IgG antibodies (Abcam Inc.) were added at different concentrations (0.1, 1, 10, 100, 1000. Mu.g/mL) at 500. Mu.l each; experimental group: 500. Mu.l of HP5A12-1, HP5A12-2 or HP5A12-3 antibodies were added at various concentrations (0.1, 1, 10, 100, 1000. Mu.g/mL), respectively;

5) Placing the laid 24-well plate in an incubator for culturing for 4 days;

6) Centrifuging, sucking supernatant, and storing in a refrigerator at-80deg.C; cells were washed with serum-free medium.

7) Flow cytometry detection: the Mean Fluorescence Intensity (MFI) of each group of cells was measured using a 488nm excitation wavelength channel with a blank control group of cells. Experiments were repeated 3 times.

8) The inhibition ratio is calculated according to the formula:

drawing a dose-effect curve, and calculating IC ₅₀ 。

As shown in FIG. 8, antibodies HP5A12-1, HP5A12-2 and HP5A12-3 were all capable of inhibiting CD4 ⁺ CD45 ^RO+ T cell (memory T cell) proliferation and shows dose-dependent relationship, IC ₅₀ 131.1. Mu.g/mL, 137.9. Mu.g/mL, 174.3. Mu.g/mL, respectively. It was shown that the antibodies HP5A12-1, HP5A12-2 and HP5A12-3 all inhibit CD4 ⁺ CD45 ^RO+ T cells (memory T cells) proliferate.

The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Nucleotide sequence Listing electronic document

<110> Chinese people's air force medical university

<120> an anti-BASIGIN humanized antibody and use thereof

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<220> humanized antibody HP5A12-3 heavy chain variable region (VH) amino acid sequence

<400>11

E V Q L V E S G G G 10

L V Q P G G S L R L 20

S C A A S G F T F S 30

N Y W M H W V R Q A 40

P G K G L E W V A E 50

I R L K S I N Y A T 60

H Y A E S V K G R F 70

T I S R D D S K N T 80

L Y L Q M N S L K T 90

E D T A V Y Y C T S 100

Y D Y E Y W G Q G T 110

L I T V S A 116

<210>12

<211>348

<212>DNA

<213> humanization

<220> humanized antibody HP5A12-3 heavy chain variable region (VH) nucleotide sequence

<400>12

gaa gtg cag ctt gtg gag tct gga gga ggc 30

ttg gtg caa cct gga gga tcc ctg agg ctc 60

tcc tgt gcc gcc tct gga ttc act ttc agt 90

aac tac tgg atg cac tgg gtc cgc cag gct 120

cca ggg aag ggg ctt gag tgg gtt gcc gaa 150

att aga ttg aaa tct att aat tat gca aca 180

cat tat gcg gag tct gtg aaa ggg agg ttc 210

acc atc tca aga gat gat tcc aaa aac acc 240

ctg tac ctg caa atg aac tcc tta aag act 270

gaa gac act gcc gtg tat tac tgt acc agt 300

tat gat tac gaa tac tgg ggc caa ggg act 330

ctg atc act gtc tct gca 348

<210>13

<211>11

<212>PRT

<213>

<220> light chain CDR-1 amino acid sequence

<400>13

K A S E I V G T Y V 10

S 11

<210>14

<211>7

<212>PRT

<213>

<220> light chain CDR-2 amino acid sequence

<400>14

G A S N R Y T 7

<210>15

<211>9

<212>PRT

<213>

<220> light chain CDR-3 amino acid sequence

<400>15

G Q S Y S Y P F T 9

<210>16

<211>10

<212>PRT

<213>

<220> heavy chain CDR-1 amino acid sequence

<400>16

G F T F S N Y W M H 10

<210>17

<211>19

<212>PRT

<213>

<220> heavy chain CDR-2 amino acid sequence

<400>17

E I R L K S I N Y A 10

T H Y A E S V K G 19

<210>18

<211>5

<212>PRT

<213>

<220> heavy chain CDR-3 amino acid sequence

<400>18

Y D Y E Y 5

<210>19

<211>36

<212>DNA

<213>

<220> upstream primer pFL-5A12-F1

<400>19

C C A G C C G G C C 10

A T G G C C G A A G 20

T G A A G C T T G A 30

G G A G T C 36

<210>20

<211>34

<212>DNA

<213>

<220> downstream primer linker-R1

<400>20

C A A A G T T G G A 10

A A T A A A A G C G 20

G C C G C A G A A C 30

A A A A 34

<210>21

<211>34

<212>DNA

<213>

<220> upstream primer linker-F1

<400>21

T T T T G T T C T G 10

C G G C C G C T T T 20

T A T T T C C A A C 30

T T T G 34

<210>22

<211>37

<212>DNA

<213>

<220> downstream primer pFL-5A12-R2

<400>22

T T T G T T C T G C 10

G G C C G C T T T T 20

A T T T C C A A C T 30

T T G T C C C 37

<210>23

<211>43

<212>DNA

<213>

<220> upstream primer 5A12-L1_F1

<400>23

C A G C C G G C C A 10

T G G C C G A A G T 20

G C A G C T T S T G 30

G A G T C T G G A G 40

G A G 43

<210>24

<211>48

<212>DNA

<213>

<220> downstream primer 5A12-L1_R1

<400>24

G A A A G T G A A T 10

C C A G A G G C G G 20

M A C A G G A G A G 30

C Y T C A G G G A T 40

C C T C C A G G 48

<210>25

<211>53

<212>DNA

<213>

<220> upstream primer 5A12-L1_F2

<400>25

C G C C A G G C T C 10

C A G G G A A G G G 20

G C T T G A G T G G 30

G T T R S C G A A A 40

T T A G A T T G A A 50

A T C 53

<210>26

<211>47

<212>DNA

<213>

<220> downstream primer 5A12-L1_R3

<400>26

T A A G G A G T T C 10

A T T T G C A G G T 20

A C A G G R T G K T 30

T T T G G A A T C A 40

T C T C T T G 47

<210>27

<211>50

<212>DNA

<213>

<220> upstream primer 5A12-L1_F3

<400>27

C A A A T G A A C T 10

C C T T A A R G A C 20

T G A A G A C A C T 30

R C C G T G T A T T 40

A C T G T A C C A G 50

<210>28

<211>38

<212>DNA

<213>

<220> downstream primer 5A12-L1_R4

<400>28

G G G A G A T T G G 10

G T C A T C T G A A 20

T G T C G C T A G C 30

A C C G C C A C 38

<210>29

<211>39

<212>DNA

<213>

<220> upstream primer 5A12-L1_F4

<400>29

G T A G G A G A C A 10

G G G T C A C C M T 20

C W C C T G C A A G 30

G C C A G T G A G 39

<210>30

<211>51

<212>DNA

<213>

<220> downstream primer 5A12-L1_R6

<400>30

G T T G G A T G C C 10

C C G T A T A T C A 20

G C A G T T T A G G 30

G G H C T K G C C T 40

G G T T T C T G T T 50

G 51

<210>31

<211>53

<212>DNA

<213>

<220> upstream primer 5A12-L1_F6

<400>31

T C A G C A G T C T 10

G C A G Y C C G A S 20

G A C W T C G C A A 30

C C T A T T A C T G 40

T G G A C A G A G T 50

T A C 53

<210>32

<211>37

<212>DNA

<213>

<220> downstream primer 5A12-L1_R8

<400>32

T T T G T T C T G C 10

G G C C G C T T T T 20

A T T T C C A A C T 30

T T G T C C C 37

<210>33

<211>53

<212>DNA

<213>

<220> upstream primer 5A12-L1_R2

<400>33

C C C T G G A G C C 10

T G G C G G A C C C 20

A G T G C A T C C A 30

G T A G T T A C T G 40

A A A G T G A A T C 50

C A G 53

<210>34

<211>49

<212>DNA

<213>

<220> upstream primer 5A12-L1_R5

<400>34

G G T G A C C C T G 10

T C T C C T A C T G 20

A G R S G G A C A G 30

G G W G G V G G G A 40

G A T T G G G T C 49

<210>35

<211>54

<212>DNA

<213>

<220> downstream primer 5A12-L1_R7

<400>35

A G A T C C A C T G 10

C C G G W G A A G C 20

G G G M G G G G A C 30

C C C A G T G T A C 40

C G G T T G G A T G 50

C C C C 54

<210>36

<211>50

<212>DNA

<213>

<220> upstream primer 5A12-L1_F5

<400>36

C C G G C A G T G G 10

A T C T G G C A C A 20

G A T T T C A C T C 30

T G A C C A T C A G 40

C A G T C T G C A G 50

<210>37

<211>DNA

<212>41

<213>

<220> upstream primer 5A12-L2_F1

<400>37

T G G C T C C C C G 10

G G G C G C G C T G 20

T G A C A T T C A G 30

A T G A C C C A A T 40

C 41

<210>38

<211>42

<212>DNA

<213>

<220> upstream primer 5A12-L2_F3

<400>38

C T C T C C A C A G 10

G T G T A C A C T C 20

C G A A G T G C A G 30

C T T G T G G A G T 40

C T 42

<210>39

<211>41

<212>DNA

<213>

<220> upstream primer 5A12-L2_F4

<400>39

T G G C T C C C C G 10

G G G C G C G C T G 20

T G A C A T T C A G 30

G T G A C C C A A T 40

C 41

<210>40

<211>41

<212>DNA

<213>

<220> upstream primer 5A12-L2_F2

<400>40

C T C T C C A C A G 10

G T G T A C A C T C 20

C G A A G T G C A G 30

C T T C T G G A G T 40

C 41

<210>41

<211>40

<212>DNA

<213>

<220> downstream primer pCI-5A12_R8

<400>41

G G T G C A G C C A 10

C C G T A C G T T T 20

T A T T T C C A A C 30

T T T G T C C C C G 40

<210>42

<211>39

<212>DNA

<213>

<220> downstream primer new-pCI-5A12_R3

<400>42

G G G C C C T T G G 10

T C G A C G C T G C 20

A G A G A C A G T G 30

A T C A G A G T C 39

Claims (7)

1. The anti-human BASIGN humanized antibody is characterized in that the amino acid sequence of a light chain variable region of the anti-human BASIGN humanized antibody is shown as SEQ ID NO. 1, and the amino acid sequence of a heavy chain variable region of the anti-human BASIGN humanized antibody is shown as SEQ ID NO. 3.

2. The anti-human BASIGN humanized antibody is characterized in that the amino acid sequence of a light chain variable region of the anti-human BASIGN humanized antibody is shown as SEQ ID NO. 5, and the amino acid sequence of a heavy chain variable region of the anti-human BASIGN humanized antibody is shown as SEQ ID NO. 7.

3. The humanized antibody of the anti-human BASIGN is characterized in that the amino acid sequence of a light chain variable region of the humanized antibody of the anti-human BASIGN is shown as SEQ ID NO. 9, and the amino acid sequence of a heavy chain variable region of the humanized antibody of the anti-human BASIGN is shown as SEQ ID NO. 11.

4. The humanized antibody of any one of claims 1-3, wherein the light chain constant region of said humanized antibody is kappa or lambda type and the heavy chain constant region is IgG2, igG1, igG3, igG4, igM, igA1, igA2, igD or IgE.

5. A basign antigen binding fragment comprising the anti-human basign humanized antibody of any one of claims 1-4 and Fab, fv, scFv or single chain antibody.

6. Use of an anti-human BASIGIN humanized antibody according to any one of claims 1-3 for the preparation of a medicament for the treatment of a disease associated with expression of anti-CD 147;

the CD147 expression related diseases are selected from lung cancer, liver cancer and colon cancer.

7. A gene expression vector, wherein the vector is selected from pcDNA3.3-LC-104new-M and pOptivec-HC-104new-M;

The carrier embedded in the claim of right 1-3 any one of the anti human BASIGN humanized antibody nucleotide sequence.

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