CN104086654A - Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof - Google Patents

Abstract

The invention provides a humanized modified anti-CD147 chimeric antibody HcHAb18 and an application thereof. The anti-CD147 human-rat chimeric antibody is prepared on the basis of obtaining hybridoma cells of an anti-human CD147 monoclonal antibody and antibody HcHAb18 genes of the hybridoma cells, wherein the variable region of light chains of the anti-CD147 human-rat chimeric antibody has the amino acid sequence shown in SEQ ID NO:1; the variable region of heavy chains of the anti-CD147 human-rat chimeric antibody has the amino acid sequence shown in SEQ ID NO:2; fucose are not combined in N-acetylglucosamine in a reducing end of a carbohydrate chain at a Fc segment of the anti-CD147 monoclonal antibody in a specific host cell; the glycoform of the anti-CD147 monoclonal antibody is of a mannose type (MAN 5); the anti-CD147 chimeric antibody has antibody dependent cell-mediated cytotoxicity (ADCC). The invention further provides an expression vector and an engineering cell strain of the anti-CD147 chimeric antibody and the application of the anti-CD147 chimeric antibody taken as a cancer treating medicine.

Description

Humanization modification type anti-CD147 chimeric antibody HcHAb18 and application thereof

Technical field

The present invention relates to biological technical field.Specifically, the present invention relates to a kind of new anti-CD147 mono-clonal of humanization modification type chimeric antibody, this antibody can suppress growth and metastasis of tumours, and relates to the nucleotide sequence of aminoacid sequence and encoding antibody.The present invention also comprises that this antibody is as the purposes of the medicine for cancer therapy.

Background of invention

CD147 molecule is a kind of cell transmembrane glycoprotein of wide expression, and in the mankind, CD147 has 269 amino acid and forms, and can be divided into extracellular region, cross-film district and intracellular region.It is signal peptide that N holds front 21 residues after initial translation, and 22～205 form extracellular regions, and 206～229Wei cross-film district, has typical leucine zipper structure, and C end 230～269 is intracellular region.

CD147 belongs to immunoglobulin superfamily (immunoglobulin superfamily, IgSF) member, and outer 4 halfcystines of born of the same parents form 2 disulfide linkage, form two Ig spline structure territories.The analysis of X ray crystalline diffraction shows, this molecule extracellular region crystalline structure is by two Ig spline structure territories, and wherein the structural domain of N-terminal is IgC2 ?set, and belongs to IgI ?set near the C end structure territory of after birth.

CD147 is also a kind of glycoprotein, at Asp44, Asp152, that an Asp1863 site can N-occur is glycosylation modified, makes its molecular weight rise to 35～60kDa by the 30kDa under de-glycosylation state.

The glycosylation of CD147 is very important to its function maturation, relevant with progress, invasion and attack, the transfer of tumour.

Research both at home and abroad all shows, CD147 molecule is specificity overexpression in the malignant tumor tissue of epithelial origin, as lung cancer, mammary cancer, esophagus cancer, liver cancer, cancer of the stomach, bladder cancer, ovarian cancer, prostate cancer, rodent ulcer, oral squamous cell carcinoma etc.

" CD147 detection kit (Immunohistochemical Method) " of the applicant's research and development (patent No.: ZL200910022400.1) the immunohistochemical staining result in 3045 routine clinical tissue samples shows CD147 molecule remarkable high expression level (total positives rate 85.30% in the tumor tissues such as lung cancer, liver cancer, cancer of the stomach, the esophageal carcinoma, colorectal cancer, mammary cancer, ovarian cancer, 1694/1986), and (the total positives rate 7.27% of low expression in healthy tissues and benign disease tissues, 77/1059), especially in lung cancer, show higher sensitivity (95.36%) and specific degree (97.96%).

Previously research shows, CD147 molecule is the important functional membrane albumen of tumor development process, participates in multiple and phenomenon related to cancer, as:

1. adhesion and the motion of CD147 mediation tumour cell: the CD147 of tumor cell surface expression and vinclin interact, promotes tumour cell pseudopodium to form, and sprawls and adheres to; Interact with annexin annxin II, promote tumour cell MMP-2 secretion, strengthen Tumor Cell Migration ability and Invasion Potential; By stimulating tumour cell self and inoblast around thereof, secrete multiple matrix metalloproteinase (extracellular matrix metalloproteinase, MMP), comprise MMP-1, MMP-2, MMP-3, MMP-11 and MT1-MMP and MT2-MMP etc., thereby quickening tumour is the degraded of matrix around, has promoted the transfer of tumour.In the allograft mouse model of tumour, the expression of inhibition tumor cell CD147 or function, when in significantly lowering tumor tissue, MMPs expresses, can also the transfer of inhibition tumor cell in Mice Body.CD147 promotes the mechanism of MMPs secretion, may be by activation intracellular signal albumen ERK1/2, MAPK and FAK, and in promotion born of the same parents, in Ca2+, stream is realized.

2. CD147 participates in the anaerobic metabolism of tumour cell: CD147 is the important molecule companion of monocarboxylate transporter body (monocarboxylate transporter) MCT-1 and MCT-4.CD147 can mutually combine with MCT-1 and MCT-4, assists them on cytolemma, correctly to locate, and continues to regulate their transport functions to lactic acid metabolism product.Because tumour cell mainly relies on anaerobic metabolism generate energy, so CD147 can be indirectly by affecting the expression of MCT and the energy metabolism of function regulate tumor cell.

3. CD147 promotes drug resistance of tumor cell: resistance is the major reason of malignant tumour clinical treatment failure.The tumour cell of multidrug resistance (multi-drug resistance, MDR) pumps cell to escape killing and wounding of chemotherapeutic by overexpression P-glycoprotein (P-glycoprotein, P-g) by the chemotherapeutics that enters tumour cell.CD147 molecule, when promoting MMPs secretion, affect transcribing of MDR gene by coexpression regulation mechanism, the expression of promotion P-g, thereby multidrug resistance that can induced tumor.The confirmation CD147 such as Kanekura cause MDR by P-g, so lower the expression of CD147, may be target spots that effectively resists MDR.Wang etc. find in experimental study in vitro, and the expression of reticent cancer of the stomach SGC7901 clone CD147 can make its chemosensitivity to cis-platinum increase.Zou etc. confirm in human ovarian cancer cell line HO-8910 system, by suppressing the expression of CD147, can increase the susceptibility to taxol.Kuang is same in human oral cavity epithelial squama cancer finds that CD147 plays an important role in resistance process.Tang proof CD147 promotes p-TFII-I cell nuclear localization, the key factor Bip that raises unfolded protein reaction (UPR) expresses, cause tumour cell er stress and UPR, thereby apoptosis inhibit and the insensitivity to medicine, suppress CD147 molecule and can promote hepatoma cell apoptosis, and can strengthen the susceptibility of tumour cell to existing antitumor drug.

4. CD147 raises vegf expression promotion tumor angiogenesis: VEGF affects the key factor that tumour forms, grows and shift.The expression that CD147 can raise VEGF promotes tumor vascular growth; Suppress the expression of CD147, can significantly suppress secretion and the tumor vascular generation of VEGF.

5. CD147 participates in interacting in conjunction with different kinds of molecules: the cross-film section of CD147 albumen exists the negative charge L-glutamic acid of a high conservative on evolving, that it can interactional architecture basics occur with various kinds of cell membranin, also point out CD147 by the interaction with multiple protein, to affect the multiple physiological activity of cell simultaneously.CD147 can, by interacting with the multiple protein such as CD98, Integrin, caveolin-1, cyclophilins (CyP), affect growth and the transfer process of tumour cell from many aspects such as energy metabolism, cell-matrix interaction, signal conduction.

In addition, multinomial retrospective study also shows, between the expression intensity and the prognosis of tumour patient of CD147 molecule in tumor tissues, to exist dependency closely.In nonsmall-cell lung cancer patient, the level that CD147 expression is increased and patient's prognosis situation are closely related.

Therefore, CD147 molecule has become the novel targets of oncotherapy, and succeeding in developing of antibody drug " Li Kating " wherein proved security and the validity of this target spot patent medicine.

Li Kating (iodine [ ¹³¹i] U.S. appropriate former times monoclonal antibody injection liquid (Iodine[ ¹³¹i] Metuximab Injection)), be to take the first monoclonal antibody immunity targeted drug for primary hepatocyte hepatocarcinoma that CD147 molecule is target spot.This medicine is by the CD147 antigen of the high expression level on U.S. appropriate former times monoclonal antibody specific binding hepatoma cell membrane, by the radioiodine of monoclonal antibody load [ ¹³¹i] be transported to tumor locus, by " cross fire " effect of high energy β ion, performance local tumor lethal effect.

Multi-center clinical trial result shows: to liver cancer post-transplantation, recurrence has reduced by 21% to Li Kating; Radiofrequency ablation among hepatocellular carcinoma patients postoperative recurrence has been reduced to 34%.Treatment primary hepatocarcinoma clinic control rate 86.30%, clinical effective rate 27.40%, median survival time 20 months, has shown good security and validity.But Li Kating due to load have iodine radioisotope [ ¹³¹i], therefore in links such as clinical application, Medical Personnel Protection, offal treatments, there is certain limitation.

Monoclonal antibody (McAb) with its high specific, high-affinity, toxic side effect is little, immunogenicity is low, long action time in body, can utilize in body the advantages such as autoimmunization system performance curative effect, in the diagnosis and treatment of numerous disease, be used widely, become an effective way of newtype drug development.

Yet, numerous researchs show, mouse monoclonal antibody has the relatively short transformation period, and during for the mankind, the basic function characteristic that lacks some immunoglobulin (Ig)s, as the cell-mediated cytotoxicity (Antibody Dependent Cell mediated Cytotoxicity, ADCC) of Complement Dependent R cytotoxicity (Complement Dependent Cytotoxicity, CDC) and antibody dependence.

In addition, mouse source McAb repeats to inject in human body and can cause that patient brings out human antimouse antibody (human anti mouse antibody, HAMA) reaction, occurs the performance of systemic anaphylaxis toxic reaction blocking antibody effect.

Therefore, in order to reduce the immunogenicity of mouse source McAb in human body, avoid as much as possible patient to occur HAMA reaction, thereby implement treatment can to patient's repeated application McAb, improve the effector function of antibody simultaneously, the lethal effect of reinforcement to tumour target cell, becomes the focus that current antibody bio-pharmaceutical is researched and developed.

By being mosaic gene by the C district gene splicing of the V district gene of mouse antibody and people's antibody, then insertion vector, proceed to engineering cell, the chimeric antibody obtaining, because it has reduced mouse source gene element, thus the untoward reaction that satisfying reduction mouse antibody causes, on the other hand, retain the complementary determinant of antigen-antibody on mouse antibody, maintain the specificity of humanization modified antibody recognition antigen; The 3rd, the effect district of humanized antibody can interact with the other parts of human immune system better, by ADCC or CDC effect, more effectively kills and wounds target cell.

Although immunoglobulin (Ig) has broad variety and subclass thereof in human body, current monoclonal antibody medicine is mainly IgG1, because they have longer transformation period and stronger effector function in serum.

The main path that tumor therapeutic antibody plays a role in vivo comprises: ADCC, CDC, the direct cell death inducing of antibody and maintain salvage pathway of antibody concentration etc.ADCC effect is the main approach of antibody killing tumor cells.

After ADCC is antibody recognition target cell surface antigen, (Fc γ RI, Fc γ R II a and Fc γ R III be acceptor a) in conjunction with NK cell surface activated form Fc γ Rs for antibody Fc, wherein Fc γ R III a is principal recipient, this combination has triggered the conduction of cell interior signal, and causes NK cell to discharge pore-forming protein/granzyme mixture cracking target cell.IgG molecule its Fc section have two N ?the oligosaccharide site that connects, be on 297 l-asparagines (Asn297) of heavy chain.The structure of this glycosylation site is heterogeneous relevant to the effector function of antibody.After previously research shows to remove Fucose, can strengthen the binding ability of antibody Fc to Fc γ RIIIa, thereby improve the ADCC effect of antibody.

Summary of the invention

For defect or the deficiency of prior art, one of the object of the invention is to provide the anti-CD147 chimeric antibody of a kind of humanization modification type.

For this reason, the anti-CD147 chimeric antibody of humanization modification type provided by the invention HcHAb18 specific binding people CD147 molecule, this antibody comprises variable region of heavy chain and variable region of light chain, it is characterized in that: variable region of light chain has the aminoacid sequence of SEQ ID NO:1, variable region of heavy chain has the aminoacid sequence of SEQ ID NO:2.

The anti-CD147 chimeric antibody of humanization modification type provided by the invention HcHAb18 feature is also that heavy chain and constant region of light chain sequence are corresponding to people's isotype IgG1.

The anti-CD147 chimeric antibody of humanization modification type provided by the invention HcHAb18 feature is also that it comprises the light-chain amino acid sequence that contains SEQ ID NO:3, and comprises the heavy chain amino acid sequence that contains SEQ ID NO:4.

The anti-CD147 chimeric antibody of humanization modification type provided by the invention HcHAb18 feature is also that antibody has the compound sugar chain Fc district of N-glucosides-connection, wherein Fucose or wood sugar not the 2-Acetamido-2-deoxy-D-glucose in described sugar chain reducing end be combined.

Another object of the present invention has been to provide the DNA molecular of the above-mentioned antibody of encoding.The nucleotide sequence that this DNA molecular contains the described monoclonal antibody of the coding variable region of light chain shown in SEQ ID NO:5, and the nucleotide sequence of the described monoclonal antibody of the coding variable region of heavy chain shown in SEQ ID NO:6.Coding light chain nucleotide sequence shown in SEQ ID NO:7, the encoding heavy chain nucleotide sequence shown in SEQ ID NO:8.

Three of object of the present invention has been to provide a kind of expression vector.This expression vector is pQD-Hyg-GSeu-cHAb18, the expression regulation sequence that this expression vector contains above-mentioned DNA sequence dna and is connected with this series of operations.

Four of object of the present invention is to provide a kind of host cell.This host cell is transformed by above-mentioned expression vector, and preferred, this host cell derives from CHO-K1 cell.

Five of object of the present invention is to provide the application of above-mentioned antibody in the medicine for the preparation for the treatment of cancer, and described cancer is to be selected from lung cancer, liver cancer, cervical cancer, colorectal carcinoma, mammary cancer, ovarian cancer, the esophageal carcinoma, hemopathy or cancer of the stomach.

For overcoming the mouse antibody HAb18 (patent No.: the ZL02114471.0) limitation in clinical application, the present invention clones from hybridoma by the method for RT-PCR that it is light, heavy chain variable region gene, be connected with people's constant region gene respectively, built the chimeric antibody gene of anti-CD147, and built corresponding expression vector, by the Fucose defective type CHO-K1 cell (MAGE1.5) (Eureka Therapeutics) of transfection chemical induction, obtained the anti-CD147 chimeric antibody-HcHAb18 of ADCC effect enhancement type, this antibody Fucose in particular host cell be not incorporated into N in antibody Fc section sugar chain reducing end ?acetylglucosamine end, antibody Fc section sugar type is seminose type (MAN5).Experiment in vitro shows, this chimeric antibody has retained the avidity similar to mouse parental antibody and specificity, and can bring out ADCC effect, has the ability of inhibition tumor cell invasion and attack migration simultaneously; In body, experiment shows, this chimeric antibody has the energy for growth that suppresses tumour.

Accompanying drawing explanation

Fig. 1 is matter class pQD-Hyg-GSeu-cHAb18 carrier schematic diagram;

Fig. 2 is HcHAb18 antibody sugar spectrum analysis

Fig. 3 is that HcHAb18 antibody and target antigen CD147 molecule avidity are measured, and in figure, A figure is HcHAb18 measurement result; B figure is mouse parental antibody HAb18 measurement result;

Fig. 4 is the immunohistochemical staining result of HcHAb18 monoclonal antibody in tissue;

Fig. 5 is that HcHAb18 is to the external fragmentation test of lung cancer cell line ADCC;

Fig. 6 is the impact of HcHAb18 on lung carcinoma cell motor capacity;

Fig. 7 is that HcHAb18 suppresses lung carcinoma cell invasion ability;

Fig. 8 is that HcHAb18 suppresses NCI-H520 tumor-bearing mice tumor growth in vivo.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.Yet following examples, experimental example are only further described the present invention, rather than be used for limiting the present invention.

Embodiment 1: the anti-CD147 monoclonal antibody in mouse source is light, the Cloning and sequencing of heavy chain variable region gene

Press Trizol Reagent test kit (Invitrogen company) specification sheets and extract 2 * 10 ⁶secrete the hybridoma (patent No.: total RNA 02114471.0) of anti-human CD147 monoclonal antibody HAb18.After 1% agarose electrophoresis is identified total RNA quality, use PrimeScript ^tMrT reagent Kit (TaKaRa) reverse transcription test kit carries out reverse transcription, obtains the cDNA of total RNA.According to patent " Anti-human Liver Cancer Monoclonal Antibody Hab18, heavy chain variable region gene and application thereof ", (patent No.: the HAb18 monoclonal antibody 02114471.0) announced is light, heavy chain variable region gene sequence information, the synthetic corresponding primer amplification HAb18 monoclonal antibody of design is light, heavy chain variable region gene.Primer is synthetic by Shanghai Sheng Gong bio-engineering corporation, and sequence is as follows:

VL-5’：5’-GGGGATATCCACCATGAACTTCGGGCTGAGCTGG-3’；VL-3’：5’-GGATACAGTTGGTGGTGCAGTCGACTTACGTTT(GT)GTTTCA(AG)CTT-3’；VH-5’：5’-GGGGATATCCACCATGGACTCACATACTCAGGTC-3‘；VH-3’：5’-GAC(ACT)CATGGGG(CG)TGT(TC)GTGCTAGCTG(AD)(AG)GAGAC(AGT)GTGA-3’。

Reaction conditions: 94 ℃, 1min; 55 ℃, 1min; 72 ℃, 1min; 40 circulations, last 72 ℃ of extensions, 10min.After PCR reaction finishes, utilize 1% agarose gel electrophoresis purifying reclaim PCR product and be connected into pMD18-T carrier (TaKaRa), screening positive clone sequence verification, result proves this sequence and the patent (patent No.: 02114471.0) announce sequence consistent.By order-checking correct result difference called after pMD18T-VL or pMD18T-VH.

The optimization of embodiment 2:VL and VH gene and chimeric antibody build

After obtaining VL and VH sequencing result.First, adopt the codon bias of hamster to transform light, heavy chain gene, lower to solve the protein expression amount producing because of rare codon in Chinese hamster ovary celI in translation process, its principle is to transport amino acid whose tRNA bias based on solving in amino acid building-up process, adopt institute's more codon of corresponding tRNA content, accelerate synthesizing of protein.According to the codon availability table of hamster, the VL obtaining in embodiment mono-and the codon bias in VH nucleotide sequence are optimized, obtain light, weight chain variable region amino acid sequence and the nucleotide coding sequence thereof of monoclonal antibody as shown in SEQ ID NO:1 to SEQ ID NO:8.

Secondly, synthetic antibody is light, the oligonucleotide fragment of heavy chain gene, and synthesizes respectively NheI and BamH1 restriction enzyme site at the two ends of heavy chain gene, at the two ends of light chain gene, synthesizes respectively HindIII and XbaI enzyme cutting site.With NheI and BamH1, HindIII and XbaI, respectively the heavy chain of antibody of synthetic and variable region of light chain DNA sequence dna are carried out to enzyme and cut, after 1% agarose electrophoresis separation, with Gel Extraction Kit (Omega bio-tek) purifying endonuclease bamhi.

Then, the T4DNA ligase enzyme for plasmid pcDNA3.1 (Invitrogen) (TaKaRa company) that the endonuclease bamhi that purifying is obtained is cut with same enzyme is connected, be built into the carrier for expression of eukaryon pQD-Hyg-GSeu-cHAb18 containing heavy chain of antibody and light chain full-length gene, as shown in Figure 1.

The transfection of embodiment 3:CHO cell and the screening of recombinant clone

With the expression vector pQD-Hyg-GSeu-cHAb18 that contains humanized antibody gene of above-mentioned structure, transform intestinal bacteria DH-5 α bacterial strain, be inoculated on 100ml LB substratum and increase.

With ultrapure plasmid DNA purification kit (Qiagen), by the requirement of test kit specification sheets, extract expression vector pQD-Hyg-GSeu-cHAb18 plasmid DNA.

Employing electroporation is Chinese hamster ovary celI (MAGE1.5 the cell) (patent: 200980145664.9) to fucosyl transferase defect by pQD-Hyg-GSeu-cHAb18 plasmid DNA transfection.

Adopting final concentration is 500ug/ml hygromycin B (Invitrogen, be called for short HyB) the MAGE1.5 cell of transfection is carried out to pressure screening, the sub-alum amine of METHIONINE (L-Methionine Sulfoximine, MSX) that rear employing final concentration is 25uM (Sigma) is proceeded cell screening and is cultivated; And adopt ClonePix FL (Genentix, Inc), the cell of still surviving after the screening of MSX pressure is carried out to repeatedly cloning screening.By aforesaid operations, obtained humanized antibody cell strain HcH18-M.

On CD OptiCHO (Invitrogen) substratum, cultivate the monoclonal cell strain HcH18-M filtering out, with Protein A affinity chromatography separation and purification antibody HcHAb18 (being the anti-CD147 chimeric antibody of humanization modification type) from culture supernatant.

The N-of embodiment 4:HcHAb18 antibody connects oligosaccharides spectrum analysis

Get the chimeric antibody HcHAb18 of 2mg purifying after desalting treatment, put into the long test tube of thin-walled, dry through 45 ℃, vacuum-drying instrument, add through 2M trifluoroacetic acid (TFA) 4mL, 100 ℃ of Water Under solution 2h, are resuspended in Milli-Q level water after being again dried.Sample, after desalting treatment again, adds PNGase F, and 37 ℃ of reactions are spent the night, and cut the N-connecting-type sugar chain being connected on antibody, and membrane filtration obtains trial-product N-and connects oligosaccharides and analyze.

N-connects oligosaccharides and analyzes by High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD) method.Chromatographic condition is as follows: chromatographic column Shimpack CLC-ODS column (60 * 150mm; Japan), chromatographic column is through leacheate A (10mM sodium radio-phosphate,P-32 solution (pH3.8)) balance, flow velocity 1.0ml/min, 55 ℃; Add N-to connect after oligosaccharides sample 25 μ l, with leacheate B (10mM sodium radio-phosphate,P-32 solution (pH3.8) is containing 0.5% propyl carbinol) and leacheate A, carry out gradient elution, the ratio of leacheate B and leacheate A is increased to 60:40 in 80min internal linear.With fluorescence (excitation wavelength 320nm, wavelength of transmitted light 400nm), detect effluent liquid.The oligosaccharides peak that detection obtains compares by the oligosaccharides standard substance (TaKaRa) of PA mark.

Result as shown in Figure 2, compare with the control sample cHAb18 that is expressed in wild-type Chinese hamster ovary celI, the HcHAb18 that is expressed in MAGE1.5 cell only has single oligosaccharides peak, further carrying out monose analysis shows, the sugared type of HcHAb18 antibody is high mannose (Man5), Fucose or wood sugar do not detected.

Embodiment 5: the biological function of recombinant humanized modification type chimeric antibody

(1) affinity of antibody is measured

Through the avidity of ProteOn XPR36 protein interaction systems measurement HcHAb18 antibody and target antigen CD147 molecule, utilize Langmuir kinetics (Kinetic ?Langmuir) model analysis data.Experimental result shows: the avidity K of HcHAb18 antibody and Antigens CD14 7 _d=3.62*10 ^?10m.HAb18 compares with mouse antibody, through the avidity of improved HcHAb18 antibody and target antigen without significant difference.Result as shown in Figure 3.

(2) antibodies specific is measured

With immunohistochemical method, detect the specific binding capacity of HcHAb18 monoclonal antibody and tumor tissues, investigate immuning tissue's cross reaction of this antibody.Concrete operations are as follows: conventional dimethylbenzene dewaxing, gradient alcohol dehydration, aquation organization chip; 3%H ₂o ₂blocking-up deactivating endogenous peroxydase; The sealing of normal sheep serum working fluid; Take HcHAb18 antibody as primary antibodie, and the anti-human Fc antibody of biotin labeled rabbit is two anti-, and the Streptomycin sulphate avidin working fluid of horseradish peroxidase-labeled is three anti-, DAB colour developing, and haematoxylin redyeing, transparent rear mounting, microscopy dewater.Result as shown in Figure 4 and Table 1, HcHAb18 monoclonal antibody visible specificity painted (total positives rate 84.66%) coloring degree in the Several Kinds of Malignancy tissues such as lung cancer, liver cancer, cervical cancer, colorectal carcinoma, ovarian cancer, mammary cancer, the esophageal carcinoma and cancer of the stomach is " ++ " or " +++ ", and its coloring site is cancer cells after birth; And few in conjunction with (total positives rate 13.70%, 22/159) with healthy tissues, in lung cancer, show higher sensitivity 88.24%) and specific degree (91.67%).

Table 1HcHAb18 monoclonal antibody and tumor tissues immunity crossing research

(3) ADCC effect test

With separated human spleen cell action effect cell, relatively various dose HcHAb18 monoclonal antibody killing effect in vitro to lung carcinoma cell under effector cell participates in, imitates target cell than being 50:1.After the coated tumour cell of MTS method detection human spleen cell and HcHAb18 is hatched altogether, the tumor-killing effect that HcHAb18 mediates.Result demonstration, the ADCC effect of HcHAb18 generation that monoclonal antibody is induced is significantly higher than contrast monoclonal antibody (cHAb18).HcHAb18 monoclonal antibody external to Lung Squamous Carcinoma Cells NCI ?H520, lung adenocarcinoma cell A549 and small cell carcinoma of lung NCI ?the EC50 of H446 be respectively 0.04 μ g/ml, 0.06 μ g/ml and 0.11 μ g/ml.Result as shown in Figure 5.(4) cell in vitro scratch experiment

After 0.1 μ g/ml, 1 μ g/ml or 10 μ g/ml HcHAb18 processing, NCI ?H520 cell migration speed by 18.8 μ m/hr (substratum contrast), be reduced to respectively 15.0 μ m/hr, 12.4 μ m/hr, 10.5 μ m/hr, rate of migration contrasts and compares with substratum, reduced respectively 20.2%, 34.0% and 44.1% (Fig. 7), one-way analysis of variance (one ?way ANOVA) result shows that result has statistical significance (P<0.01, P<0.001, P<0.001); A549 cell migration speed is by 20.1 μ m/hr (substratum contrast), be reduced to respectively 15.2 μ m/hr, 12.4 μ m/hr and 10.1 μ m/hr, rate of migration contrasts and compares with substratum, reduced respectively 24.4%, 38.3% and 50.0% (Fig. 6), one-way analysis of variance (one ?way ANOVA) shows that result has statistical significance (P<0.05, P<0.001, P<0.001).

(5) cell invasion test

After 0.1 μ g/ml, 1 μ g/ml or 10 μ g/ml HcHAb18 processing, compare with blank, NCI ?H520 cell-penetrating cell count significantly reduced respectively 30.0% (P<0.01), 48.0% (P<0.001), 66.2% (P<0.001) (Fig. 6); Similarly, the A549 cell that film is worn in invasion and attack has reduced respectively 53.5% (P<0.01), 61.5% (P<0.001), 74.6% (P<0.001), and result as shown in Figure 7.

(6) in tumor-bearing mice body, suppress tumor growth analyses

The cell in vitro of learning from else's experience is cultivated the subcutaneous portable human lung carcinoma cell NCI of the well-grown people ?H520 tumor nodule that goes down to posterity in nude mouse, and tumor cell suspension is made in aseptic technique, is inoculated in nude mice armpit subcutaneous, and cell density is 8 * 10 ⁶/ mouse, treats that tumor growth is to being greater than 100mm ³after, be divided at random 6 groups: 10 every group of control group (aseptic 0.9% sodium chloride injection 10mL/kg), HcHAb18 low dose group (2mg/kg), middle dosage group (10mg/kg), high dose group (30mg/kg), chemotherapeutics group (cis-platinum 2mg/kg+ Gemcitabine 100mg/kg) and Combined Preparation groups (HcHAb1810mg/kg+ cis-platinum 2mg/kg+ gemzar 100mg/kg).During administration, observe the general clinical symptom of animal, with vernier caliper measurement meter record tumour major diameter and minor axis, according to following formula, calculate gross tumor volume: gross tumor volume (V)=(major diameter * minor axis ²)/2.After administration, the 28th day de-neck put to death mouse, and peels off tumour, weighs.

Result is as shown in Figure 8:

(1) knurl volume inhibiting rate

Compare with control group, three dosage group gross tumor volumes of HcHAb18 monoclonal antibody are all significantly less than control group, and low dose group knurl volume inhibiting rate is respectively 47.31% (P<0.01); Middle dosage group knurl volume inhibiting rate is respectively 49.97% (P<0.01); High dose group knurl volume inhibiting rate is respectively 55.21% (P<0.01);

Compare with chemotherapy group (47.31%), each dosage group of HcHAb18 and combine group statistics there are no significant difference, but high dose group and the tumour inhibiting rate of combining group be all higher than chemotherapy group, combines group tumor killing effect the most obvious, and tumour inhibiting rate is respectively 61.05%.

(2) the heavy inhibiting rate of knurl

Compare with control group, three dosage group tumor weights of HcHAb18 monoclonal antibody are all significantly less than control group, and the heavy inhibiting rate of low dose group knurl is respectively 47.12% (P<0.01); The heavy inhibiting rate of middle dosage group knurl is respectively 48.73% (P<0.01); The heavy inhibiting rate of high dose group knurl is respectively 53.28% (P<0.01);

Compare with chemotherapy group (44.58%), each dosage group of HcHAb18 and combine group statistics there are no significant difference, but high dose group and the heavy tumour inhibiting rate of knurl of combining group be all higher than chemotherapy group, combines group tumor killing effect the most obvious, and tumour inhibiting rate is respectively 61.06%.

Above result shows: HcHAb18 monoclonal antibody can significantly suppress the growth of tumor-bearing mice in-vivo tumour; The tumor killing effect of high dose group is better than routine clinical consumption chemotherapy effect; With chemotherapeutics coupling, can significantly improve the inhibition of lung cancer growth.

Claims (10)

1. the anti-CD147 chimeric antibody of a humanization modification type HcHAb18, this antibodies specific is in conjunction with people's tumor associated antigen CD147 molecule, it comprises variable region of light chain and variable region of heavy chain, it is characterized in that, described light chain variable region amino acid sequence is as shown in SEQ ID NO:1, described weight chain variable region amino acid sequence as shown in SEQ ID NO:2, constant region behaviour antibody constant region.

2. the anti-CD147 chimeric antibody of humanization modification type as claimed in claim 1 HcHAb18, is characterized in that, this antibody is IgG1 people's isotype.

3. the anti-CD147 chimeric antibody of humanization modification type as claimed in claim 1 HcHAb18, it is characterized in that, this antibody comprises the compound sugar chain Fc district with N-glucosides-connection, and wherein Fucose or wood sugar are not incorporated into the 2-Acetamido-2-deoxy-D-glucose in described sugar chain reducing end.

4. the anti-CD147 chimeric antibody of humanization modification type as claimed in claim 1 HcHAb18, is characterized in that, the aminoacid sequence of light chain is as shown in SEQ ID NO:3, and heavy chain amino acid sequence is as shown in SEQ IDNO:4.

5. a nucleic acid molecule, its anti-CD147 chimeric antibody HcHAb18 claimed in claim 1 that encodes, it is characterized in that, the nucleotide sequence of encoded light chain variable region is as shown in SEQ ID NO:5, and the nucleotide sequence of encoding heavy chain variable region is as shown in SEQ ID NO:6.

6. nucleic acid molecule as claimed in claim 5, is characterized in that, coding light chain nucleotide sequence is as shown in SEQ ID NO:7, and encoding heavy chain nucleotide sequence is as shown in SEQ ID NO:8.

7. an expression vector, is characterized in that, this expression vector contains the nucleic acid molecule described in claim 5 or 6, and this expression vector is pQD-Hyg-GSeu-cHAb18.

8. a host cell, is characterized in that, this host cell is transformed by the expression vector of claim 7.

9. host cell claimed in claim 8, is CHO-K1 cell.

10. the application of antibody HcHAb18 claimed in claim 1 in the medicine for the preparation for the treatment of cancer, described cancer is to be selected from lung cancer, liver cancer, cervical cancer, colorectal carcinoma, mammary cancer, ovarian cancer, the esophageal carcinoma, hemopathy or cancer of the stomach.