CN104086654B - The anti-CD147 chimeric antibody HcHAb18 of humanization modification type and application thereof - Google Patents

Abstract

The present invention provides a kind of anti-CD147 chimeric antibody HcHAb18 of humanization modification type and application thereof. Specifically on the basis of the hybridoma and antibody HAb18 gene thereof that obtain anti-human CD147 monoclonal antibody, prepare anti-CD147 human mouse chimeric antibody, does its variable region of light chain have SEQ? ID? aminoacid sequence shown in NO:1, does its variable region of heavy chain have SEQ? ID? aminoacid sequence shown in NO:2, the N acetylglucosamine that Fucose is not incorporated in antibody Fc section sugar chain reducing end in particular host cell, sugar-type is sweet dew sugar-type (MAN5), and this antibody has the cytotoxicity (ADCC) that antibody-mediated cell relies on. The present invention also comprises the expression vector of described anti-CD147 chimeric antibody, engineering cell strain and the application as cancer treatment drugs thereof.

Description

The anti-CD147 chimeric antibody HcHAb18 of humanization modification type and application thereof

Technical field

The present invention relates to biological technical field. Specifically, the present invention relates to a kind of anti-CD147 mono-clonal chimeric antibody of new humanization modification type, this antibody can grow and transfer by Tumor suppression, and relates to the nucleotide sequence of aminoacid sequence and encoding antibody. The present invention also comprises the purposes of this antibody as the medicine for cancer therapy.

Background of invention

CD147 molecule is the cell transmembrane glycoprotein of a kind of wide expression, and in the mankind, CD147 has 269 amino acid compositions, can be divided into extracellular region, Ji Baonei district of cross-film district. After N end initiation of translation, front 21 residues are signal peptide, and 22��205 formation extracellular regions, 206��229 is cross-film district, has typical leucine zipper structure, and C end 230��269 is Bao Nei district.

CD147 belongs to immunoglobulin superfamily (immunoglobulinsuperfamily, IgSF) member, and outer 4 halfcystines of born of the same parents form 2 disulfide linkage, form two Ig spline structure territories. X-ray crystalline diffraction analysis 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 born of the same parents' film.

CD147 is also a kind of glycoprotein, N-can be occurred glycosylation modified in Asp44, Asp152, Asp1863 site so that it is molecular weight rises to 35��60kDa by the 30kDa under de-glycosylation state.

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

Domestic and international research all shows, CD147 molecule is specificity overexpression in the malignant tumor tissue of epithelial origin, such 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.

The immunohistochemical staining result display CD147 molecule of " CD147 detection kit (immunohistochemical methods method) " (patent No.: the ZL200910022400.1) of applicant's research and development in 3045 example clinical tissue samples is in lung cancer, liver cancer, cancer of the stomach, the esophageal carcinoma, colorectal cancer, mammary cancer, remarkable high expression level (total positive rate 85.30% in the tumor tissues such as ovarian cancer, 1694/1986), and (the total positive rate 7.27% of low expression in healthy tissues and optimum pathological 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 the multiple phenomenon with related to cancer, as:

1. CD147 mediates adhesion and the motion of tumour cell: CD147 and the vinclin of tumor cell surface expression interacts, and promotes that tumour cell pseudopodium is formed, sprawls and adhere to; Interact with annexin annxinII, promote that tumour cell MMP-2 secretes, strengthen tumour cell motor capacity and Invasion Potential; By stimulating tumour cell self and inoblast around thereof, secrete multiple matrix metalloproteinase (extracellularmatrixmetalloproteinase, MMP), comprise MMP-1, MMP-2, MMP-3, MMP-11 and MT1-MMP and MT2-MMP etc., thus accelerate the degraded of tumour surrounding substrate, facilitate the transfer of tumour. In the allograft mouse model of tumour, the expression of inhibition tumor cell CD147 or function, while in significantly downward tumor tissue, MMPs expresses, it is also possible to the transfer of inhibition tumor cell in mouse body. CD147 promotes the mechanism of MMPs secretion, it may be that by activation intracellular signal albumen ERK1/2, MAPK and FAK, and promotes what the interior stream of Intracellular Ca2+ realized.

2. CD147 participates in the anaerobic metabolism of tumour cell: CD147 is the important molecule companion of monocarboxylate transporter body (monocarboxylatetransporter) MCT-1 and MCT-4. CD147 can be combined with each other with MCT-1 and MCT-4, assists them correctly to locate on cytolemma, and continues to regulate them to the transport function of lactic acid metabolism product. Owing to tumour cell mainly relies on anaerobic metabolism generate energy, therefore CD147 can indirectly by the energy metabolism of the expression and function point analysis tumour cell that affect MCT.

3. CD147 promotes drug resistance of tumor cell: resistance is the major reason of malignant tumour clinical treatment failure. The chemotherapeutics entering tumour cell is pumped cell to escape killing and wounding of chemotherapeutic by overexpression P-glycoprotein (P-glycoprotein, P-g) by the tumour cell of multidrug resistance (multi-drugresistance, MDR). CD147 molecule, when promoting MMPs to secrete, affects transcribing of MDR gene by coexpression regulation mechanism, promotes the expression of P-g, thus can induce the multidrug resistance of tumour. Kanekura etc. confirm that CD147 causes MDR by P-g, so the expression lowering CD147 may be a target spot effectively resisting MDR. Wang etc. test in vitro in research and find, the expression of reticent cancer of the stomach SGC7901 clone CD147, can make it be increased by chemosensitivity along platinum. 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 in human oral cavity epithelial squama cancer it is again seen that CD147 plays an important role in resistance process. Tang proves that CD147 promotes p-TFII-I nucleus inner position, the key factor Bip raising Non-adhesion inhibition index (UPR) expresses, cause tumour cell er stress and UPR, thus apoptosis inhibit and the insensitivity to medicine, suppress CD147 molecule can promote hepatoma cell apoptosis, and tumour cell can be strengthened to the susceptibility of existing antitumor drug.

4. CD147 raises vegf expression promotion tumor angiogenesis: VEGF is the key factor affecting tumour formation, growth and transfer. The expression that CD147 can raise VEGF promotes tumor vascular growth; Suppress the expression of CD147, it is possible to significantly suppress the secretion of VEGF and tumor vascular generation.

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

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

Therefore, CD147 molecule has become the novel targets of oncotherapy, wherein the succeeding in developing of antibody drug " Li Kating ", and demonstrates 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] MetuximabInjection)), it is the first monoclonal antibody immunity targeted drug for primary hepatocyte hepatocarcinoma by target spot of CD147 molecule. This medicine by the CD147 antigen of the high expression level on U.S. appropriate former times monoclonal antibody specific binding liver cancer cell film, by the radioiodine of monoclonal antibody load [¹³¹I] it is transported to tumor locus, acted on by " cross fire " of high energy �� ion, play local tumor lethal effect.

Multi-center clinical trial result shows: the recurrence of liver cancer post-transplantation is reduced 21% by Li Kating; Liver cancer radio-frequency (RF) ablation postoperative recurrence is reduced 34%. Treatment primary hepatocarcinoma clinic control rate 86.30%, clinical effective rate 27.40%, median survival time 20 months, shows good security and validity. But Li Kating due to load have iodine radioisotope [¹³¹I], therefore there is certain limitation in links such as clinical application, Medical Personnel Protection, offal treatments.

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

But, numerous research shows, mouse monoclonal antibody has the relatively short transformation period, and during for the mankind, lack the basic function characteristic of some immunoglobulin (Ig)s, R cytotoxicity (ComplementDependentCytotoxicity, CDC) and the cell mediated cytotoxicity (AntibodyDependentCellmediatedCytotoxicity, ADCC) of antibody dependence is relied on such as complement.

In addition, mouse source McAb repeats to cause patient to bring out human antimouse antibody (humanantimouseantibody, HAMA) reaction in injection human body, systemic anaphylaxis toxic reaction occurs and the performance of blocking antibody effect.

Therefore, in order to reduce the immunogenicity of mouse source McAb in human body, patient is avoided to occur that HAMA reacts as much as possible, such that it is able to implement treatment to patient repeated application McAb, improve the effector function of antibody simultaneously, strengthen the lethal effect to tumor target cell, become the focus of current antibody bio-pharmaceutical research and development.

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

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

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

ADCC is after antibody recognition target cell surface antigen, antibody Fc is in conjunction with NK cell surface activation type Fc �� Rs (Fc �� RI, Fc �� R II a and Fc �� R III a) acceptor, wherein Fc �� R III a is principal recipient, this kind of combination triggers cell interior intracellular signaling, and causes NK cell release pore-forming protein/particle enzyme complex cracking target cell. An IgG molecule has the oligosaccharide site of two N connections in its Fc section, is on 297 l-asparagines (Asn297) of heavy chain. The heterogeneous property of the structure of this glycosylation site is relevant to the effector function of antibody. Previously research can strengthen antibody Fc to the binding ability of Fc �� RIIIa after showing to remove Fucose, thus improves 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 a kind of anti-CD147 chimeric antibody of humanization modification type.

For this reason, humanization modification type provided by the invention anti-CD147 chimeric antibody HcHAb18 specific binding people's 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 SEQIDNO:1, variable region of heavy chain has the aminoacid sequence of SEQIDNO:2.

Humanization modification type provided by the invention anti-CD147 chimeric antibody HcHAb18 feature is also that heavy chain and constant light chain sequences are corresponding to people isotype IgG1.

Humanization modification type provided by the invention anti-CD147 chimeric antibody HcHAb18 feature is also that it comprises the light-chain amino acid sequence containing SEQIDNO:3, and comprises the heavy chain amino acid sequence containing SEQIDNO:4.

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

Another object of the present invention there is provided the DNA molecular encoding above-mentioned antibody. This DNA molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in SEQIDNO:5, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in SEQIDNO:6. Coding light chain nucleotide sequence shown in SEQIDNO:7, the encoding heavy chain nucleotide sequence shown in SEQIDNO:8.

The three of the object of the present invention there is provided 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 sequence being operational.

The four of the object of the present invention are to provide a kind of host cell. This host cell is transformed by above-mentioned expression vector, it is preferable that, this host cell resources is in CHO-K1 cell.

The five of the object of the present invention are to provide above-mentioned antibody for the preparation of the application in the medicine of Therapeutic cancer, described cancer is 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 limitation of murine antibody HAb18 (patent No.: ZL02114471.0) in clinical application, it is light that the present invention clones it by the method for RT-PCR from hybridoma, heavy chain variable region gene, it is connected with the constant region gene of people respectively, construct the chimeric antibody gene of anti-CD147, and construct corresponding expression vector, by Fucose defective type CHO-K1 cell (MAGE1.5) (EurekaTherapeutics) of transfection chemical induction, obtain the anti-CD147 chimeric antibody HcHAb18 of ADCC effect enhancement type, the N acetylglucosamine end that this antibody Fucose in particular host cell is not incorporated in antibody Fc section sugar chain reducing end, antibody Fc section sugar-type is sweet dew sugar-type (MAN5). experiment in vitro shows, this chimeric antibody remains 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, experiment in vivo shows, this chimeric antibody has the energy for growth of Tumor suppression.

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 measure, and in figure, A figure is HcHAb18 measurement result; B figure is mouse parental antibody HAb18 measurement result;

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

Fig. 5 is that HcHAb18 is to lung cancer cell line ADCC killing experiment in vitro;

Fig. 6 is that HcHAb18 is on the impact of 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. But the present invention is only further described by following examples, experimental example, instead of it is 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

2 �� 10 are extracted by TrizolReagent test kit (Invitrogen company) specification sheets⁶Secrete the hybridoma (patent No.: total serum IgE 02114471.0) of anti-human CD147 monoclonal antibody HAb18. After 1% agarose electrophoresis qualification total serum IgE quality, use PrimeScript^TMRTreagentKit (TaKaRa) reverse transcription test kit carries out reverse transcription, obtains the cDNA of total serum IgE. 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 corresponding primer amplification HAb18 monoclonal antibody of design and synthesis is light, heavy chain variable region gene. Primer is by the synthesis of 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 DEG C, 1min; 55 DEG C, 1min; 72 DEG C, 1min; 40 circulations, last 72 DEG C of extensions, 10min. After PCR reaction terminates, 1% agarose gel electrophoresis purifying is utilized to reclaim PCR primer and be connected into pMD18-T carrier (TaKaRa), screening positive clone sequence verification, result proves this sequence and the patent (patent No.: 02114471.0) announced sequence consistent.By result correct for order-checking called after pMD18T-VL or pMD18T-VH respectively.

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

After obtaining VL and VH sequencing result. First, the codon bias of hamster is adopted light, heavy chain gene to be transformed, with solve in translation process because of in Chinese hamster ovary celI rare codon produce protein expression amount lower, its principle transports amino acid whose tRNA bias based on solving in Amino acid synthesis process, namely the codon adopting corresponding tRNA content more, accelerates the synthesis of protein. Codon availability table according to hamster, codon bias in VL and the VH nucleotide sequence obtained in embodiment one is optimized, obtains light, heavy chain variable amino acid sequence and the nucleotide coding sequence thereof of the monoclonal antibody as shown in SEQIDNO:1 to SEQIDNO:8.

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

Then, the plasmid pcDNA3.1 (Invitrogen) that the endonuclease bamhi that purifying obtains is cut with same enzyme is connected with T4DNA ligase enzyme (TaKaRa company), it is 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 transformation of E. coli DH-5 �� bacterial strain containing humanized antibody gene of above-mentioned structure, it is inoculated on 100mlLB substratum and increases.

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

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

Final concentration is adopted to be 500ug/ml hygromycin B (Invitrogen, it is called for short HyB) the MAGE1.5 cell of transfection is carried out pressure screening, the METHIONINE sub-alum amine (L-MethionineSulfoximine, MSX) (Sigma) that rear employing final concentration is 25uM proceeds cell screening cultivation; And adopting ClonePixFL (Genentix, Inc), the cell still survived after being screened by MSX pressure carries out repeatedly cloning screening. Humanized antibody cell strain HcH18-M is obtained by aforesaid operations.

CDOptiCHO (Invitrogen) substratum cultivates the monoclonal cell strain HcH18-M filtered out, with ProteinA affinity chromatography separation and purification antibody HcHAb18 (i.e. anti-CD147 chimeric antibody of humanization modification type) from culture supernatant.

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

The chimeric antibody HcHAb18 getting 2mg purifying is after desalting treatment, put into the long test tube of thin-walled, through the 45 DEG C of dryings of vacuum-drying instrument, add through 2M trifluoroacetic acid (TFA) 4mL, it is hydrolyzed 2h under 100 DEG C of conditions, again it is resuspended in Milli-Q level water after drying. Sample, after desalting treatment again, adds PNGaseF, and 37 DEG C of reactions are spent the night, and cuts the N-being connected on antibody and connects type sugar chain, membrane filtration, obtains trial-product N-connection oligosaccharides and analyzes.

N-is connected oligosaccharides and is analyzed by high performance anion exchange chromatography-Pulse amperometric detection method (HPAEC-PAD) method. Chromatographic condition is as follows: chromatographic column ShimpackCLC-ODScolumn (60 �� 150mm; Japan), chromatographic column balances through leacheate A (10mM sodium radio-phosphate,P-32 solution (pH3.8)), flow velocity 1.0ml/min, 55 DEG C; After adding N-connection oligosaccharides sample 25 �� l, carrying out gradient elution with leacheate B (10mM sodium radio-phosphate,P-32 solution (pH3.8) is containing 0.5% propyl carbinol) and leacheate A, the ratio of leacheate B and leacheate A is increased to 60:40 in 80min internal linear. Effluent liquid is detected with fluorescence (excitation wavelength 320nm, wavelength of transmitted light 400nm). The oligosaccharides standard substance (TaKaRa) that the oligosaccharides peak that detection obtains is marked by PA compare.

Result is as shown in Figure 2, compared with the control sample cHAb18 being expressed in wild-type CHO cells, it is expressed in the HcHAb18 only single oligosaccharides peak of MAGE1.5 cell, carry out monose analysis further to show, the sugar-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 measures

Through the avidity of ProteOnXPR36 protein interaction systems measurement HcHAb18 antibody and target antigen CD147 molecule, utilize Lang Miaoer kinetics (Kinetic Langmuir) model analysis data. Experimental result shows: the avidity K of HcHAb18 antibody and Antigens CD14 7_D=3.62*10^�\10M. Compared with murine antibody HAb18, through the avidity of improved HcHAb18 antibody and target antigen without significant difference. Result is as shown in Figure 3.

(2) antibodies specific measures

With immunohistochemical method, the specific binding capacity of detection HcHAb18 monoclonal antibody and tumor tissues, investigates immuning tissue's cross reaction of this antibody. Concrete operation is as follows: conventional dimethylbenzene dewaxing, gradient alcohol dehydration, aquation organization chip; 3%H₂O₂Block deactivating endogenous peroxydase; Normal sheep serum working fluid is closed; Taking HcHAb18 antibody as primary antibodie, the anti-human Fc antibody of the rabbit of biotin labeling be two resist, the Streptomycin sulphate avidin working fluid of horseradish peroxidase-labeled be three resist, DAB develop the color, haematoxylin redyeing, dewater transparent after seal sheet, mirror inspection. Result is as shown in Figure 4 and Table 1, HcHAb18 monoclonal antibody visible specificity painted (total positive rate 84.66%) coloring degree in the multiple malignant tumor 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 born of the same parents' film; And (total positive rate 13.70%, 22/159) is seldom combined with healthy tissues, lung cancer shows higher sensitivity 88.24%) and specific degree (91.67%).

Table 1HcHAb18 monoclonal antibody and the research of tumor tissues immunological cross

(3) ADCC effect test

To be separated human spleen cell action effect cell, comparing the Cytotoxicity in vitro effect to lung carcinoma cell under effector cell participates in of various dose HcHAb18 monoclonal antibody, effect target cell is than being 50:1. After the tumour cell of MTS method detection human spleen cell and HcHAb18 bag quilt is hatched altogether, the tumor-killing effect that HcHAb18 mediates. Result shows, and HcHAb18 monoclonal antibody is induced the ADCC effect of generation to be significantly higher than comparison monoclonal antibody (cHAb18). The external EC50 to Lung Squamous Carcinoma Cells NCI H520, human umbilical vein endothelial cell and small cell carcinoma of lung NCI H446 of HcHAb18 monoclonal antibody is respectively 0.04 �� g/ml, 0.06 �� g/ml and 0.11 �� g/ml.Result is as shown in Figure 5. (4) cell in vitro scratch experiment

After 0.1 �� g/ml, 1 �� g/ml or 10 �� g/mlHcHAb18 process, NCI H520 cell migration speed is by 18.8 ��m/hr (substratum comparison), be reduced to respectively 15.0 ��m/hr, 12.4 ��m/hr, 10.5 ��m/hr, rate of migration is compared with substratum comparison, reduce 20.2%, 34.0% and 44.1% (Fig. 7) respectively, one-way analysis of variance (one wayANOVA) result display result has statistical significance (P < 0.01, P < 0.001, P < 0.001); A549 cell migration speed is by 20.1 ��m/hr (substratum comparison), be reduced to respectively 15.2 ��m/hr, 12.4 ��m/hr and 10.1 ��m/hr, rate of migration is compared with substratum comparison, reduce 24.4%, 38.3% and 50.0% (Fig. 6) respectively, one-way analysis of variance (one wayANOVA) shows that result has statistical significance (P < 0.05, P < 0.001, P < 0.001).

(5) cell invasion test

After 0.1 �� g/ml, 1 �� g/ml or 10 �� g/mlHcHAb18 process, compared with blank, NCI H520 cell-penetrating cell count significantly reduces 30.0% (P < 0.01), 48.0% (P < 0.001), 66.2% (P < 0.001) (Fig. 6) respectively; Equally, the A549 cell that film is worn in invasion and attack decreases 53.5% (P < 0.01), 61.5% (P < 0.001), 74.6% (P < 0.001) respectively, and result is as shown in Figure 7.

(6) Tumor suppression growth analysis in tumor-bearing mice body

Learning from else's experience the Cell culture invitro portable human lung carcinoma cell NCI H520 tumor nodule of the people of the subcutaneous well-grown that goes down to posterity in nude mouse, tumor cell suspension is made in aseptic technique, is inoculated in naked mouse armpit subcutaneous, and cell density is 8 �� 10⁶/ mouse, treats that tumor growth is to being greater than 100mm³After, it is divided into 6 groups at random: 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 (along platinum 2mg/kg+ Gemcitabine 100mg/kg) and Combined Preparation group (HcHAb1810mg/kg+ is along platinum 2mg/kg+ gemzar 100mg/kg) often organize 10. Observe the general clinical symptom of animal during administration, with vernier caliper measurement and count the record long footpath of tumour and short footpath, according to following formulae discovery gross tumor volume: gross tumor volume (V)=(long footpath �� short footpath²)/2. After administration, the 28th day de-neck puts to death mouse, and peels off tumour, weighs.

Result is as shown in Figure 8:

(1) knurl volume inhibiting rate

Compared with control group, HcHAb18 monoclonal antibody three dosage group gross tumor volumes 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);

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

(2) tumor-like hyperplasia

Compared with control group, HcHAb18 monoclonal antibody three dosage group tumor weights are all significantly less than control group, and low dose group tumor-like hyperplasia is respectively 47.12% (P < 0.01); Middle dosage group tumor-like hyperplasia is respectively 48.73% (P < 0.01); High dose group tumor-like hyperplasia is respectively 53.28% (P < 0.01);

Compared with chemotherapy group (44.58%), the 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 the knurl combining group are all higher than chemotherapy group, combine 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 clinical conventional amount used chemotherapy effect; With chemotherapeutics coupling, the inhibition of lung cancer growth can be significantly improved.

Claims (7)

1. the anti-CD147 chimeric antibody HcHAb18 of humanization modification type, 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 chain variable region amino acid sequence is as shown in SEQIDNO:1, described heavy chain variable amino acid sequence as shown in SEQIDNO:2, constant region behaviour antibody constant region.

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

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

4. the anti-CD147 chimeric antibody HcHAb18 of humanization modification type as claimed in claim 1, it is characterised in that, the aminoacid sequence of light chain is as shown in SEQIDNO:3, and heavy chain amino acid sequence is as shown in SEQIDNO:4.

5. a nucleic acid molecule, it encodes anti-CD147 chimeric antibody HcHAb18 according to claim 1, it is characterised in that, the nucleotide sequence of encoded light chain variable region is as shown in SEQIDNO:5, and the nucleotide sequence of encoding heavy chain variable region is as shown in SEQIDNO:6.

6. nucleic acid molecule as claimed in claim 5, it is characterised in that, coding light chain nucleotide sequence is as shown in SEQIDNO:7, and encoding heavy chain nucleotide sequence is as shown in SEQIDNO:8.

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