CN1420128A - Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof - Google Patents
Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof Download PDFInfo
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Abstract
A monoclonic antibody of HER2, the DNA moleculae for coding it, its preparing process, and its medical composition are disclosed. Its advantages are high bioactivity and high expression in host cell.
Description
Invention field
The present invention relates to biological technical field.Specifically, the present invention relates to a kind of new Humanized anti-HER 2 monoclonal antibody.In addition, the pharmaceutical composition that the invention still further relates to this MONOCLONAL ANTIBODIES SPECIFIC FOR method and contain this monoclonal antibody.
Background of invention
Mammary cancer is one of modal malignant tumour of women.According to statistics, ten thousand women suffered from breast cancer surplus the whole world had 120 every year approximately, and 500,000 women die from mammary cancer.Developed country's breast cancer incidences such as North America, West Europe, Northern Europe are the highest, and African sickness rate is minimum.In recent years, the morbidity of mammary cancer worldwide is obvious ascendant trend.No matter still hang down a district in the district occurred frequently, breast cancer incidence all rises with the speed of 5-20%.Along with Asian countries's growth in the living standard, the rising tendency of Asia women breast cancer morbidity becomes one of area of ascensional range maximum apparently higher than American-European countries.Although China compares with other countries and area, still belong to the low area of sending out of mammary cancer, the sickness rate of mammary cancer also is in ascent stage.In China, mammary cancer has developed into the first place of women's malignant tumour morbidity in some cities, the sickness rate of 1972～1974 years women with breast cancer in Shanghai be 18.3/10 ten thousand, 1987～1989 years be 25.1/10 ten thousand, increase by 37.6%, year increase by 2.3%.According to scholarly forecast, Shanghai female mammary gland cancer morbidity will reach 28.8/10 ten thousand in 2000, occupy the first place of women's malignant tumour.
HER2 (human epidermal growth factor receptor 2, human epidermal growth factor receptor2) acceptor is that molecular weight is the transmembrane protein of 185kD, tool tyrosine kinase activity, after part and the HER2 receptors bind, make HER2 receptor autophosphorylation phosphorylation and activate its tyrosine kinase activity, thereby promote cell proliferation.Certain HER2 expression of gene is arranged among the normal people, but its overexpression just can cause cell hyperproliferation and phenotype vicious transformation, has also just formed tumour.Discover that the overexpression of HER2 acceptor gene is arranged in the cancerous tissue of 25%～30% advanced breast cancer, and the overexpression of this gene be one of mammary cancer independently the prognosis mala factor (Slamon etc., Science, 235:177-182[1987]; Slamon etc., Science, 244:707-712[1989]).It is rapid that the mammary cancer characteristics of HER2 acceptor overexpression show as disease progression, and the chemotherapy catabasis is short, and tamoxifen is easily produced resistance, and disease free survival rate and total survival rate all are lower than the mammary cancer of HER2 feminine gender.
Discover that both at home and abroad the monoclonal antibody of anti-HER2 can suppress the growth of HER2 male breast cancer cell or other type HER2 positive cancer cell in external, body.But, be subjected to the restrictions such as immunogenicity of mouse endogenous antibody with mouse endogenous antibody treatment human body diseases.Clinical experience in the past is verified, and mouse source antibody is difficult to obtain the ideal curative effect.In recent years, the appearance of humanized antibody technology and development are for the clinical possibility that provides is provided in this monoclonal antibody immunotherapy at the HER2 acceptor.But, existing commercial anti-HER 2 monoclonal antibody Herceptin
The biological activity of (Genentech company) still remains to be improved.
Therefore, still need a kind of new higher anti-anti-HER 2 monoclonal antibody of humanization of biological activity in this area.
Goal of the invention
The purpose of this invention is to provide the monoclonal antibody of a specific specificity in conjunction with the HER2 acceptor.
Another object of the present invention provides the dna molecular of the above-mentioned anti-HER 2 monoclonal antibody of coding.
Another object of the present invention provides a kind of pharmaceutical composition that contains said monoclonal antibody.
A further object of the invention provides the method for preparing said monoclonal antibody.
To achieve the above object of the invention, one aspect of the present invention provides a kind of Humanized monoclonal antibodies, this protein-specific is in conjunction with people HER2 acceptor, this albumen comprises variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:5 or the SEQ ID NO:9, and variable region of light chain has the aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:6 or the SEQ ID NO:10.
Preferably, the variable region of heavy chain in this monoclonal antibody has the aminoacid sequence shown in the SEQ ID NO:1, and variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:2.
The present invention provides the dna molecular of the above-mentioned Humanized monoclonal antibodies of encoding on the other hand.
In a preferable example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in SEQ ID NO:3, SEQ ID NO:7 or the SEQ IDNO:11, and the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in SEQ ID NO:4, SEQ IDNO:8 or the SEQ ID NO:12.
Third aspect present invention provides a kind of expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations.
Fourth aspect present invention provides a kind of host cell, it is characterized in that, it is transformed by above-mentioned expression vector.In a preferable example, this host cell is selected from intestinal bacteria and COS cell.
Fifth aspect present invention provides a kind of pharmaceutical composition, it is characterized in that, it contains the pharmaceutically above-mentioned human monoclonal antibodies and the pharmaceutically acceptable carrier of significant quantity.
Sixth aspect present invention provides a kind of method for preparing above-mentioned Humanized monoclonal antibodies, it is characterized in that, this method comprises:
A) provide an expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations;
B) with the described expression vector transformed host cell of step a);
C) host cell of gained culturing step b under the condition that is fit to described Humanized monoclonal antibodies expression); With
D) separation and purification obtains described humanized monoclonal antibody.
The invention has the advantages that the avidity of monoclonal antibody of the present invention and the expression amount in host cell all are significantly increased than existing commercial Herceptin monoclonal antibody.Other purpose of the present invention and advantage can be learnt by following detailed.
Description of drawings
Fig. 1 has shown the restriction enzyme mapping of the used pMG18 carrier of the present invention.Ck represents the constant region gene of antibody kappa light chain among the figure; The IgG1 constant region is represented the weight chain constant area gene of people's IgG antibody 1; PA represents that SV40 adds poly A site.
Fig. 2 has shown the position relation between the primer when making up the humanized antibody gene.
Detailed Description Of The Invention
The present invention relates to a kind of Humanized monoclonal antibodies, this protein-specific is in conjunction with people HER2 acceptor, this albumen bag Contain variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of heavy chain has SEQ ID NO:1, SEQ ID Amino acid sequence shown in NO:5 or the SEQ ID NO:9, variable region of light chain has SEQ ID NO:2, SEQ ID Amino acid sequence shown in NO:6 or the SEQ ID NO:10.
Term used herein " monoclonal antibody (monoclonal antibody) " refers to namely should from the antibody of the basic homogeneous antibody acquisition of a class The single antibody that comprises in the colony is identical, the sudden change of the natural generation that may exist except minority. Monoclonal antibody With high specificity for single antigen site. And, (normally have for different from conventional Anti-TNF-α body preparation The different antibodies of determinant) difference, each monoclonal antibody are for the single determinant on the antigen. Except their spy Outside the opposite sex, the benefit of monoclonal antibody is that also they are next synthetic by the hybridoma cultivation, can be by other immunity Globulin pollutes. Modifier " monoclonal " has represented the characteristic of antibody, is to obtain from the antibody population of basic homogeneous, This should not be interpreted into and need to produce antibody with any specific process. For example, being used for monoclonal antibody of the present invention can Make with hybridoma method (by Kohler etc., Nature, 256:495 (1975) at first proposes), or available recombinant DNA Method (for example referring to U.S. Patent No. 4,816,567) makes. " monoclonal antibody " is also available such as Clackson etc., Nature, 352:624-628 (1991) and Marks etc., J.Mol.Biol., the described technology of 222:581-597 (1991) is from biting Separate in the somatic antibody library and obtain.
Inhuman (such as mouse) antibody of " humanization " form be chimeric immunoglobulin (Ig), immunoglobulin chain or its fragment (such as Fv, Fab, Fab ', F (ab ')2Or other antigen binding sequence of antibody), they contain non-human immunoglobulin Minmal sequence. For most applications, humanized antibody is complementary determining region (CDR) residue in the human immunoglobulin(HIg) CDR by non-human antibody (required specificity, affinity and active mouse, rat or rabbit antibody are for example arranged) is residual Base replaces. In some cases, the Fv framework region (FR) of human immunoglobulin(HIg) is replaced by corresponding inhuman residue. In addition Outward, humanized antibody also can comprise and neither is present in receptor's antibody, is not present in CDR or the frame sequence of importing again In residue. Make the performance that these modifications can further improve and maximize antibody. Usually, humanized antibody comprises base All on the basis (at least one, common two) variable region, wherein all or basically all CDR district be inhuman The CDR district of immunoglobulin (Ig), all or basically all FR district be the FR district of human immunoglobulin(HIg) sequence.
Term used herein " antibody " and " immunoglobulin (Ig) " are about 150000 Dao Er that the same structure feature is arranged The different four glycan albumen that pause, it is made up of with two identical heavy chains (H) two identical light chains (L). Every light chain is logical Cross a covalent disulfide bonds and link to each other with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is not With. Every heavy chain and light chain be the intrachain disulfide bond at regular interval also. One end of every heavy chain has variable region (VH), its After be a plurality of constant regions. One end of every light chain has variable region (VL), and the other end has constant region; The constant region of light chain with First constant region of heavy chain is relative, and the variable region of light chain is relative with the variable region of heavy chain. Special amino acid residue exists Form the interface between the variable region of light chain and heavy chain.
Some part of variable region is different on sequence in term used herein " variable " the expression antibody, its shape Become various specific antibodies to combination and the specificity of its specific antigen. Yet changeability is not evenly distributed in whole In the individual antibody variable region. It concentrates on and is called in light chain and the variable region of heavy chain in complementary determining region (CDR) or the hypervariable region In three fragments. Part conservative in the variable region is called framework region (FR). In the variable region of natural heavy chain and light chain each Self-contained four FR districts, they are the beta sheet configuration haply, are linked to each other by three CDR that form connecting ring, at certain But forming section β-pleated sheet structure in a little situations. CDR in every chain closely be close together by the FR district and with other The CDR of one chain formed together antibody antigen-binding site (referring to Kabat etc., NIH Publ.No.91-3242, Volume I, 647-669 page or leaf (1991)). Constant region is not participated in the combination of antibody and antigen directly, but they show difference Effector function, for example participate in the cytotoxicity that depends on antibody of antibody.
" light chain " of vertebrate antibody (immunoglobulin (Ig)) can be classified as obviously not according to the amino acid sequence of its constant region Class in two classes (being called κ and λ) together. According to the amino acid sequence of its CH, immunoglobulin (Ig) can divide Be different kinds. Mainly contain 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can be advanced One step was divided into subclass (isotype), such as IgG1, and IgG2, IgG3, IgG4, IgA and IgA2. Corresponding to the inhomogeneity immunity The CH of globulin is called α, δ, ε, γ and μ. The subunit structure of inhomogeneity immunoglobulin (Ig) and 3-d modelling is well-known.
The invention still further relates to the dna molecular of the above-mentioned Humanized monoclonal antibodies of coding. In a better example, This dna molecular contains the described monoclonal antibody of coding shown in SEQ ID NO:3, SEQID NO:7 or the SEQ ID NO:11 The nucleotide sequence of variable region of heavy chain, and shown in SEQ ID NO:4, SEQ ID NO:8 or the SEQ ID NO:12 The nucleotide sequence of the described monoclonal antibody variable region of light chain of coding.
Humanized monoclonal antibodies of the present invention can be prepared usually by the following method.
At first, provide the nucleotide sequence that contains code book invention humanization monoclonal antibody and linking to each other with this series of operations The expression vector of expression regulation sequence.
Term used herein " expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed. Express Regulating and controlling sequence comprises promoter and the termination signal that links to each other with target nucleotide sequence operability. They also comprise nuclear usually Nucleotide sequence is the required sequence of translation suitably. " operability links to each other " refers to that some part of linear DNA sequence can Affect the activity of same other parts of linear DNA sequence. For example, if promoter or enhancer have increased coding Transcribing of sequence, then it is that operability links to each other with coded sequence.
The dna sequence dna of code book invention humanization monoclonal antibody can be used conventional means well known to those skilled in the art, such as root Manually synthetic or increase with the PCR method and to obtain according to dna sequence dna disclosed by the invention. Then, available well known in the art The whole bag of tricks is connected into this sequence in the suitable expression vector. Used expression vector is the art technology people among the present invention The known various commercially available expression vector of member is for example available from the expression vector of Qiagen and Promega company.
Subsequently, the expression vector with above-mentioned acquisition transforms suitable host cell. " host cell " generally comprises protokaryon Cell and eukaryotic. The example of prokaryotic host cell commonly used comprises Escherichia coli, hay bacillus etc. Commonly used is true The nuclear host cell comprises yeast cells, insect cell and mammalian cell. In the present invention, better host is thin Born of the same parents are Escherichia coli and COS cell.
At last, under the condition that is fit to monoclonal antibody expression of the present invention, cultivate the host cell that transforms gained, then With conventional separation and purification means well known to those skilled in the art such as ion-exchange chromatography, hydrophobic chromatography and sieve chromatographies Purifying obtains Humanized monoclonal antibodies of the present invention.
The present invention also provides a kind of pharmaceutical composition, said composition contain effective dose pharmaceutically monoclonal antibody of the present invention and Pharmaceutically acceptable carrier. Term used herein " pharmaceutically acceptable " refers to when molecule body and composition When suitably giving the animal or human, they can not produce disadvantageous, irritated or other bad reaction. Used herein " pharmaceutically acceptable carrier " should be compatible with mutain of the present invention, can be with its blend can be not usually The effect of decrease pharmaceutical composition in the situation. Can be used as some things of pharmaceutically acceptable carrier or its component The object lesson of matter is carbohydrate, such as lactose, dextrose plus saccharose; Starch is such as cornstarch and potato starch; Fiber Element and derivative thereof are such as sodium carboxymethylcellulose, ethyl cellulose and methylcellulose; The tragacanth powder; Fructus Hordei Germinatus; Gelatin; Talcum; Kollag is such as stearic acid and dolomol; Calcium sulfate; Vegetable oil is such as peanut oil, cottonseed Oil, sesame oil, olive oil, corn oil and cupu oil; Polyalcohol is such as propane diols, glycerine, D-sorbite, sweet dew Sugar alcohol and polyethylene glycol; Alginic acid; Emulsifying agent is such as Tween ; Wetting agent is such as NaLS; Colouring agent; Flavor enhancement; Tablet agent, stabilizing agent; Antioxidant; Anticorrisive agent; Apirogen water; Deng oozing salting liquid; Slow with phosphate Rush liquid etc.
Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, Body weight and roughly the factor such as disease condition, administering mode determine the useful dosage of patient is used.
Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.
The clone and the order-checking of the anti-Her2 variable region of mab in embodiment 1. mouse sources
A) immune mouse
The people's mammary tumour cell SK-Br-6 that adopts the Her2 high expression level is as immunogen.Adopting total amount is 5 * 10
7People's mammary tumour cell SK-Br-6 cell of fresh culture adds complete Freund's adjuvant, fully be ground to complete emulsification after, subcutaneous multi-point injection 10 age in days Balb/c mouse, every some injection 50 microlitres, totally 6 points.Every 1 week injection booster immunization, repeat for totally 5 times then, simultaneously the serum antibody level is monitored.Last intravenous injection 5 * 10
6New fresh cell/PBS mixed solution killed mouse after 3 days, took out spleen.
B) cytogamy
It is unicellular to isolate spleen, and definite cytoactive is greater than 90%.Spleen cell and SP2/0 murine myeloma cell with the centrifugal co-precipitation of 10: 1 mixed, are added PEG (1400MW) and enter cytogamy in 37 ℃ of water-baths, time of fusion is 1 minute, 2 minutes, 5 minutes and 10 minutes.Add 1 milliliter of fresh serum free medium, 2 milliliters, 5 milliliters and 10 milliliters at each time point.After the centrifugation cell is joined in the 20%FCS RPM1-1640 nutrient solution, change 96 orifice plates over to and cultivate.Cultivate after 24 hours for 37 ℃, the nutrient solution HAT that brings Selection In, to final concentration be 1 *.Changed fresh culture 1 time in per 3 days to growing the clone.
C) colony screening
Select mono-clonal to form the hole, took out supernatant liquor, carry out the dyeing of SK-Br-6 cell fluorescence at the 14th day.Keep positive hole, select 5 high expressing cell strains behind the subclone 3 times, respectively called after GH0, GH1, GH2, GH3 and GH4.
Through identifying, confirm that these 5 cell strains are the high expressing cell strain.These 5 clonal growths are stable, and expression amount is greater than 1 mg/litre, under 96 hole culture condition, stablely clone to the 5th generation.
The type that identifies 5 clones is: GH0 is IgG1; GH1 is IgG2a, and GH2 is IgG1, and GH3 is IgG2b, and GH4 is IgG2a.
D) bioactivity research of anti-Her2 monoclonal antibody
With the culture supernatant of a-protein method purifying said monoclonal antibody cell strain, measure the biological activity of monoclonal antibody.Measurement result sees the following form 1.Surveying the viable cell strain is SK-BR-6, and index is the differentiation retarding effect (unit represents with (being suppressed cell count/total cellular score) * 100%) to this cell line cell.The experiment triplicate, the parameter as a comparison of averaging.Not add any antibody as negative control, positive control 4D5 is mouse resource monoclonal antibody (available from Genentech).Positive control Herceptin is humanized 4D5 (available from a Genentech company).
Each cell strain product of table 1 monoclonal antibody is to the inhibition (%) of SK-Br-6 cytodifferentiation
As can be seen from Table 1, the monoclonal antibody that five strain cell strains produce all can obviously suppress the differentiation of SK-BR-6 cell, and wherein the restraining effect of GH0, GH2 and GH4 three strain cell strains obviously surpasses positive control.Because GH0 and GH4 have higher biological activity, will be that carry out on the basis with these two cell strains in following chimeric and humanization monoclonal antibody gene and cell strain.
E) clone of anti-Her2 variable region of mab and order-checking
According to the conservative property of human immunoglobulin gene, design following primer to clone the variable region encoding sequence of this monoclonal antibody
V
L-justice arranged: 5 '-tcctctagacagctgacccagtctcca-3 ' (SEQ ID NO:13)
V
L-antisense: 5 '-gttgctagcccagcttggtacchscdccgaa-3 ' (SEQ ID NO:14)
V
H-justice arranged: 5 '-aggaagctttgcagsagtcwgg-3 ' (SEQ ID NO:15)
V
H-antisense: 5 '-tgacgtacgggtgaccgtggtcccttggccccat-3 ' (SEQ ID NO:16)
In above-mentioned primer, H=a, c or t; S=c or g; D=a, g or t; R=a or g; W=a or t.
Adopt the Trizol test kit of Invitrogen company, the total RNA of extracting and purifying from the GH0 of above-mentioned acquisition and two hybridoma cell strains of GH4, the entire operation step is carried out according to the specification sheets of producer.
After the quality of identifying the total RNA of above-mentioned purifying on 1% agarose electrophoresis, carry out reverse transcription according to manufacturer's specification sheets with the reverse transcription test kit of Pharmacia company, obtain the cDNA of total RNA.
Carry out pcr amplification respectively with above-mentioned primer and cDNA and obtain VL and VH fragment.Actual conditions is as follows:
Reaction system:
VL (microlitre) VH (microlitre)
10 * PCR reaction buffer 55
DNTP (10 mmoles/liter) 55
MgCl
2(25 mmoles/liter) 66
cDNA 10 10
Adopted primer (10 micromoles per liter) 55 is arranged
Antisense primer (10 micromoles per liter) 55
Pfu (5U/ microlitre) 11
Add water to 50 microlitres, 50 microlitres
Temperature cycle:
Pre-sex change: 94 ℃ 2 minutes;
Major cycle: 94 ℃ 45 seconds, 55 ℃ 45 seconds, 70 ℃ 1 minute, totally 30 circulations;
Extend the back: 70 ℃ 5 minutes.
After finishing the said temperature circulation, on 1.2% agarose electrophoresis, identify amplified production, find that the single band of about a 300～350bp of molecular weight all appears in VL and VH reaction system.Glue with Promega company reclaims two kinds of amplified productions of test kit recovery, be connected in pUC18-3K cloning vector (" carrying out the too development of environmental monitoring according to the INCP-9 plasmid sequence " (DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASEDON INCP-9 PLASMIDS SEQUENCES), A.Greated, R.Krasowiak, M.Titok, C.M.Thomas School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus StateUniversity Scorina Av.4, Minsk 220080 Belarus) on, transformed into escherichia coli DH5a bacterial strain.Carry out blue hickie screening then, cut evaluation through enzyme again, obtain 6 and 8 positive colonies respectively.Through sequence verification, confirm that respectively 2 and 3 clones are correct clone.
Sequencing result shows that the aminoacid sequence of the product of the antibody gene coding of GH0 cell strain is in full accord with the aminoacid sequence of the Herceptin that has delivered, but its dna sequence dna is different with it.The aminoacid sequence of the product of the antibody gene coding of GH4 cell strain is inconsistent with the aminoacid sequence of the Herceptin that has delivered.SEQ IDNO:17, SEQ ID NO:18 are respectively the variable region of light chain dna sequence dnas of Herceptin monoclonal antibody and GH0 monoclonal antibody of the present invention.SEQ ID NO:19 has shown both light chain variable region amino acid sequences.SEQ IDNO:20, SEQ ID NO:21 are respectively the variable region of heavy chain dna sequence dnas of Herceptin monoclonal antibody and GH0 monoclonal antibody of the present invention.SEQ ID NO:22 has shown both weight chain variable region amino acid sequences.SEQ ID NO:29 and SEQ ID NO:30 have shown the aminoacid sequence and the nucleotide sequence of GH4 monoclonal antibody variable region of heavy chain.SEQ ID NO:31 and SEQ ID NO:32 have shown the aminoacid sequence and the nucleotide sequence of GH4 monoclonal antibody variable region of light chain.
The structure of embodiment 2 mosaic genes
According to following method, VH and VL gene clone that above-mentioned sequence verification is crossed are arrived on the pMG18-3K carrier (restriction enzyme mapping of this carrier is as shown in Figure 1), make these two genes and corresponding constant region constitute the heavy chain of antibody and the light chain gene of total length.
According to ordinary method (referring to " molecular cloning lab guide ", Jin Dongyan etc., 1992, Science Press), respectively above-mentioned heavy chain and variable region of light chain dna sequence dna are carried out enzyme with XbaI and NheI, HindIII and BsiWI and cut, with 1% agarose electrophoresis purifying endonuclease bamhi.Then, the endonuclease bamhi that purifying is obtained is connected respectively on the pMG18 carrier that XbaI and NheI, HindIII and Bsi WI enzyme are cut.
By aforesaid operations, VH-IgG1Fc fusion gene and VL-Ck fusion gene have been obtained.Cut evaluation through enzyme, obtained the right-on clone of sequence.
The humanization of embodiment 3 VL and VH gene
To carry out humanization to the anti-Her2 monoclonal antibody in mouse source, at first need the CDR district of heavy chain and the CDR district of light chain are grafted directly on the human antibody framework.Yet simple CDR transplants the avidity of often losing or reduced original antibody.Therefore, must be the avidity that mouse source residue could be rebuild antibody with some critical sites amino-acid residue reverse mutation of people FR.According to humanized the requirement (" humanization of mouse-anti CD3 monoclonal antibody OKT3., people's antibody hybridoma " (humanization of the murine anti-human CD3 monoclonal antibody OKT3.HumAntibod Hybridoma) 1994,5 (1,2): 41), above-mentioned VL and VH gene are carried out the humanization design, variable region of heavy chain and the light chain variable region amino acid sequence and the nucleotide coding sequence thereof of three Humanized monoclonal antibodies of acquisition shown in SEQ ID NO:1 to SEQ ID NO:12, wherein: SEQ ID NO:1-4 is respectively the weight chain variable region amino acid sequence of humanized antibody hGH0/1, the light chain variable region amino acid sequence, variable region of heavy chain encoding sequence and variable region of light chain encoding sequence; SEQ ID NO:5-8 is respectively weight chain variable region amino acid sequence, light chain variable region amino acid sequence, variable region of heavy chain encoding sequence and the variable region of light chain encoding sequence of humanized antibody hGH0/2: SEQID NO:9-12 is respectively weight chain variable region amino acid sequence, light chain variable region amino acid sequence, variable region of heavy chain encoding sequence and the variable region of light chain encoding sequence of humanized antibody hGH4/6.
Synthesizing of embodiment 4 humanized antibody variable region of heavy chain and chain variable region gene
It is 28～85 oligonucleotide fragment that the variable region of heavy chain encoding sequence (SEQ ID NO:3) of whole humanized antibody huGH0-1 is divided into 6 cover length as follows, the two ends of each oligonucleotide respectively have 8～9 with the good overlapping region of template complementation:
1.ctttctagacaggtgcagctgcagcgctccggccccgagctggtgaagcccg(SEQ?ID?NO：23)
2.ccccgagctggtgaagcccggcgcctccctgaagacctcctgcaccgcctcccacttcaacatcaaggacacctacatccacaaggtg(SEQ?ID?NO：24)
3.gacacctacatccacaaggtgaagcagcgccccgagcagtccctggagtggatcggccgcacctaccccaccaacggccgcacc(SEQ?ID?NO：25)
4.ccccaccaacggccgcacccgctacgaccccaagttccagtccaaggccaccatcaccgccgacacctcctccaacaccgcctacctg(SEQ?ID?NO：26)
5.cctccaacaccgcctacctgcaggtgtcccgcctgacctccgagaccaccgccgtgaagtactgctcccgctggggccgcgacg(SEQ?ID?NO：27)
6.caagctagcggaggacacggtcacggaggcgccgtggccccagtagtccatggagtagaagccgtcgcggccccagcgggag(SEQ?ID?NO：28)
Relation between the above-mentioned primer as shown in Figure 2.
With these 6 primers mutually each other template carry out PCR.PCR adopts the high-fidelity amplification system of Roche company, and reaction conditions is: 95 ℃ 5 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 55 seconds, 30 circulations; 72 ℃ 3 minutes.Article 6, the concentration of primer is: the final concentration of primer 1 and primer 6 is 100nm, and the final concentration of all the other each primers is 20nM.Products therefrom separates through agarose gel electrophoresis, reclaims test kit recovery purpose band and is cloned into (Promega company product, clone's process is carried out with reference to the specification sheets of producer) among the pGEM-T vector with the DNA glue of Promega company.Identify through blue hickie screening and XbaI/NheI double digestion, confirm to have 2～5 positive colonies.With American AB I automatic sequencer the positive colony of confirming is checked order.The right-on clone of results verification sequence according to dna sequencing.The building-up process of variable region of light chain dna sequence dna is identical with above-mentioned steps.
The method of describing according to embodiment 2 obtains aforesaid method among heavy chain and the variable region of light chain dna sequence dna insertion expression vector pMG18.The right-on clone's note of sequence is made pHuGH0/1.With obtaining expression vector pHuGH0/2 and pHuGH4/6 with quadrat method.
The transfection of embodiment 5 Chinese hamster ovary celIs and the screening of recombinant clone
With above-mentioned structure contain humanized antibody expression carrier pHuGH0/1, pHuGH0/2 and pHuGH4/6 transformed into escherichia coli DH5a bacterial strain, be inoculated in 100 milliliters of LB substratum and increase.According to manufacturer's specification sheets, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNAPurification Kit) the extracting and purifying plasmid DNA of Qiagen company.With the liposome method test kit of the Invitrogen company plasmid DNA transfection Chinese hamster ovary celI with above-mentioned purifying, operation is carried out with reference to the specification sheets of producer.
Select on the substratum Chinese hamster ovary celI of transfection to be carried out the selection in continuous 9 weeks at HAT, cultivate in the enterprising limit by row dilution of 96 orifice plates at last, carry out continuously 3 times, carry out mono-clonalization.Humanized antibody cell strain hGH0/1, hGH0/2 and hGH4/6 have been obtained by aforesaid operations.
On the RPM1641 substratum, cultivate the monoclonal cell system that chooses, supernatant is carried out the test of Western trace, judge expression intensity according to staining reaction.Found that two cell strains of hGH0/1, hGH0/2 and hGH4/6 have obviously higher expression intensity, can be used as the candidate clone of further research.
With Protein A affinity column direct separation and purification antibody hGH0/1, hGH0/2 and hGH4/6 from the culture supernatant of above-mentioned 3 clones.SDS-PAGE electrophoretic examinations proves that products therefrom purity is greater than 90%.
The affinity chromatography product carried out sieve chromatography once more, obtained purity greater than 98% sample.The gained sample can be used for following further researching and analysing.
The bioactivity research 1 of embodiment 6 recombinant humanized monoclonal antibodies) specificity fluorescent dyeing
5 strain antibody GH, GH1, GH2, GH3 and GH4 specificity dye to SK-Br-6, and control cells (HER-2/null feminine gender) strain is reactionless to dyeing.Wherein, GH0 can be by reference standard antibody (Herceptin) specific inhibition, GH1, and GH2, GH3 and GH4 can not be blocked by Herceptin, and GH0～GH5 can not block each other.This illustrates that the antibody institute bonded immunologic determinants that above-mentioned 5 cell strains produce is different.Except GH0 bonded antigenic determinant and Herceptin are the same antigenic determinant, all the other 4 cell strain institute bonded antigenic determinants are different with Herceptin.
2) immunoprecipitation experiment
The test of Western trace shows that above-mentioned 5 kinds of antibody are the same with contrast Herceptin, can and combine with Her2 protein-specific precipitation.
3) cell toxicity test
Evidence, above-mentioned 5 kinds of monoclonal antibodies are the same with 4D5 and Herceptin, the concentration of 10～100 micrograms per litre external can selective induction SK-Br-6 cell growth-inhibiting and cause apoptosis.
4) biological activity test
Carry out biological activity test according to the method for describing among the embodiment 1.Test-results is listed in the following table 2.Unit represents with (being suppressed cell count/total cellular score) * 100%.
Chimeric and the humanization monoclonal antibody of table 2 is to the restraining effect (%) of SK-Br-6 cytodifferentiation ability
Antibody concentration negative control positive control monoclonal antibody to be measured
(mcg/ml) (CD3 antibody) Herceptin hGH0/1 hGH0/2 hGH4/6
0 0.0 0.4 0.9 0.7 2.4
1 8.1 2.3 12.6 8.6 7.4
10 0.0 8.9 48.8 37.6 40.6
100 0.9 18.7 90.6 79.6 88.7
The The above results explanation, three strain Humanized monoclonal antibodies all have the ability of fairly obvious inhibition SK-Br-6 cytodifferentiation, and its action intensity is apparently higher than positive control Herceptin.Under 100 mcg/ml concentration, biological activity ratio's positive control of hGH0/1 is high 5 times, and the hGH0/2 comparison is according to high 4.2 times, and hGH4/6 is higher 4.6 times than positive control.Humanized two kinds of its corresponding chimeric antibodies of monoclonal antibody are compared, active not obviously reduction.This illustrates that the humanization that we carry out is successful, and above-mentioned Humanized monoclonal antibodies has the biological activity that obviously is better than existing goods Herceptin.
6) research of expression amount
Identify the expression amount of above-mentioned Herceptin, hGH0/1, hGH0/2 and four cell strains of hGH4/6 with the ELISA method.Concrete grammar is to adopt rpm1641 substratum, 37 ℃ of 5%CO
2Shake-flask culture, rotating speed are 88rpm.The sampling on the 7th of inoculation back is measured with the ELISA standard measure, takes a sample and is determined as 3 repetitions.In the following table 33 multiple mean values.
The comparison (mg/ml) of table 3 three strain humanized antibodies and positive control expression amount
Herceptin hGH0/1 hGH0/2 hGH4/6
23.24±1.12 227.44±0.37 179.63±0.31 309.21±0.37
As can be seen from Table 3, the comparison of the expression amount of the Humanized monoclonal antibodies that makes of the present invention is according to having improved nearly 10 times～14 times.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Sequence table＜110〉Lansheng Guojian Pharmaceutic Ind. Co., Ltd., Shanghai＜120〉Humanized anti-HER 2 monoclonal antibody and method for making and pharmaceutical composition＜130〉016015＜160〉32＜170〉PatentIn version 3.0＜210〉1＜211〉120＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉1Gln Val Gln Leu Gln Arg Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1,5 10 15Ser Leu Lys Thr Ser Cys Thr Ala Ser His Phe Asn Ile Lys Asp Thr
20 25 30Tyr?Ile?His?Lys?Val?Lys?Gln?Arg?Pro?Glu?Gln?Ser?Leu?Glu?Trp?Ile
35 40 45Gly?Arg?Thr?Tyr?Pro?Thr?Asn?Gly?Arg?Thr?Arg?Tyr?Asp?Pro?Lys?Phe
50 55 60Gln?Ser?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr65 70 75 80Leu?Gln?Val?Ser?Arg?Leu?Thr?Ser?Glu?Thr?Thr?Ala?Val?Lys?Tyr?Cys
85 90 95Ser?Arg?Trp?Gly?Arg?Asp?Gly?Phe?Tyr?Ser?Met?Asp?Tyr?Trp?Gly?His
100 105 110Gly?Ala?Ser?Val?Thr?Val?Ser?Ser
115 120＜210〉2＜211〉109＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉2Ala Asp Ile Val Met Lys Gln Ser His Lys Phe Asn Ser Thr Ser Val1,5 10 15Gly Asp Arg Ser Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Asn
20 25 30Thr?Ala?Val?Ala?Ser?Tyr?Gln?Gln?Lys?Pro?Gly?His?Ser?Pro?Lys?Leu
35 40 45Leu?Gly?Tyr?Ser?Ala?Ser?Phe?Arg?Tyr?Thr?Gly?Val?Gln?Asp?Arg?Phe
50 55 60Thr?Gly?Asn?Arg?Leu?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Val?Ser?Ser?Val65 70 75 80Gln?Ala?Glu?Asp?Leu?Ala?Val?Ile?Tyr?Cys?Gln?Gln?His?Tyr?Thr?Arg
85 90 95Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Arg?Val?Glu?Ile?Lys
100 105＜210〉3＜211〉360＜212〉DNA＜213〉 ( Homo sapiens )＜400〉3caggtgcagc tgcagcgctc cggccccgag ctggtgaagc ccggcgcctc cctgaagacc 60tcctgcaccg cctcccactt caacatcaag gacacctaca tccacaaggt gaagcagcgc 120cccgagcagt ccctggagtg gatcggccgc acctacccca ccaacggccg cacccgctac 180gaccccaagt tccagtccaa ggccaccatc accgccgaca cctcctccaa caccgcctac 240ctgcaggtgt cccgcctgac ctccgagacc accgccgtga agtactgctc ccgctggggc 300cgcgacggct tctactccat ggactactgg ggccacggcg cctccgtgac cgtgtcctcc 360＜210〉4＜211〉327＜212〉DNA＜213〉 ( Homo sapiens )＜400〉4gccgacatcg tgatgaagca gtcccacaag ttcaactcca cctccgtggg cgaccgctcc 60tccatcacct gcaaggcctc ccaggacgtg atcaacaccg ccgtggcctc ctaccagcag 120aagcccggcc actcccccaa gctgctgggc tactccgcct ccttccgcta caccggcgtg 180caggaccgct tcaccggcaa ccgcctgggc accgacttca ccttcaccgt gtcctccgtg 240caggccgagg acctggccgt gatctactgc cagcagcact acacccgccc ccccaccttc 300ggcggcggca cccgcgtgga gatcaag 327＜210〉5＜211〉120＜212〉PRT＜213〉 ( Homo sapiens )＜400〉5Gln Val Gln Leu Trp Arg Ser Gly Pro Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Leu Lys Thr Ser Cys Ala Tyr Ser His Phe Asn Ile Lys Asp Thr
20 25 30Tyr?Ile?His?Lys?Gly?Lys?Gln?Arg?Pro?Glu?Gln?Ser?Leu?Tyr?Trp?Ile
35 40 45Gly?Arg?Thr?Tyr?Pro?Thr?Asn?Ser?Arg?Thr?Arg?Tyr?Asp?Pro?Lys?Phe
50 55 60Thr?Ser?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr65 70 75 80Leu?Arg?Val?Ser?Arg?Leu?Thr?Ser?Glu?Thr?Thr?Ala?Val?Lys?Ser?Cys
85 90 95Ser?Arg?Trp?Gly?Arg?Asp?Gly?Phe?Thr?Ser?Met?Asp?Tyr?Trp?Gly?Lys
100 105 110Gly?Ala?Ser?Val?Thr?Val?Ser?Ser
115 120＜210〉6＜211〉109＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉6Ala Asp Ile Val Met Lys Gln Ser Arg Lys Phe Asn Ser Thr Ser Val1,5 10 15Gly Asp Arg Ser Ser Ile Lys Cys Lys Ala Ser Gln Ser Val Ile Asn
20 25 30Thr?Ala?Val?Ala?Ser?Tyr?Gln?Asp?Lys?Pro?Gly?His?Ser?Pro?Lys?Leu
35 40 45Gly?Gly?Tyr?Ser?Ala?Ser?Phe?Thr?Tyr?Thr?Gly?Val?Gln?Asp?Arg?Phe
50 55 60Lys?Gly?Asn?Arg?Leu?Gly?Thr?Asp?Phe?Thr?Trp?Thr?Val?Ser?Ser?Val65 70 75 80Gln?Ala?Glu?Thr?Leu?Ala?Val?Ile?Tyr?Cys?His?Gln?His?Tyr?Thr?Arg
85 90 95Pro?Ser?Thr?Phe?Gly?Ala?Gly?Thr?Arg?His?Glu?Ile?Lys
100 105＜210〉7＜211〉360＜212〉DNA＜213〉 ( Homo sapiens )＜400〉7caggtgcagc tgtggcgctc cggccccgag ctggtgcgcc ccggcgcctc cctgaagacc 60tcctgcgcct actcccactt caacatcaag gacacctaca tccacaaggg caagcagcgc 120cccgagcagt ccctgtactg gatcggccgc acctacccca ccaactcccg cacccgctac 180gaccccaagt tcacctccaa ggccaccatc accgccgaca cctcctccaa caccgcctac 240ctgcgcgtgt cccgcctgac ctccgagacc accgccgtga agtcctgctc ccgctggggc 300cgcgacggct tcacctccat ggactactgg ggcaagggcg cctccgtgac cgtgtcctcc 360＜210〉8＜211〉327＜212〉DNA＜213〉 ( Homo saPiens )＜400〉8gccgacatcg tgatgaagca gtcccgcaag ttcaactcca cctccgtggg cgaccgctcc 60tccatcaagt gcaaggcctc ccagtccgtg atcaacaccg ccgtggcctc ctaccaggac 120aagcccggcc actcccccaa gctgggcggc tactccgcct ccttcaccta caccggcgtg 180caggaccgct tcaagggcaa ccgcctgggc accgacttca cctggaccgt gtcctccgtg 240caggccgaga ccctggccgt gatctactgc caccagcact acacccgccc ctccaccttc 300ggcgccggca cccgccacga gatcaag 327＜210〉9＜211〉120＜212〉PRT＜213〉 ( Homo sapiens )＜400〉9Gln Val Gln Leu Trp Arg Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser Leu Lys Thr Ser Cys Thr Ala Ser His Phe Asn Ile Lys Asp Arg
20 25 30Tyr?Ile?His?Lys?Val?Lys?Gln?Arg?Pro?Glu?Gln?Ser?Leu?Glu?Trp?Ile
35 40 45Gly?Arg?Thr?Tyr?Pro?Thr?Asn?Gly?Arg?Thr?Arg?Gly?Asp?Pro?Lys?Phe
50 55 60Gln?Ser?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Ser?Ala?Tyr65 70 75 80Leu?Gln?Val?Ser?Arg?Leu?Thr?Ser?Glu?Thr?Thr?Ala?Val?Arg?Tyr?Cys
85 90 95Ser?Arg?Trp?Gly?Arg?Asp?Gly?Phe?Tyr?Ser?Pro?Asp?Tyr?Trp?Gly?Glu
100 105 110Gly?Ala?Ser?Val?Thr?Val?Ser?Ser
115 120＜210〉10＜211〉110＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉10Ala Asp Ile Val Met Lys Gln Ser Arg Lys Phe Asn Ser Thr Ser Val1,5 10 15Gly Asp Arg Ser Lys Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Asn
20 25 30Thr?Ala?Val?Ala?Ser?Tyr?Gly?Leu?Gln?Lys?Pro?Gly?His?Arg?Pro?Lys
35 40 45Leu?Leu?Gly?Tyr?Ser?Ala?Ser?Phe?Arg?Tyr?Thr?Gly?Val?Gln?Asp?Arg
50 55 60Phe?Thr?Arg?Asn?Arg?Leu?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Val?Ser?His65 70 75 80Val?Gln?Ala?Glu?Asp?Leu?Ala?Val?Ile?Tyr?Cys?Gln?Gln?His?Tyr?Thr
85 90 95Thr?Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Arg?Val?Glu?Ile?Lys
100 105 110＜210〉11＜211〉360＜212〉DNA＜213〉 ( Homo sapiens )＜400〉11caggtgcagc tgtggcgctc cggccccgag ctggtgaagc ccggcacctc cctgaagacc 60tcctgcaccg cctcccactt caacatcaag gaccgctaca tccacaaggt gaagcagcgc 120cccgagcagt ccctggagtg gatcggccgc acctacccca ccaacggccg cacccgcggc 180gaccccaagt tccagtccaa ggccaccatc accgccgaca cctcctccaa ctccgcctac 240ctgcaggtgt cccgcctgac ctccgagacc accgccgtgc gctactgctc ccgctggggc 300cgcgacggct tctactcccc cgactactgg ggcgagggcg cctccgtgac cgtgtcctcc 360＜210〉12＜211〉330＜212〉DNA＜213〉 ( Homo sapiens )＜400〉12gccgacatcg tgatgaagca gtcccgcaag ttcaactcca cctccgtggg cgaccgctcc 60aagatcacct gcaaggcctc ccaggacgtg atcaacaccg ccgtggcctc ctacggcctg 120cagaagcccg gccaccgccc caagctgctg ggctactccg cctccttccg ctacaccggc 180gtgcaggacc gcttcacccg caaccgcctg ggcaccgact tcaccttcac cgtgtcccac 240gtgcaggccg aggacctggc cgtgatctac tgccagcagc actacaccac cccccccacc 300ttcggcggcg gcacccgcgt ggagatcaag 330＜210〉13＜211〉27＜212〉DNA＜213〉＜400〉13tcctctagac agctgaccca gtctcca 27＜210〉14＜211〉31＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜223〉h＝a,ct； S=c or g; D=a, g or t;＜400〉14gttgctagcc cagcttggta cchscdccga a 31＜210〉15＜211〉22＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜223〉s=c or g; w＝at＜400〉15aggaagcttt gcagsagtcw gg 22＜210〉16＜211〉34＜212〉DNA＜213〉＜400〉16tgacgtacgg gtgaccgtgg tcccttggcc ccat 34＜210〉17＜211〉324＜212〉DNA＜213〉 ( Homo sapiens )＜400〉17gctgatatcg tgatgaccca gtcccacaag ttcatgtcca cctctgtggg cgatagggtc 60agcatcacct gcaaggccag tcaggatgtg aatactgctg tagcctggta tcaacagaaa 120ccaggacatt ctccgaaact actgatttac tcggcatcct tccggtacac tggagtccct 180gatcgcttca ctggcaatag atctgggacg gatttcactt tcaccatcag cagtgtgcag 240gctgaagacc tggcagttta ttactgtcag caacattata ctactcctcc cacgttcgga 300gggggtacca aggtggagat caaa 324＜210〉18＜211〉324＜212〉DNA＜213〉 ( Mus musculus )＜400〉18gccgacatcg tgatgaccca gtcccacaag ttcatgtcca cctccgtggg cgaccgcgtg 60tccatcacct gcaaggcctc ccaggacgtg aacaccgccg tggcctggta ccagcagaag 120cccggccact cccccaagct gctgatctac tccgcctcct tccgctacac cggcgtgccc 180gaccgcttca ccggcaaccg ctccggcacc gacttcacct tcaccatctc ctccgtgcag 240gccgaggacc tggccgtgta ctactgccag cagcactaca ccaccccccc caccttcggc 300ggcggcacca aggtggagat caag 324＜210〉19＜211〉108＜212〉PRT＜213〉 ( Homo sapiens )＜400〉19Ala Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val1 5 10 15Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr
20 25 30Ala?Val?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?His?Ser?Pro?Lys?Leu?Leu
35 40 45Ile?Tyr?Ser?Ala?Ser?Phe?Arg?Tyr?Thr?Gly?Val?Pro?Asp?Arg?Phe?Thr
50 55 60Gly?Asn?Arg?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Val?Gln65 70 75 80Ala?Glu?Asp?Leu?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?His?Tyr?Thr?Thr?Pro
85 90 95Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
100 105＜210〉20＜211〉369＜212〉DNA＜213〉 ( Homo sapiens )＜400〉20gcgtacgctc aggttcagct gcagcagtct ggccctgagc tggtgaagcc aggggcctca 60ctcaagttgt cctgtacagc ttctggcttc aacattaaag acacctatat acactgggtg 120aaacagaggc ctgaacaggg cctggaatgg attggaagga tttatcctac gaatggttat 180actagatatg acccgaagtt ccaggacaag gccactataa cagcagacac atcctccaac 240acagcctacc tgcaggtcag ccgcctgaca tctgaggaca ctgccgtcta ttattgttct 300agatggggag gggacggctt ctatgctatg gactactggg gtcaaggagc ctcggtcacc 360gtctcctcg 369＜210〉21＜211〉369＜212〉DNA＜213〉 ( Mus musculus )＜400〉21gcataggccc aggtgcagct gcagcagtcc ggccccgagc tggtgaagcc cggcgcctcc 60ctgaagctgt cctgcaccgc ctccggcttc aacatcaagg acacctacat ccactgggtg 120aagcagcgcc ccgagcaggg cctggagtgg atcggccgca tctaccccac caacggctac 180acccgctacg accccaagtt ccaggacaag gccaccatca ccgccgacac ctcctccaac 240accgcctacc tgcaggtgtc ccgcctgacc tccgaggaca ccgccgtgta ctactgctcc 300cgctggggcg gcgacggctt ctacgccatg gactactggg gccagggcgc ctccgtgacc 360gtgtcctcc 369＜210〉22＜211〉120＜212〉PRT＜213〉 ( Homo sapiens )＜400〉22Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30Tyr?Ile?His?Trp?Val?Lys?Gln?Arg?Pro?Glu?Gln?Gly?Leu?Glu?Trp?Ile
35 40 45Gly?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr?Arg?Tyr?Asp?Pro?Lys?Phe
50 55 60Gln?Asp?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr65 70 75 80Leu?Gln?Val?Ser?Arg?Leu?Thr?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala?Met?Asp?Tyr?Trp?Gly?Gln
100 105 110Gly?Ala?Ser?Val?Thr?Val?Ser?Ser
115 120＜210〉23＜211〉52＜212〉DNA＜213〉＜400〉23ctttctagac aggtgcagct gcagcgctcc ggccccgagc tggtgaagcc cg 52＜210〉24＜211〉88＜212〉DNA＜213〉＜400〉24ccccgagctg gtgaagcccg gcgcctccct gaagacctcc tgcaccgcct cccacttcaa 60catcaaggac acctacatcc acaaggtg 88＜210〉25＜211〉84＜212〉DNA＜213〉＜400〉25gacacctaca tccacaaggt gaagcagcgc cccgagcagt ccctggagtg gatcggccgc 60acctacccca ccaacggccg cacc 84＜210〉26＜211〉88＜212〉DNA＜213〉＜400〉26ccccaccaac ggccgcaccc gctacgaccc caagttccag tccaaggcca ccatcaccgc 60cgacacctcc tccaacaccg cctacctg 88＜210〉27＜211〉84＜212〉DNA＜213〉＜400〉27cctccaacac cgcctacctg caggtgtccc gcctgacctc cgagaccacc gccgtgaagt 60actgctcccg ctggggccgc gacg 84＜210〉28＜211〉82＜212〉DNA＜213〉＜400〉28caagctagcg gaggacacgg tcacggaggc gccgtggccc cagtagtcca tggagtagaa 60gccgtcgcgg ccccagcggg ag 82＜210〉29＜211〉120＜212〉PRT＜213〉 ( Mus musculus )＜400〉29Gln Val Gln Leu Trp Arg Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser Leu Lys Thr Ser Cys Thr Ala Ser His Phe Asn Ile Lys Asp Arg
20 25 30Tyr?Ile?His?Lys?Val?Lys?Gln?Arg?Pro?Glu?Gln?Ser?Leu?Glu?Trp?Ile
35 40 45Gly?Arg?Thr?Tyr?Pro?Thr?Asn?Gly?Arg?Thr?Arg?Gly?Asp?Pro?Lys?Phe
50 55 60Gln?Ser?Lys?Ala?Thr?Ile?Thr?Ala?Asp?Thr?Ser?Ser?Asn?Ser?Ala?Tyr65 70 75 80Leu?Gln?Val?Ser?Arg?Leu?Thr?Ser?Glu?Thr?Thr?Ala?Val?Arg?Tyr?Cys
85 90 95Ser?Arg?Trp?Gly?Arg?Asp?Gly?Phe?Tyr?Ser?Pro?Asp?Tyr?Trp?Gly?Glu
100 105 110Gly?Ala?Ser?Val?Thr?Val?Ser?Ser
115 120＜210〉30＜211〉360＜212〉DNA＜213〉 ( Mus musculus )＜400〉30caagttcaat tgtggagatc tggtccagaa ttggttaagc caggtacttc tttgaagact 60tcttgtactg cttctcactt caacattaag gatagataca ttcacaaggt taagcaaaga 120ccagaacaat ctttggaatg gattggtaga acttacccaa ctaacggtag aactagaggt 180gatccaaagt tccaatctaa ggctactatt actgctgata cttcttctaa ctctgcttac 240ttgcaagttt ctagattgac ttctgaaact actgctgtta gatactgttc tagatggggt 300agagatggtt tctactctcc agattactgg ggtgaaggtg cttctgttac tgtttcttct 360＜210〉31＜211〉110＜212〉PRT＜213〉 ( Mus musculus )＜400〉31Ala Asp Ile Val Met Lys Gln Ser Arg Lys Phe Asn Ser Thr Ser Val1 5 10 15Gly Asp Arg Ser Lys Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Asn
20 25 30Thr?Ala?Val?Ala?Ser?Tyr?Gly?Leu?Gln?Lys?Pro?Gly?His?Arg?Pro?Lys
35 40 45Leu?Leu?Gly?Tyr?Ser?Ala?Ser?Phe?Arg?Tyr?Thr?Gly?Val?Gln?Asp?Arg
50 55 60Phe?Thr?Arg?Asn?Arg?Leu?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Val?Ser?His65 70 75 80Val?Gln?Ala?Glu?Asp?Leu?Ala?Val?Ile?Tyr?Cys?Gln?Gln?His?Tyr?Thr
85 90 95Thr?Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Arg?Val?Glu?Ile?Lys
100 105 110＜210〉32＜211〉330＜212〉DNA＜213〉mouse (Mus musculus)＜400〉32gctgatattg ttatgaagca atctagaaag ttcaactcta cttctgttgg tgatagatct 60aagattactt gtaaggcttc tcaagatgtt attaacactg ctgttgcttc ttacggtttg 120caaaagccag gtcacagacc aaagttgttg ggttactctg cttctttcag atacactggt 180gttcaagata gattcactag aaacagattg ggtactgatt tcactttcac tgtttctcac 240gttcaagctg aagatttggc tgttatttac tgtcaacaac actacactac tccaccaact 300ttcggtggtg gtactagagt tgaaattaag 330
Claims (9)
1. Humanized monoclonal antibodies, this antibodies specific is in conjunction with people HER2 acceptor, it comprises variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:5 or the SEQID NO:9, and variable region of light chain has the aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:6 or the SEQ IDNO:10.
2. according to the Humanized monoclonal antibodies shown in the claim 1, it is characterized in that its variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:1, variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:2.
3. a dna molecular is characterized in that, the described Humanized monoclonal antibodies of its coding claim 1.
4. dna molecular according to claim 3, it is characterized in that, this dna molecular contains the nucleotide sequence of the described Humanized monoclonal antibodies variable region of heavy chain of coding shown in SEQ IDNO:3, SEQ ID NO:7 or the SEQ ID NO:11, and the nucleotide sequence of the described Humanized monoclonal antibodies variable region of light chain of coding shown in SEQ ID NO:4, SEQ ID NO:8 or the SEQ ID NO:12.
5. an expression vector is characterized in that, the expression regulation sequence that it contains the described dna sequence dna of claim 3 and links to each other with this series of operations.
6. a host cell is characterized in that, it is transformed by the described expression vector of claim 5.
7. host cell according to claim 6 is characterized in that, it is selected from intestinal bacteria and COS cell.
8. a pharmaceutical composition is characterized in that, it contains the pharmaceutically described human monoclonal antibodies of claim 1 and the pharmaceutically acceptable carrier of significant quantity.
9. a method for preparing the described Humanized monoclonal antibodies of claim 1 is characterized in that, this method comprises:
A) provide an expression vector, the expression regulation sequence that this expression vector contains the described dna sequence dna of claim 3 and links to each other with this series of operations;
B) with the described expression vector transformed host cell of step a);
C) host cell of gained culturing step b under the condition that is fit to described Humanized monoclonal antibodies expression); With
D) separation and purification obtains described humanized monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132225 CN1210308C (en) | 2001-11-16 | 2001-11-16 | Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132225 CN1210308C (en) | 2001-11-16 | 2001-11-16 | Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1420128A true CN1420128A (en) | 2003-05-28 |
| CN1210308C CN1210308C (en) | 2005-07-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01132225 Expired - Lifetime CN1210308C (en) | 2001-11-16 | 2001-11-16 | Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof |
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| CN (1) | CN1210308C (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318591C (en) * | 2004-07-08 | 2007-05-30 | 杨安钢 | Immune tbid gene and its coded protein and application |
| CN102046195A (en) * | 2008-04-02 | 2011-05-04 | 宏观基因有限公司 | HER2/neu-specific antibodies and methods of using same |
| CN102153644A (en) * | 2010-12-31 | 2011-08-17 | 广东药学院 | Antigenic peptide for dimerization of epidermal growth factor receptor |
| CN102167742A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human monoclonal antibody against HER2, preparation method and purpose thereof |
| CN101165068B (en) * | 2006-10-18 | 2011-09-21 | 上海复旦张江生物医药股份有限公司 | Monoclonal antibody for resisting HER2/ErbB2 antigen, preparation method and medicament composition thereof |
| CN102321175A (en) * | 2011-09-21 | 2012-01-18 | 天津胜发生物技术有限公司 | Nano-antibody or polypeptide aiming at breast cancer Her2/new |
| CN104447993A (en) * | 2013-09-13 | 2015-03-25 | 上海海思太科药业有限公司 | Human anti-HER2 monoclonal antibody with high activity, and preparation method and application thereof |
| CN105017425A (en) * | 2014-04-30 | 2015-11-04 | 京天成生物技术(北京)有限公司 | Anti-HER2-neutrality activity monoclonal antibody and application thereof |
| CN108610419A (en) * | 2016-12-09 | 2018-10-02 | 泰州迈博太科药业有限公司 | A kind of Humanized anti-HER 2 monoclonal antibody |
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2001
- 2001-11-16 CN CN 01132225 patent/CN1210308C/en not_active Expired - Lifetime
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318591C (en) * | 2004-07-08 | 2007-05-30 | 杨安钢 | Immune tbid gene and its coded protein and application |
| CN101165068B (en) * | 2006-10-18 | 2011-09-21 | 上海复旦张江生物医药股份有限公司 | Monoclonal antibody for resisting HER2/ErbB2 antigen, preparation method and medicament composition thereof |
| CN102046195A (en) * | 2008-04-02 | 2011-05-04 | 宏观基因有限公司 | HER2/neu-specific antibodies and methods of using same |
| EP2540745A1 (en) * | 2010-02-25 | 2013-01-02 | Shanghai Biomabs Pharmaceuticals Co., Ltd | Fully humanized anti-her2 antibody, preparation method and use thereof |
| EP2540745A4 (en) * | 2010-02-25 | 2013-09-11 | Shanghai Biomabs Pharmaceuticals Co Ltd | Fully humanized anti-her2 antibody, preparation method and use thereof |
| CN102167742A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human monoclonal antibody against HER2, preparation method and purpose thereof |
| US8974785B2 (en) | 2010-02-25 | 2015-03-10 | Shanghai Biomabs Pharmaceuticals Co., Ltd. | Fully humanized anti-HER2 antibody, preparation method and use thereof |
| WO2011103700A1 (en) * | 2010-02-25 | 2011-09-01 | 百迈博药业有限公司 | Fully humanized anti-her2 antibody, preparation method and use thereof |
| CN102167742B (en) * | 2010-02-25 | 2014-05-14 | 上海百迈博制药有限公司 | Human monoclonal antibody against HER2, preparation method and purpose thereof |
| CN102153644B (en) * | 2010-12-31 | 2012-11-28 | 广东药学院 | Antigenic peptide for dimerization of epidermal growth factor receptor |
| CN102153644A (en) * | 2010-12-31 | 2011-08-17 | 广东药学院 | Antigenic peptide for dimerization of epidermal growth factor receptor |
| CN102321175B (en) * | 2011-09-21 | 2013-08-07 | 天津胜发生物技术有限公司 | Nano-antibody or polypeptide aiming at breast cancer Her2/new |
| CN102321175A (en) * | 2011-09-21 | 2012-01-18 | 天津胜发生物技术有限公司 | Nano-antibody or polypeptide aiming at breast cancer Her2/new |
| CN104447993A (en) * | 2013-09-13 | 2015-03-25 | 上海海思太科药业有限公司 | Human anti-HER2 monoclonal antibody with high activity, and preparation method and application thereof |
| CN105017425A (en) * | 2014-04-30 | 2015-11-04 | 京天成生物技术(北京)有限公司 | Anti-HER2-neutrality activity monoclonal antibody and application thereof |
| CN105017425B (en) * | 2014-04-30 | 2018-02-16 | 京天成生物技术(北京)有限公司 | Anti- HER2 neutralization activities monoclonal antibody and its application |
| CN108610419A (en) * | 2016-12-09 | 2018-10-02 | 泰州迈博太科药业有限公司 | A kind of Humanized anti-HER 2 monoclonal antibody |
| CN108610419B (en) * | 2016-12-09 | 2020-08-18 | 泰州迈博太科药业有限公司 | Humanized anti-HER 2 monoclonal antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1210308C (en) | 2005-07-13 |
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