CN104726462A - Anti-HER2 humanized antibody MIL41, and preparation method and applications thereof - Google Patents
Anti-HER2 humanized antibody MIL41, and preparation method and applications thereof Download PDFInfo
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Abstract
The invention relates to an anti-HER2 humanized antibody MIL41, and nucleic acid molecules used for coding the anti-HER2 humanized antibody. The invention also relates to a preparation method of the anti-HER2 humanized antibody, applications of the nucleic acid molecules used for preparing the anti-HER2 humanized antibody, and applications of the anti-HER2 humanized antibody in preparation of antitumor drugs. Compared with conventional anti-HER2 humanized antibody, the anti-HER2 humanized antibody is high in purity, and is more beneficial for ensuring of antibody product quality in large scale production.
Description
Technical field
The present invention relates to a kind of anti-HER 2 humanized antibody, and the nucleic acid molecule of described humanized antibody of encoding.The invention still further relates to the purposes of described nucleic acid molecule for the preparation of described anti-HER 2 humanized antibody, and described humanized antibody is for the preparation of the purposes of antitumor drug.
Background technology
HER2(ERBB2) be I type transmembrane tyrosine kinase receptor family member, process LAN in the tumour cell of many epithelial origins, as mammary cancer, the ovarian cancer of 25-32%, the Primary Renal Cell Carcinoma etc. of 30-40% of 25-30%, it is the important target spot of tumor-targeting drug development.
Appropriate pearl (the Pertuzumab of humanized antibody handkerchief of Genentech company research and development, also 2C4 is referred to as, trade(brand)name Perjeta), by in conjunction with HER2, block contacting between HER2 and HER3, and the strongest dimeric forms of HER2 and HER3 to be HER family initial oncogenic signals conduction, this interaction is in the propagation of many dissimilar tumours and all play an important role in being formed.The tumour cell of Pertuzumab to HER2 low expression level also has effect.Handkerchief trastuzumab have passed the certification of U.S. FDA on June 8th, 2012, is used for the treatment of the metastatic breast cancer of the HER2 positive.
By retrieving relevant document and patent, (Chinese invention patent application number: 200580031905.4), we learn that Pertuzumab exists the problem of sequence isomer VHS in process of production, and this can affect the purity of the finished product.The object of the present invention's research is the antibody product obtained without VHS isomer or low VHS content of isomer.
Summary of the invention
The present inventor is groped by Computer Design, optimization and a large amount of experiments, be surprised to find the nucleotide sequence by changing the appropriate pearl of antibody handkerchief, and ensuing clone, expression strategy, the VHS isomer phenomenon that this antibody occurs in expression can be overcome, thus greatly improve the purity of product.Particularly, the present invention includes the following aspects:
First aspect present invention relates to the nucleic acid molecule of coding anti-HER 2 humanized antibody, and the nucleotide sequence of its light chain is as shown in SEQ ID NO:1, and the nucleotide sequence of its heavy chain is as shown in SEQID NO:2.
Second aspect present invention relates to recombinant vectors, and it contains the nucleic acid molecule of any one of first aspect present invention.
In the present invention, described carrier can be cloning vector or expression vector.Described carrier contains the nucleic acid molecule described in first aspect present invention, and described carrier by such as above-mentioned nucleic acid molecule insertion cloning vector or expression vector being obtained, or can be obtained by synthetic.
Described expression vector is such as prokaryotic expression carrier, carrier for expression of eukaryon, phage vector or virus vector.Wherein said prokaryotic expression carrier is such as pet vector, PGEX carrier, described carrier for expression of eukaryon is such as pcDNA3.1, pEGFP-C1, pPIC9K, described phage vector is such as λ phage vector λ gt, λ gt-λ B, and described virus vector is such as retrovirus, slow virus, adenovirus or gland relevant viral vector.
In embodiments of the invention, described carrier is carrier for expression of eukaryon pTGS-FRT-DHFR.
In embodiments of the invention, carrier for expression of eukaryon pTGS-FRT-DHFR has been done further transformation.In embodiments of the invention, described remodeling method selects label for removing Totomycin, and adds glutamine synthase expression box.In specific embodiment of the invention scheme, described expression vector is carrier GS.
In the present invention, connect the sequence of SEQ ID NO:1 and SEQ ID NO:2 in described carrier for expression of eukaryon pTGS-FRT-DHFR or carrier GS after, namely recombinant vectors is obtained.
Third aspect present invention relates to reconstitution cell, and it contains the recombinant vectors of any one of second aspect present invention.
The reconstitution cell of any one according to a third aspect of the present invention, it is mammalian cell.In embodiments of the invention, described mammalian cell is be applicable to expressing the cell of humanized antibody, such as, derive from the mammalian cell of people, mouse or monkey.In embodiments of the invention, described mammalian cell is Chinese hamster ovary celI.
In embodiments of the invention, described mammalian cell is CHO-K1 cell.
In specific embodiment of the invention scheme, described CHO-K1 cell is through the cell after the domestication of target substratum.
In embodiments of the invention, the Zooblast culture medium that described target substratum refers to serum-free, chemical composition is determined.
In embodiments of the invention, Pluronic F-68, glucose, culture medium dry powder Maxgrow202, sodium bicarbonate, sodium-chlor and HEPES is contained in described target substratum;
In specific embodiment of the invention scheme, the composition of described target substratum is PluronicF-681.0g/L, glucose 8.8g/L, culture medium dry powder Maxgrow2027.44g/L, sodium bicarbonate 1.98g/L, sodium-chlor 3.47g/L and 1M HEPES15ml/L, and regulates pH to 7.0 ± 0.1.In specific embodiment of the invention scheme, described target substratum is used for cell domestication and seed culture.In embodiments of the invention, described target substratum is the seed culture medium of table 1-1.
In embodiments of the invention, the method that described domestication is cultivated is well known in the art, such as first by cell adherent culture in the target substratum containing 10% calf serum, serum is removed step by step according to the ratio of 50%, such as calf serum concentration is down to 5%, 2.5%, 1.25% until completely not containing serum step by step from 10%, then continues to go down to posterity for several times, suspends completely to host cell, be doubled and redoubled stable, finally obtain the stable host cell that can grow in target substratum.
A fourth aspect of the present invention relates to anti-HER 2 humanized antibody, and it is prepared by the reconstitution cell of the nucleic acid molecule of any one of first aspect present invention, the recombinant vectors of any one of second aspect or any one of the third aspect.
In embodiments of the invention, in described anti-HER 2 humanized antibody, the content of VHS isomer is 0 ~ 0.4%, such as, be 0 ~ 0.2%, 0 ~ 0.1%.
In embodiments of the invention, in described anti-HER 2 humanized antibody, the content of NGHC isomer is 0.2% ~ 1%, such as, be 0.3% ~ 0.75%.
In embodiments of the invention, the quantivative approach of described VHS isomer is electric charge Analysis of Variable (being also cation exchange analysis method).
In embodiments of the invention, the quantivative approach of described NGHC isomer is the capillary gel electrophoresis analytical method (CE-SDS) of reduced form.
A fifth aspect of the present invention relates to the preparation method of the anti-HER 2 humanized antibody of any one of fourth aspect present invention, and it comprises the step utilizing the recombinant vectors of the nucleic acid molecule of any one of first aspect present invention, any one of second aspect or the reconstitution cell of any one of the third aspect to prepare this anti-HER 2 humanized antibody.
The preparation method of any one according to a fifth aspect of the present invention, it specifically comprises the following steps:
1) nucleotide sequence of the nucleic acid molecule of first aspect present invention is cloned in expression vector, obtain recombinant expression vector, preferably, described recombinant expression vector is that transformation obtains on the basis of carrier pTGS-FRT-DHFR, preferably, described transformation refers to that removing Totomycin selects label, and adds glutamine synthase expression box;
2) recombinant expression vector is proceeded to host cell, such as, in CHO-K1 cell, obtain recombinant host cell, preferably, described host cell is through the CHO-K1 cell after the domestication of target substratum;
3) by step 2) recombinant host cell that obtains cultivates in target substratum, and obtaining can the monoclonal cell strain of high expression antibody.
4) monoclonal cell strain of progressively amplification culture step 3) acquisition, results culture supernatant;
5) culture supernatant that step 4) obtains is carried out purifying, obtain the anti-HER 2 humanized antibody of any one of fourth aspect present invention.
In embodiments of the invention, described recombinant expression vector is GS carrier.
In embodiments of the invention, the Zooblast culture medium that described target substratum refers to serum-free, chemical composition is determined.
In embodiments of the invention, Pluronic F-68, glucose, culture medium dry powder Maxgrow202, sodium bicarbonate, sodium-chlor and HEPES is contained in described target substratum;
In specific embodiment of the invention scheme, the composition of described target substratum is PluronicF-681.0g/L, glucose 8.8g/L, culture medium dry powder Maxgrow2027.44g/L, sodium bicarbonate 1.898g/L, sodium-chlor 3.47g/L and 1M HEPES15ml/L, and regulates pH to 7.0 ± 0.1.In specific embodiment of the invention scheme, described target substratum is used for cell domestication and seed culture.In embodiments of the invention, described target substratum is the seed culture medium of table 1-1.
In embodiments of the invention, the method that described domestication is cultivated is well known in the art, such as first by cell adherent culture in the target substratum containing 10% calf serum, serum is removed step by step according to the ratio of 50%, such as calf serum concentration is down to 5%, 2.5%, 1.25% until completely not containing serum step by step from 10%, then continues to go down to posterity for several times, suspends completely to host cell, be doubled and redoubled stable, finally obtain the stable host cell that can grow in target substratum.
In embodiments of the invention, the concrete grammar of step 3) is: by step 2) recombinant host cell that obtains cultivates in target substratum, MSX(such as 75 μMs of MSX of proper concn are added) in substratum, for some time is cultivated in incubator, pick out and can survive in this substratum and express the cell of antibody, then carry out subclone screening, obtaining can the monoclonal cell strain of high expression antibody.
In embodiments of the invention, the concrete grammar of step 4) is: can the monoclonal cell strain of high expression antibody through target substratum multistep enlarged culturing, substratum is for producing substratum: seed culture medium is 1:1, culture cycle is 12-14 days, cultivate the fed-batch medium that the 3rd, 6,9 day adds 10% volume, cultivate and terminate rear results supernatant.
In specific embodiment of the invention scheme, described seed culture medium is target substratum.
In embodiments of the invention, sodium hydroxide, culture medium dry powder Maxpro302, vitamin B12, ferrous sulfate, biphosphate sodium-hydrate, glucose, L-cysteine hydrochloride monohydrate, Pluronic F-68, sodium-chlor, HCl, sodium bicarbonate and HEPES is contained in described productive culture base.
In specific embodiment of the invention scheme, containing sodium hydroxide 0.8g/L, culture medium dry powder Maxpro30211.5g/L, 1g/L vitamin B12 stock solution (1-2) ml/L, 10g/L ferrous sulfate stock solution (0.4-0.6) ml/L, biphosphate sodium-hydrate 0.35g/L, glucose 8.8g/L, L-cysteine hydrochloride monohydrate (0.3-0.375) g/L, Pluronic F-681g/L, sodium-chlor 1.55g/L, 5M HCl5.6ml/L, sodium bicarbonate 1.22g/L and 1M HEPES7.5ml/L in described productive culture base, and regulate pH to 7.0 ± 0.1.In specific embodiment of the invention scheme, described productive culture base is used for antibody producing.In embodiments of the invention, described productive culture base is the productive culture base of table 1-2.
In embodiments of the invention, sodium hydroxide, disodium hydrogen phosphate,anhydrous, fed-batch medium dry powder Maxfeed402, TYR disodium monocalcium salt thing, L-cysteine hydrochloride one water thing, l-asparagine, glucose, vitamin B12, ferrous sulfate, Pluronic F-68, sodium-chlor, HCl, sodium bicarbonate is contained in described fed-batch medium.
In specific embodiment of the invention scheme, containing 5M sodium hydroxide 7.325mL, disodium hydrogen phosphate,anhydrous 3.09g/L, fed-batch medium dry powder Maxfeed40239.03g/L, 50g/L TYR disodium monocalcium salt thing 23.8mL, 50g/L L-cysteine hydrochloride one water thing 23.2mL, glucose 50g/L, 1.75g/L vitamin B12 stock solution 0.3ml/L, 5g/L iron vitriol stock solution 0.3ml/L, Pluronic F-680.3g/L, sodium-chlor 0.24g/L and sodium bicarbonate 0.366g/L in described fed-batch medium.In specific embodiment of the invention scheme, described fed-batch medium is used for feeding culture.In embodiments of the invention, described fed-batch medium is the fed-batch medium of table 1-3.
In embodiments of the invention, the purifying of described step 5) comprises affinity chromatography, anion-exchange chromatography and cation-exchange chromatography successively.
In specific embodiment of the invention scheme, the purification step of described step 5) specifically comprises: first collected the elutriant of protein A affinity chromatography post pH 3.4-3.6 scope (monitoring with 280nm), adjust pH to 7 ~ 9, be loaded to anion-exchange chromatography post, 280nm carries out monitoring and collecting sample, collection liquid pH value is adjusted to 5 ~ 7, is loaded to cation-exchange chromatography post, collect sample, after ultrafiltration and concentration, obtain anti-HER 2 humanized antibody.
In embodiments of the invention, described protein A affinity chromatography post is MabselectSuRe or ProSep Ultra Plus or ProSep vA Ultra or MabCapture A or the protein A affinity chromatography medium with similar functions.
In embodiments of the invention, described anion-exchange chromatography post is Q SepharoseFF or Fractogel TMAE or Poros HQ or Captol Q or the anion-exchange chromatography medium with similar functions.
In embodiments of the invention, described cation-exchange chromatography post is Poros HS or Poros XS or SP Sepharose FF or Fractogel SO3-or Fractogel SH Hicap or the cation-exchange chromatography medium with similar functions.
Sixth aspect present invention relates to composition, and it contains the anti-HER 2 humanized antibody of any one of fourth aspect present invention;
In embodiments of the invention, in described anti-HER 2 humanized antibody, the content of VHS isomer is 0 ~ 0.4%, such as, be 0 ~ 0.2%, 0 ~ 0.1%; Such as, and/or in embodiments of the invention, in described anti-HER 2 humanized antibody, the content of NGHC isomer is 0.2% ~ 1%, is 0.3% ~ 0.75%.
The invention still further relates to the nucleic acid molecule of any one of first aspect present invention, the recombinant vectors of any one of second aspect or the reconstitution cell of any one of the third aspect and prepare the purposes in anti-HER 2 humanized antibody.
In embodiments of the invention, the aminoacid sequence of wherein said anti-HER 2 humanized antibody variable region of light chain is as shown in SEQ ID NO:3, and the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID NO:4.
The anti-HER 2 humanized antibody that the invention still further relates to any one of fourth aspect present invention is preparing the purposes in antitumor drug; Preferably, described tumour is such as mammary cancer, ovarian cancer, prostate cancer, esophagus cancer or cancer of the stomach.
In the present invention, described VHS isomer refers in the modification after protein expression, the isomer that antibody products albumen is cut not exclusively due to N-terminal leader peptase and obtained, described enzyme is cut incomplete N-terminal leader peptide and is contained VHS-sequence, or be made up of VHS-sequence, described VHS-sequence refers to α-amino-isovaleric acid-His-Ser-sequence.
In the present invention, the content of described VHS isomer refers to that VHS isomer accounts for the per-cent of general antibody protein product content.
In the present invention, described NGHC(non-glycosylated heavy chain, the heavy chain of not glycosyafated modification) refer to that in modification, antibody products ferritin heavy chain does not complete glycosylation modified isomer after protein expression.
In the present invention, the content of described NGHC refers to that NGHC isomer accounts for the per-cent of general antibody protein product content.
In the present invention, the described domestication to cell cultivate be instigate cell can in new substratum the process of growth and breeding.
In the present invention, as do not specialized, then per-cent (%) refers to weight percent.
The beneficial effect of the invention
The present invention is by improving nucleotide sequence and the carrier design of encoding antibody, preferred reconstitution cell, the preparation method of optimization aim product antibodies in serum-free, chemical composition determinacy substratum, thus the posttranslational modification mode changed when to express antibody in mammalian cell, obtain not containing VHS isomer with not containing the anti-HER 2 humanized antibody of NGHC isomer, because it has higher purity, more be conducive to the quality and the activity that ensure antibody, substantially increase the production level of anti-HER 2 humanized antibody.
Accompanying drawing explanation
The agarose electrophoresis result of Fig. 1 pcr amplification MIL41/VH, MIL41/V kappa gene fragment; Wherein swimming lane 2,5 is molecular weight Marker DL2000, and swimming lane 1 is the PCR primer of MIL41/V κ, and swimming lane 3,4 is the PCR primer of MIL41/VH.
Schema is cultivated in the domestication of Fig. 2 host cell.
Cell growth curve figure in Fig. 3 host cell domestication process
The preparation of Fig. 4 antibody MIL41, purifying schema.
The IEC color atlas of the appropriate pearl of Fig. 5 handkerchief.
The IEC color atlas of Fig. 6 MIL41.
The overlapping collection of illustrative plates of IEC of the appropriate pearl of Fig. 7 handkerchief and MIL41
The appropriate pearl of Fig. 8 MIL41, handkerchief, irrelevant antibody suppress the experiment knot of MDA-MB-175 propagation
Really.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
The acquisition of embodiment 1 antibody MIL41 nucleotide sequence
Antibody amino acids sequence is the antibody sequence of recombinant human antibody rhuMab2C4, from international monopoly PCT/US2003/021590.Select the codon that frequency of utilization is higher in mammalian cell to carry out reverse translation, by bioinformatics technique reasonably optimizing, utilize molecule modeling and molecular biology mutating experiment, obtain the nucleotide sequence of antibody MIL41 of the present invention.
The light chain nucleotide sequence of antibody MIL41 is:
gatatccagatgacccagagcccctccagcctgtccgccagcgtgggcgaccgcgt gaccatc acctgcaaggccagccaggacgtgagcatcggcgtggcctggtaccagcagaagcc cggcaaggcc cccaagctgctgatctacagcgcctcctaccgctacaccggcgtgccctcccgctt cagcggctccggc agcggcaccgactttaccctgaccatctccagcctgcagcccgaggactttgccac ctactactgccag cagtactacatctatccctataccttcggccagggcaccaaggtggagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaa atctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtc acagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgag caaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcc tgagctcgcccgtcacaaagagcttcaacaggggagagtgt(SEQ ID NO:1), the sequence wherein with underscore is variable region sequences;
The heavy chain nucleotide sequence of antibody MIL41 is:
Gaggtgcagctggtggagagcggcggcggcctggtgcagcccggcggcagcctgcg cctgtc Ctgcgccgccagcggcttcacctttaccgactacaccatggactgggtgcgccagg ctcccggcaagg Gcctggagtgggtggccgacgtgaaccccaacagcggcggcagcatctacaaccag cgcttcaagg Gccgcttcaccctgagcgtggaccgcagcaagaacaccctgtacctgcagatgaac agcctgcgcgc Cgaggacaccgccgtgtactactgcgcccgcaacctgggccccagcttctacttcg actattgggggc AgggcaccctggtcaccgtgagcagcGctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgactgtgccctctagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaagagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa（SEQ ID NO：2), in which the underlined sequences for the variable region sequence.
The light-chain amino acid sequence of antibody MIL41 is:
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQ ID NO：3）；
The heavy chain amino acid sequence of antibody MIL41 is:
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO：4）。
The clone of embodiment 2 antibody MIL41 gene
experiment material:
Phusion polysaccharase, Taq archaeal dna polymerase, restriction enzyme, pGEM-T-easy carrier, Pyrobest DNA polymerase are NEB Products;
DNTP, DNA Marker standard is TaKaRa product;
It is Qiagen Products that DNA reclaims test kit;
Trans2-Blue competence is Quan Shi King Company product;
Littlely carry middle amount plasmid kit (DP107-02) for Tian Gen biochemical corp product;
Carry greatly plasmid kit (DP117) for Tian Gen biochemical corp product;
OPD is Sigma Products;
The goat anti-human antibody of FITC mark: be Pierce Products;
Design of primers is Biosun software;
Primer synthesis (Genewiz), gold is intelligence biotechnology (Beijing) company limited only;
Gene sequencing is completed by Sinogenomax Co., Ltd..
experimental technique and result:
By the weight chain variable region gene of the method synthetic antibody of PCR, be the weight chain variable region gene of MIL41 antibody.Check order correct clone, assembling expression plasmid.Use round pcr complete synthesis MIL41/VH, MIL41/V kappa gene fragment, and introduce suitable restriction enzyme site, at antibody chain variable region gene 5, with 3, end introduces ClaI and BsiwI site, at antibody heavy chain variable region gene 5, with 3, end introduces EcoR I and Nhe I site.
2.1 design of primers
According to the principle of total gene synthesis, utilize computer aided design software, design primer, considers the correlation parameter such as secondary structure, GC content of primer.Every bar gene design 10 primers, are numbered P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10, respectively for total gene synthesis.
2.2 full genome synthesis
1) dilute with sterilized water after primer synthesis, method is as follows:
A) get the centrifugal 2min of primer pipe 12000rpm, primer presses the concentration dilution of 100 μMs ,-20 DEG C of preservations;
B) it is that 10 μMs of conducts use liquid that the primer P2 ~ P9 that takes a morsel is mixed into final concentration;
C) the primer P1 that takes a morsel becomes final concentration to be that 10 μMs of conducts use liquid P1/P10 with P10 mixed diluting;
2) total gene synthesis, adopt Pyrobest DNA polymerase, method is as follows:
A) get 1 μ L P2 ~ P9 mix primer as masterplate, be formulated as follows Overlap PCR system:
It is 50uL that sterilized water supplies cumulative volume
B) above PCR system is reacted as follows:
After reaction terminates, drop to room temperature.
Add primer P1/P10, carry out following PCR reaction:
After reaction terminates, be down to room temperature.
C) 1 ~ 2% agarose gel electrophoresis is separated PCR primer, and antibody heavy chain gene reclaims 440bp size fragment, and light chain gene reclaims 400bp size fragment.
D) reclaim tailing (Pyrobest DNA polymerase can not hold tailing A, so directly can not connect carrier T in PCR primer 3 '), make following 10 μ L systems:
Reaction conditions is as follows: 72 DEG C of 20mins, after reaction terminates, drops to room temperature.
E) get tailing product, connect pGEM-TEasy carrier:
Room temperature connects a 2h or 4 DEG C connection and spends the night, and connects product conversion JM109 intestinal bacteria, is coated on the LB nutrient agar containing 100 μ g/mL penbritins (final concentration).Cultivate in the LB liquid nutrient medium being cloned in containing 100 μ g/mL penbritins (final concentration) obtained, extract plasmid with plasmid extraction kit (vast Imtech), the plasmid of acquisition carries out nucleic acid sequencing qualification.
In above-mentioned steps, through agarose gel electrophoresis analysis after Overlap pcr amplification, obtain the object band (Fig. 1) of special size; Product is through recovery, tailing, clone's equimolecular biology techniques, and successful clone enters pGEM-TEasy carrier; Through order-checking qualification, the gene of synthesis is consistent with aim sequence.
Agarose electrophoresis result: VH is about 440bp goal gene fragment, and V κ is about 400bp object segment, respectively called after MIL41/VH and MIL41/V κ.
The preparation of embodiment 3MIL41 antibody
experiment material
Entrust the dehydrated medium of Hyclone processing, for culturing cell in the domestication of host cell, cell strain screening and antibody preparation process after preparation, in the substratum of configuration, methionine(Met) sulfoximide (the Methionine sulfoximine bought from Sigma is added during cell screening, MSX), for carrying out cell counting after oneself being mixed with solution with the trypan blue dry powder bought from sigma.
method and result
The structure of 3.1 antibody carrier for expression of eukaryon
Select the expression vector pTGS-FRT-DHFR (license number: ZL200510064335.0) that the applicant obtains, remove Totomycin and select label, GS (glutamine synthetase is added by PshA1 and Xho1 restriction enzyme site, glutamine synthetase) express box, as selection markers; Wherein GS cDNA is obtained by RT-PCR from the clone CHO expressing GS.Through improved carrier called after GS carrier.After obtaining the corresponding endonuclease digestion of MIL41 antibody weight chain variable region gene cloning vector (chain variable region gene ClaI and BsiwI digests, and heavy chain variable region gene EcoR I and Nhe I digests), connect with the carrier through identical endonuclease digestion.Through transforming equimolecular biology common technology, building and obtaining carrier for expression of eukaryon.Specifically be implemented as follows:
A) in example 2, MIL41 weight chain variable region gene is building up in pGEM-TEasy carrier, carrier called after MIL41/V κ and MIL41/VH respectively of acquisition;
B) get MIL41/V κ ClaI and BsiwI to digest, obtain MIL41 chain variable region gene;
C) get 1 μ g GS carrier, digest with ClaI and BsiwI.The GS carrier through ClaI and BsiwI digestion connecting gained with T4DNA ligase enzyme and the antibody MIL41 chain variable region gene digested with ClaI and BsiwI.Connect product conversion XLI-blue intestinal bacteria, be coated on the LB nutrient agar containing 100 μ g/mL penbritins (final concentration).Cultivate in the LB liquid nutrient medium being cloned in containing 100 μ g/mL penbritins (final concentration) obtained, extract plasmid with plasmid extraction kit (Tian Gen biochemical corp).The plasmid extracted, after ClaI and BsiwI digestion, with 1% agarose gel electrophoresis analysis, selects a clone carrying antibody MIL41 chain variable region gene.The plasmid called after pTGS-MIL41V κ carrying antibody MIL41 chain variable region gene obtained.
D) get MIL41/VH EcoR I and Nhe I to digest, obtain MIL41 heavy chain variable region gene;
E) get 1 μ g pTGS-MIL41V κ carrier, digest with EcoR I and Nhe I.The pTGS-MIL41V κ carrier digested through EcoR I and Nhe I of gained and the antibody MIL41 heavy chain variable region gene by MIL41/VH vector digestion is connected with T4DNA ligase enzyme.Connect product conversion XLI-blue intestinal bacteria, be coated on the LB nutrient agar containing 100 μ g/mL penbritins (final concentration).Cultivate in the LB liquid nutrient medium being cloned in containing 100 μ g/mL penbritins (final concentration) obtained, extract plasmid with plasmid extraction kit (Tian Gen biochemical corp).The plasmid extracted, after EcoR I and Nhe I digests, with 1% agarose gel electrophoresis analysis, selects a clone carrying antibody MIL41 heavy chain variable region gene.
The plasmid called after MIL41 carrying antibody MIL41 heavy chain variable region gene that pTGS-MIL41V κ basis obtains.
3.2 host cell screening
By the CHO-K1 cell bought from ATCC, carry out domestication by flow process shown in Fig. 2 to cultivate, host cell CHO-K1 is carried out adherent culture in seed culture medium (see table 1-1) (containing 10% calf serum), remove serum (10%-->5%-->2.5%--GreatT.Grea T.GT1.25%--> is not completely containing serum) step by step, transposition shake-flask culture, continue to go down to posterity for several times, host cell suspends completely, be doubled and redoubled stable, finally obtain the stable host cell that can grow in seed culture medium.In host cell domestication process, the result of Growth of Cells as shown in Figure 3.
The preparation of 3.3 peculiar substratum
The preparation of substratum is carried out according to table 1-1,1-2,1-3 composition.After 0.22 μm of film sterile filtration, for cell cultures.
Table 1-1: seed culture medium
Table 1-2: productive culture base
Table 1-3: fed-batch medium
The preparation of 3.4MIL41 antibody
The MIL41 antibody carrier for expression of eukaryon using electrotransfection method step 3.2 to be obtained proceeds in object host cell (host cell that the screening of embodiment 3 step 3.2 obtains), 75 μMs of MSX are added in seed culture medium, cultivate 2 ~ 4 weeks in 37 DEG C of CO2 incubators, pick out the cell can survived in this substratum, detected the cell obtaining and can express antibody by ELISA method.Carry out subclone screening through limiting dilution assay, through cultivation and the screening of 6 ~ 8 weeks, obtaining can the monoclonal cell strain of high expression MIL41 antibody.
Cell strain is through the enlarged culturing of substratum multistep, inoculum density is 0.5 × 106cells/ml, within every three days, go down to posterity once, fermention medium (substratum is for producing substratum: seed culture medium (1:1)) is transferred to after extending to enough cell concentrations, culture cycle is in the fermentation medium 12-14 days, cultivate the fed-batch medium that the 3rd, 6,9 day adds 10% volume, cultivate and terminate rear results supernatant, supernatant is carried out purifying.
Adopt AKTA(GE company) carry out the separation and purification of MIL41 antibody.First the elutriant of protein A affinity chromatography post (MabSelect SuRe) pH 3.4-3.6 scope (monitoring with 280nm) was collected, adjust pH to 8.0, be loaded to anion-exchange chromatography post (Q-Sepharose FF), 280nm carries out monitoring and collecting sample.Collection liquid pH value is adjusted to 5.5, is loaded to cation-exchange chromatography post (Poros) and collects sample.MIL41 antibody is obtained after ultrafiltration and concentration.Idiographic flow as shown in Figure 4.
Prepare antibody MIL41 according to the method described above for following embodiment.
The analysis of embodiment 4MIL41 antibody and activity identification
experiment material
Carry out ion-exchange chromatography (being called for short IEC) with the ion-exchange chromatography bought from Thermo (model: Propac WCX-10,4.0mm × 250mm, manufacturer: Dai An company) to analyze, the MES bought from sigma and the Na from traditional Chinese medicines institutional purchase
2eDTA, NaCl configure IEC moving phase, the carboxypeptidase (CpB) that Shanghai Yaxin Biotech Co., Ltd. buys carries out sample preparation, beta-mercaptoethanol (Sangon Biotech (Shanghai) Co., Ltd., article No.: 10482-100mL), purity detecting test kit (Beckman Coulter, article No.: A10663), do not wrap by kapillary (Beckman Coulter, article No.: 338451), commercially available CCK8 developer, Perjeta is purchased from Haizheng Medicine Stock Co., Ltd., Zhejiang Prov.
methods and results
The analysis of 4.1MIL41 antibody
4.1.1MIL41 the electric charge variable (IEC) of antibody is analyzed
4.1.1.1 sample preparation
The sequence of the C-terminal of the heavy chain of MIL41 monoclonal antibody is-Ser-Pro-Gly-Lys, and in mammalian cell fermenting process, in this sequence, the Methionin (Lys) of C-terminal often can excise by the carboxypeptidase (CpB) entrained by host cell.
And the incomplete removal of this Methionin can cause the electric charge of antibody heterogeneous, in order to inquire into the impact of C-terminal Methionin on the electric charge heterogeneity of test antibodies, this experiment utilizes the carboxypeptidase (CpB) of restructuring by after C-terminal Lys excision residual for test antibodies, then carries out electric charge variable analysis.
After antibody to be measured and standard substance Perjeta are diluted to 1mg/mL, according to 1.5:100(w/w) ratio add 1mg/mL CpB(as: adding 15 μ L1mg/mL CpB to 1000 μ L concentration is in the sample of 1mg/mL), hatch 30 ± 2min in 37 ± 2 DEG C.
4.1.1.2 experimentation
Chromatographic column is Thermo Propac WCX-10,4.0 × 250mm; Mobile phase A: 20mM MES, 1mM Na
2eDTA, pH6.0 ± 0.05; Mobile phase B: 20mM MES, 1mM Na
2eDTA, 250mM NaCl, pH6.0 ± 0.05; Flow velocity is 0.8ml/min; Determined wavelength is 280nm;
HPLC sample introduction, after maintaining starting condition 3min, NaCl concentration is linearly increased to 70mM by 5min afterwards, and NaCl concentration is linearly increased to 145mM by 60min afterwards, and applied sample amount is 50 μ g, detects, calculate each peak area by area normalization method at wavelength 280nm place.
4.1.1.3 experimental result
The IEC collection of illustrative plates of the product P erjeta of listing, result as shown in Figure 5.
According to Chinese invention patent, (application number: report 200580031905.4), A is acid peak, and B is main peak, and C, D, E, F and G peak is alkaline peak.Partial Protein wherein in C peak causes due to a heavy chain having remained C-terminal Lys, and Lys is basic aminoacids, and in weakly strictly diagonally dominant matrix, the existence of Lys residue can make the more difficult wash-out of albumen, and appearance time is more late; Partial Protein in D peak causes due to two heavy chains having remained C-terminal Lys; E peak is mainly because a light chain N holds containing VHS(Val-His-Ser) cause, in Val-His-Ser, the iso-electric point of Histidine is 7.6, belong to the amino acid of alkalescence relatively, N holds the existence of VHS can strengthen the interaction of antibody and weak cation filler, thus more difficult wash-out; F peak is held containing VHS(Val-His-Ser due to light chain N) and a heavy chain C hold and cause containing Lys.
Antibody MIL41 of the present invention analyzes through IEC, and result as shown in Figure 6.
Two are analyzed the result after collection of illustrative plates superposition, as shown in Figure 7.
Fig. 7 is the IEC Overlay chromatograms of MIL41 product and commercialized product Perjeta (Pertuzumab), corresponding very good of each component peaks, does not have significantly new peak to occur, and not containing E, F and G component in MIL41 standard substance.
After collection of illustrative plates is carried out integration, acquired results is as shown in table 2:
Table 2
| Title | VHS content (accounting for the per-cent of total collection of illustrative plates) |
| Perjeta | 7.2% |
| MIL41 | 0.1% |
Note: in table, VHS content refers to the per-cent shared by E and F peak sum.
Visible by above result, MIL41 product and commercialized product Perjeta have the heterogeneous kind of identical electric charge, find no the new peak inconsistent with Perjeta; Compared with Perjeta, main peak ratio is almost consistent; Acid peak A and alkaline C is slightly high; And containing less alkalescence heterogeneous (E, F), this alkalescence is heterogeneous not exclusively to be caused primarily of VHS excision by document supposition.Cause the reason of this result may be difference due to nucleotide sequence, and follow-up cloning expression method, thus modifying upon translation can be different.
4.1.2MIL41 antibody reduction gel electrophoresis (CE-SDS)
4.1.2.1 sample preparation
In antibody, add SDS-MW sample buffer to volume is 95 μ L, makes trial-product final concentration be 1mg/mL, and add 5 μ L beta-mercaptoethanols, 70oC water-bath 10min, water-bath is to be measured after trial-product being down to room temperature.
4.1.2.2 experimentation
Capillary electrophoresis apparatus purchased from Beckman, model PA800+.
Capillary electrophoresis separation method arranges as shown in table 3:
Table 3
| Sample introduction voltage | 5kV | Sample injection time | 20 seconds |
| Separation voltage | 20kV | Disengaging time | 40 minutes |
Data processing software: 32Karat V9.1build10
4.1.2.3 experimental result
After integration, acquired results is as shown in table 4:
Table 4
| Title | LC(light chain, %) | HC(heavy chain, %) | NGHC（%） |
| Perjeta | 23.71 | 63.08 | 1.67 |
| MIL41 | 23.75 | 65.14 | 0.51 |
Result from upper table, MIL41 product by analysis after the content of gained NGHC isomer lower than Perjeta.
4.2MIL41 antibody activity is analyzed
By antibody, the proliferation inhibition test that Her2 positive cell (breast cancer cell MDA-MB-175, ATCC buy) carries out is illustrated to the activity of antibody.96 orifice plate inoculation HTB-25(5 × 10
4individual cells/well), at CO
2after incubator hatches 24h, abandon supernatant and add test antibodies and blank, mixing of vibrating gently, CO
2after incubator hatches 72h, add developer CCK8, CO
2after incubator hatches 2h, detect OD450 value, according to the inhibiting rate of OD450 value calculating antibody for cell proliferation.
Inhibiting rate=(1-experimental group OD450/ blank group OD450) × 100%, acquired results as shown in Figure 8.
From above result, the active indifference of MIL41 antibody and Perjeta.
Comprehensive above, the gene order of encoding antibody the present invention obtained imports in the host cell body that can grow in peculiar substratum, the MIL41 antibody VHS content of isomer produced according to existing technical process is lower, all lower than 0.4%, even reach 0.1%, than patent (Chinese invention patent application number: 200580031905.4), the 1-20% of report is much lower, NGHC content of isomer is lower, about 0.5%.By the known above-mentioned activity that these change antagonist of cell growth inhibition assay without impact.Show that the gene order of encoding antibody the present invention obtained imports in the host cell body that can grow in peculiar substratum, the MIL41 antibody produced according to existing technical process, overall quality is better than commercially available prod Perjeta.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (11)
1. the nucleic acid molecule of coding anti-HER 2 humanized antibody, the nucleotide sequence of its light chain is as shown in SEQ ID NO:1, and the nucleotide sequence of its heavy chain is as shown in SEQ ID NO:2.
2. recombinant vectors, it contains the nucleic acid molecule of claim 1.
3. reconstitution cell, it contains the recombinant vectors of claim 2;
Preferably, described cell is mammalian cell, such as, be CHO-K1 cell;
Further preferably, described mammalian cell is the cell after the domestication of target substratum.
4. anti-HER 2 humanized antibody, it is prepared by the reconstitution cell of the nucleic acid molecule of claim 1, the recombinant vectors of claim 2 or claim 3.
5. the anti-HER 2 humanized antibody of claim 4, the content of wherein said VHS isomer is 0 ~ 0.4%, such as, be 0 ~ 0.2%, 0 ~ 0.1%.
6. the anti-HER 2 humanized antibody of claim 4, the content of wherein said NGHC isomer is 0.2% ~ 1%, such as, be 0.3% ~ 0.75%.
7. the preparation method of the anti-HER 2 humanized antibody of claim 5 or 6, it comprises the step utilizing the reconstitution cell of the nucleic acid molecule of claim 1, the recombinant vectors of claim 2 or claim 3 to prepare this anti-HER 2 humanized antibody.
8. the preparation method of claim 7, it specifically comprises the following steps:
1) nucleotide sequence of the nucleic acid molecule of claim 1 is cloned in expression vector, obtain recombinant expression vector, preferably, described expression vector is that transformation obtains on the basis of carrier pTGS-FRT-DHFR, preferably, described transformation refers to that removing Totomycin selects label, and adds glutamine synthase expression box;
2) recombinant expression vector is proceeded to host cell, such as, in CHO-K1 cell, obtain recombinant host cell, preferably, described host cell is through the CHO-K1 cell after the domestication of target substratum;
3) by step 2) recombinant host cell that obtains cultivates in target substratum, and obtaining can the monoclonal cell strain of high expression antibody;
4) monoclonal cell strain of progressively amplification culture step 3) acquisition, results culture supernatant;
5) culture supernatant that step 4) obtains is carried out the anti-HER 2 humanized antibody that purifying obtains any one of claim 4-6; Preferably, described purifying comprises affinity chromatography, anion-exchange chromatography and cation-exchange chromatography.
9. composition, it contains the anti-HER 2 humanized antibody of any one of claim 4-6;
Preferably, wherein the content of VHS isomer is 0 ~ 0.4%, such as, be 0 ~ 0.2%, 0 ~ 0.1%; And/or
Preferably, wherein the content of NGHC isomer is 0.2% ~ 1%, such as, be 0.3% ~ 0.75%.
10. the reconstitution cell of the nucleic acid molecule of claim 1, the recombinant vectors of claim 2 or claim 3 is preparing the purposes in anti-HER 2 humanized antibody;
The aminoacid sequence of wherein said anti-HER 2 humanized antibody variable region of light chain is as shown in SEQ IDNO:3, and the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID NO:4.
The anti-HER 2 humanized antibody of 11. any one of claim 4-6 is preparing the purposes in antitumor drug; Preferably, described tumour is such as mammary cancer, ovarian cancer, prostate cancer, esophagus cancer or cancer of the stomach.
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