CN104045714B - Herceptin mutant IgG and its application - Google Patents
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Abstract
The invention belongs to genetic engineering and immunological technique fields, disclose Herceptin mutant IgG and its application.The present invention obtains 8 Herceptin IgG mutant by carrying out point mutation to Herceptin heavy chain coding DNA, any one in SEQ ID NO.2~SEQ ID NO.9 of the DNA sequences encoding of heavy chain.The protein purification yield that each mutant transient expression of Herceptin of the present invention obtains is very high with Herceptin.Each mutant IgG can have different degrees of raising to the ADCC effect of Herceptin under the premise of not influencing IgG yield and thermal stability, the same with Herceptin, can apply in preparing anticancer drug.
Description
Technical field
The invention belongs to genetic engineering and immunological technique fields, are related to Herceptin mutant IgG and its application.
Background technique
It appears on the market from the first time as antibody drug in 1986 of OKT3, antibody drug exploitation takes in past 25 years
Obtained mysterious progress.There are five in ten kinds of best-selling drugs in 2010 (Humira, Remicade, Enbrel,
Rituxan and Avastin) it is antibody drug or Fc fusion protein.
Her2 is that being found in the breast cancer patients of 25-30% together for the member of EGFR family has high expression [1], according to
This Her2 is considered as the important target spot that targeted therapy is carried out for breast cancer.Nineteen ninety, Fendly of Genentech etc. is from one
It is separated to monoclonal antibody 4D5 in serial mouse monoclonal antibody, and finds the extracellular domain of this antibody combination Her2, inhibition has the super table of Her2
The growth [2] of the tumour cell reached.4D5 was humanized in 1992, and the antibody after humanization is named as Trastuzumab
[3], trade name Herceptin.Herceptin ratified conduct and Taxol(paclitaxel by FDA in 1998) joint use
The first-line drug of the medicine treatment highly expressed metastatic breast cancer of Her2.Herceptin is also approved for by a wheel or more wheels
Metastatic breast cancer after chemotherapy.In terms of gastrointestinal cancer treatment, Herceptin also goes through to treat together with chemotherapeutics
Stomach and the highly expressed aggressive tumours (http://www.herceptin.com/breast) of the Her2 of stomach oesophagus combined area.
The mechanism of action of Herceptin is believed to comprise following aspects: 1) Herceptin can be by accelerating receptor
Endocytosis and degradation and reduce the level [4] of cell surface Her2, Herceptin can also be multiple by induction p27/Cdk2
The formation for closing object inhibits the progress [5,6] of cell cycle.The third mechanism of action of Herceptin is the thin of induction of antibodies mediation
Cytotoxicity (ADCC), this effect are the Fc γ RIII of the Fc section and bone marrow cell surface antibody receptor by Herceptin
With [5,7] of Fc γ RIIB regulation.In addition to this Herceptin has been also believed to that tumor neovasculature is inhibited to form [8] and tumour
Spread the effect of [5].In these mechanism of action, induction ADCC is considered playing important work in the Herceptin the effect of
With.
Changing amino acid sequence can be used to improve the ability of antibody induction ADCC.Human IgG receptor family includes three
Member Fc γ RI(CD64), Fc γ RII (CD32) and Fc γ RIII(CD16).IgG and different antibody receptors combine can band
Carry out completely different reaction.Combination Fc γ RI, Fc γ RIIA, Fc γ RIIIA combination with activity antibody receptor can bring sharp
Reaction living, leads to the generation to target cell cytotoxicity.With inhibiting antibody receptor, predominantly Fc γ RIIB, in conjunction with can band
Inhibit to react, inhibits generation [9] (Ravetch, Annual the review of to target cell cytotoxicity
Immunology2001).
By the screening in a library, Stavenhagen etc. obtains the binding ability to Fc γ RIIIA and Fc γ IIA
The Fc mutant being significantly increased, these mutant include F243L/R292P/Y300L/V305I/P396L, F243L/R292P/
Y300L/P396L, F243L/R292P ,/Y300L.These mutant in vitro ADCC experiment in show it is more obvious than wild type Fc
The ability of the induction ADCC of raising.These Fc mutant are shown at low concentrations better than wild-type antibodies in animal experiments
Inhibit the ability [10] of tumour growth.
It is tested by ELISA and AlphaScreen, Anderson etc., which has been screened, ties inhibiting antibody receptor Fc γ RIIB
Fc mutant resultant force decline or activity antibody receptor Fc γ RIIIA binding force is risen.A part of these variations is in body
Show that the antibody than wild type Fc containing wild type is about higher by the ability [11] of 10 times of induction ADCC in outer experiment.
In another trial, Tsuji etc. has only been mutated a site 295 on Fc.External knot on cellular level
It closes experiment display this mutant Fc and is improved eight times than wild type Fc with CD16 binding ability, and highest is combined and mentioned from 80%
Height is to 95%.In vitro experiment, the antibody comprising this Fc mutant, which is shown, is higher by about 1,000 than wild type Fc antibody
The ability of induction ADCC again.In addition to this, the antibody comprising this mutant includes wild lower than 0.001 μ g/ml(in concentration
Type Fc antibody effect usual concentration) when still can induce ADCC react [12].
Summary of the invention
The purpose of the present invention is being directed to the above-mentioned deficiency of the prior art, Herceptin mutant IgG is provided.
It is a further object of the present invention to provide the applications of mutant IgG.
The purpose of the present invention can be achieved through the following technical solutions:
Herceptin mutant IgG, the DNA sequences encoding of heavy chain is in SEQ ID NO.2~SEQ ID NO.9
Any one；Any one in SEQ ID NO.11~SEQ ID NO.18 of amino acid sequence, light chain is same
Trastuzumab wild type, light chain DNA sequences encoding are SEQ ID19, and protein sequence is SEQ ID20.
The recombinant expression plasmid for expressing Herceptin mutant IgG, containing SEQ ID NO.2 of the present invention~
Any one in SEQ ID NO.9.
Herceptin mutant IgG heavy chain DNA sequences encoding, wherein the heavy chain-coding sequence of mutant 1IgG is SEQ
The heavy chain-coding sequence of ID NO.2, mutant 2IgG are SEQ ID NO.3, and the heavy chain-coding sequence of mutant 3IgG is SEQ
The heavy chain-coding sequence of ID NO.4, mutant 4IgG are SEQ ID NO.5, and the heavy chain-coding sequence of mutant 5IgG is SEQ
The heavy chain-coding sequence of ID NO.6, mutant 6IgG are SEQ ID NO.7, and the heavy chain-coding sequence of mutant 7IgG is SEQ
The heavy chain-coding sequence of ID NO.8, mutant 8IgG are SEQ ID NO.9.
Herceptin mutant IgG heavy chain amino acid sequence, mutant 1IgG heavy chain amino acid sequence are SEQ ID
NO.11, mutant 2IgG heavy chain amino acid sequence are SEQ ID NO.12, and mutant 3IgG heavy chain amino acid sequence is SEQ ID
NO.13, mutant 4IgG heavy chain amino acid sequence are SEQ ID NO.14, and mutant 5IgG heavy chain amino acid sequence is SEQ ID
NO.15, mutant 6IgG heavy chain amino acid sequence are SEQ ID NO.16, and mutant 7IgG heavy chain amino acid sequence is SEQ ID
NO.17, mutant 8IgG heavy chain amino acid sequence are SEQ ID NO.18.
The expression plasmid preferably arrives any one DNA sequence dna recombination in SEQ ID NO.2~SEQ ID NO.9
Gained in carrier for expression of eukaryon pTGE5.
Host cell containing Herceptin mutant IgG coding DNA.
The host cell preferably comprises the HEK293-6E cell of recombinant expression plasmid of the present invention.
Herceptin mutant IgG of the present invention is preparing the application in anticancer drug.
Herceptin mutant IgG of the present invention is preferably preparing answering in anti-breast cancer and/or gastric cancer medicament
With.
Recombinant expression plasmid of the present invention is preparing the application in anticancer drug.
Recombinant expression plasmid of the present invention is preferably preparing the application in anti-breast cancer and/or gastric cancer medicament.
Herceptin of the present invention, that is, Trastuzumab wild type.
The utility model has the advantages that
The present invention obtains 8 Herceptin IgG mutation by carrying out point mutation to Herceptin heavy chain coding DNA
Body.(i.e. Trastuzumab is wild for the same Herceptin of protein purification yield that each mutant transient expression of Herceptin obtains
Raw type) it is very high.Under the adjusting of PBMC cell, the ADCC effect that mutant 1,2,3,6,7,8 mediates is all (i.e. than Herceptin
Trastuzumab wild type) it increases；Under the adjusting of NK92/CD16A cell, 8 mutant mediated ADCC effects all compare
Herceptin (i.e. Trastuzumab wild type) increases.The mutation as the result is shown of IgG thermal stability is surveyed with circular dichroism spectra
The thermal stability of IgG, which has no, afterwards is remarkably decreased.Therefore, Herceptin (i.e. Trastuzumab wild type) of the present invention is several
Mutant IgG can to Herceptin, (i.e. Trastuzumab be wild under the premise of not influencing IgG yield and thermal stability
Type) ADCC effect have different degrees of raising, same to Herceptin (i.e. Trastuzumab wild type) equally, can prepare
It is applied in anti-breast cancer and/or gastric cancer medicament.
Detailed description of the invention
The SDS-PAGE(A of Fig. 1 mutant 2) and Western blot(B) electrophoretogram.
Lane M is protein Marker, and Lane1 is the culture medium supernatant after transfection 6 days under reducing condition, and Lane2 is
The culture medium supernatant after transfection 6 days under non reducing conditions, Lane P be positive control Human IgG1, Kappa (Sigma,
Cat.No.I5154)。
The SDS-PAGE electrophoresis of Fig. 2 wild type of Trastuzumab and mutant after purification.
The target cell apoptosis that Fig. 3 .PBMC cell is adjusted is with the wild type of Trastuzumab and the change of each mutation bulk concentration
Change.Wherein smoothed curve is curve after fitting.
The target cell apoptosis that Fig. 4 .NK92/CD16A cell is adjusted is dense with the wild type of Trastuzumab and each mutant
The variation of degree.Wherein smoothed curve is curve after fitting.
The ellipticity variation with temperature of Fig. 5 .Trastuzumab wild type and each mutant in 202nm and 208nm is bent
Line.Wherein smoothed curve is to be fitted the curve obtained after experimental data with temperature-induced binary states fibrosis models.
Specific embodiment
The present invention is further described and explained with reference to the accompanying drawings and embodiments.
Reagent involved in following embodiment and instrument:
1, reagent
Freestyle293 culture medium (Invitrogen, Carlsbad, CA, USA)
PEI(Polysciences,Eppelheim,Germany)
Goat Anti-Human IgG–HRP(GenScript,Cat.No.A00166)
Protein A Column (GE Healthcare, Cat.No.17-0402-01)
Fetal Bovine Serum(Gibco,Invitrogen,Cat.No.10437-036)
Penicilin-Streptomycin(Gibco,Invitrogen,Cat.No.10378)
Phosphate-Buffered Saline(Gibco,Invitrogen Cat.No.10010-023)
Phenol red free MEM culture medium (Invitrogen, Cat.No.41061)
96 orifice plates (Greiner, Cat.No.655180)
Lebovitz ' s L-15 culture medium (Gibco, Invitrogen, Cat.No.11415, Lot No.862546)
PTGE5 carrier for expression of eukaryon (National Research Council of Canada, NRC)
2, cell line
HEK293-6E(National Research Council of Canada,NRC)
NK92/CD16A cell is that the stabilization of the NK92 cell (ATCC, Cat.No.CRL-2407) of GenScript production is thin
Born of the same parents system
MDA-MB-453(ATCC,Cat.No.HTB-131)
3, assay kit
Quick StartTM Bradford Protein Assay(Bio-Rad,Cat.No.500-0201)
LDH kit (Roche, Cat.No.11644793001)
ToxinSensorTM Endotoxin Removal Kit(GenScript,Cat.No.L00338)
ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit(GenScript,
Cat.No.L00350)
4, instrument
purifier10purification system(GE Healthcare)
FlexStation3(Molecular Devices)
Jasco J-815CD spectrometer(Jasco Inc.)
Embodiment 1
The building of 1.1 carrier for expression of eukaryon
It is respectively synthesized the full length DNA of eight mutant IgG and the wild type full-length DNA sequence dna of Trastuzumab,
The wild type heavy chain DNA sequence dna of Trastuzumab is SEQ ID NO.1, and the heavy chain DNA sequences of mutant 1IgG are SEQ ID
The heavy chain DNA sequences of NO.2, mutant 2IgG are SEQ ID NO.3, and the heavy chain DNA sequences of mutant 3IgG are SEQ ID
The heavy chain DNA sequences of NO.4, mutant 4IgG are SEQ ID NO.5, and the heavy chain DNA sequences of mutant 5IgG are SEQ ID
The heavy chain DNA sequences of NO.6, mutant 6IgG are SEQ ID NO.7, and the heavy chain DNA sequences of mutant 7IgG are SEQ ID
The heavy chain DNA sequences of NO.8, mutant 8IgG are SEQ ID NO.9, the protein sequence of these clones is followed successively by SEQ ID10-
18.The light chain of Trastuzumab wild type and all mutant described herein is all the same, and DNA sequence dna is SEQ ID19,
Protein sequence is SEQ ID20.It is artificial synthesized that these are cloned in GenScript, it is verified it is correct after, inserted with the method for recombination
Enter into carrier for expression of eukaryon pTGE5, obtains each expression plasmid for expressing each mutant overall length IgG.
1.2 cell culture and transient transfection
In conical flask with serum-free Freestyle293 culture medium culture HEK293-6E cell (37 DEG C, 5%CO2).Deng
After growing to suitable concentration to cell, transfected in the mixture that each expression plasmid and PEI prepared by embodiment 1 is wherein added.
After transfection six days, culture medium supernatant is collected, detects the expression quantity of each IgG with SDS-PAGE, Western blot and ELISA.
1.3IgG purification
Each IgG that 1.2 obtain is loaded to protein A column, is used purifier10purification
System purifies destination protein from cell pyrolysis liquid.Protein storage is in PBS buffer solution (pH7.4) after purification, Zhi Houyong
ToxinSensorTMKit is made a return journey the endotoxin in removing protein.The purity and the non-reduced SDS-PAGE of molecular weight of destination protein
It analyzes protein concentration and uses Quick StartTMBradford Protein Assay is calculated.
By taking mutant 2 as an example, in the culture medium supernatant of HEK293-6E cell, under the reducing conditions with SDS-PAGE and
Western blot can detect the obvious band about in 55kDa and 25kDa, the respectively heavy chain and light chain of antibody, non-
Under reducing condition, it can detect about at the band of 160kDa (such as Fig. 1), illustrate that mutant 2IgG antibody can be gone out by successful expression
Come.
1 institute of purity and yield such as Fig. 2 and table after purification of the albumen that transient expression obtains protein A chromatographic column
Show.The yield of each antibody is all very high, the yield slightly lower (44.5mg/l) prepared in addition to Trastuzumab wild type second with
Outside, for other antibody production rates all in 100mg/l or more, the yield that mutant 2 is prepared for the first time is more up to 629mg/l.After purification
Antibody purity it is higher, all 80% or more, (except mutant 6, purity is general 60%).
The purification yield of table 1.Trastuzumab wild type and mutant
1.4ADCC measurement
Respectively with peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) and NK92/
CD16A cell is as effector cell, and breast cancer MDA-MB-453 cell is as target cell.First by the anti-of target cell and various concentration
Body mixes at room temperature, and addition effector cell, which mixes gently, after incubation half an hour is placed on 37 DEG C of CO2Culture 5 is small in incubator
When.With LDH kit test result.Experimental result is analyzed using software Prism4 (GraphPad Software, Inc.).
The percentage of target cell lysis when drawing PBMC and NK92/CD16A cell as effector cell according to experimental data
It is as shown in Figure 3 and Figure 4 respectively with the curve that IgG concentration changes.The ADCC for the Trastuzumab wild type that front and back purifies twice
Effect curve can be completely coincident, it was demonstrated that the Trastuzumab of second of purifying has with the Trastuzumab of first time
Effect.Table 2 is to obtain EC after experimental data brings formula fitting into50With the numerical value of maximum cell cracking percentage.In PBMC cell
Under adjusting, the maximum cell lysis efficiency of each mutant all with the suitable of Trastuzumab wild type, mutant 1,2,3,6,
7, the EC of the 8 ADCC effects mediated50Be worth it is also more much lower than same plate Trastuzumab wild type, illustrate mutant 1,2,3,
6,7, the 8 ADCC effects mediated all increase than Trastuzumab wild type.Under the adjusting of NK92/CD16A cell, in addition to
Mutant 4,5 and Trastuzumab wild type maximum cell lysis efficiency quite other than, the maximum cell of other mutant is split
It is all higher by 50% or more than wild type to solve efficiency, and the EC of all mutant mediated ADCC effects50Value is all than same plate
Trastuzumab wild type it is low, illustrate that all mutant mediated ADCC effects are all higher than Trastuzumab wild type.
The ADCC that table 2.PBMC cell and NK92/CD16A cell are adjusted
* P1, P2, P3 and P4 are respectively the first plate (for the reference of mutant 1,2,3), and the second plate is (for mutant 4,5,6
Reference), third plate (for mutant 7,8, the reference of the mutant 2 of second of preparation), the 4th plate is (for first time preparation
The reference of Trastuzumab).
1.5IgG thermal stability determination
Each mutant IgG is changed in the buffer solution of 10mM sodium phosphate pH7.4, sample temperature from 313K (40 DEG C) by
It gradually slowly rises to 363K (90 DEG C), temperature acquires the once Far-UV circular dichroism from 180nm to 260nm every 2 DEG C.Monitoring
The ellipticity variation with temperature of 202nm and 208nm is fitted experimental data with temperature-induced binary states fibrosis models
Obtain the denaturation neutral temperature (Tm) of IgG.Data analysis uses software Prism4 (GraphPad Software, Inc.).
Trastuzumab wild type and each mutant, which vary with temperature curve in the ellipticity of 202nm and 208nm, such as schemes
Shown in 5.Since 342K(69 DEG C), the secondary structure of each IgG starts to gradually decrease, at 352K(79 DEG C) left and right IgG loses
All second-order structure becomes random coil.It is fitted experimental data with temperature-induced binary states fibrosis models, is obtained in 202nm
It is as shown in table 3 with IgG denaturation neutral temperature (Tm) of 208nm monitoring.Except the Tm value ratio Trastuzumab wild type of mutant 7
Other than slightly lower, other mutant it is all equal or higher with the Tm value of Trastuzumab wild type, illustrate mutation after IgG heat
Stability, which has no, to be remarkably decreased.
The denaturation neutral temperature of table 3.Trastuzumab wild type and mutant
[bibliography]
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Claims (10)
1. Herceptin mutant IgG, it is characterised in that the DNA sequences encoding of its heavy chain is SEQ ID NO.7；Its light chain is compiled
Code DNA sequence dna is SEQ ID NO.19.
2. Herceptin mutant IgG according to claim 1, it is characterised in that the amino acid sequence of its heavy chain is
SEQ ID NO.16。
3. expressing the recombinant expression plasmid of Herceptin mutant IgG, it is characterised in that contain the song described in claim 1
The DNA sequences encoding of trastuzumab mutant IgG.
4. the recombinant expression plasmid of expression Herceptin mutant IgG according to claim 3, it is characterised in that described
Expression plasmid be by the DNA sequences encoding of the IgG of Herceptin mutant described in claim 1 recombination to eukaryotic expression
Gained in carrier pTGE5.
5. the host cell containing the Herceptin mutant IgG DNA sequences encoding described in claim 1.
6. host cell according to claim 5, it is characterised in that the host cell is containing described in claim 3
The HEK293-6E cell of recombinant expression plasmid.
7. Herceptin mutant IgG described in claim 1 is preparing the application in anticancer drug.
8. application according to claim 7, it is characterised in that Herceptin mutant IgG described in claim 1 exists
Prepare the application in anti-breast cancer and/or gastric cancer medicament.
9. recombinant expression plasmid described in claim 3 is preparing the application in anticancer drug.
10. application according to claim 9, it is characterised in that recombinant expression plasmid described in claim 3 is preparing anti-cream
Application in gland cancer and/or gastric cancer medicament.
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Non-Patent Citations (3)
| Title |
|---|
| Asymmetrical Fc Engineering greatly enhances antibody-dependent cellular cytotoxicity(ADCC) effctor function and stability of the modified antibodies;Zhi Liu等;《The journal of biological chemistry》;20140207;第289卷(第6期);第3571-3590页 |
| Engineered antibody Fc variants with enhanced effector function;Lazar G.A等;《PNAS》;20060314;第103卷(第11期);第4005-4010页 |
| Resistance to Trastuzumab in Breast Cancer;Pohlmann PR等;《Clin Cancer Res》;20091215;第15卷(第24期);第7479-7491页 |
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