CN105017421B - A kind of anti-HER2 bispecific antibodies and preparation method and application - Google Patents
A kind of anti-HER2 bispecific antibodies and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of anti-HER2 bispecific antibodies and preparation method and application.A kind of antibody disclosed by the invention, it is made up of two identical half molecules, wherein each half molecule passes through disulfide bond by being made up of a light chain and a heavy chain for disulfide bond between two heavy chains of described two half molecules；The amino acid sequence of the heavy chain is as shown in SEQ ID No.19, and the amino acid sequence of the light chain is as shown in SEQ ID No.20.A kind of anti-HER2 bispecific antibodies disclosed by the invention have the significant breast cancer cell in-vitro multiplication activity for suppressing the high expression of HER2 and had broad application prospects in vivo into function of tumor, the antibody in the treatment of breast cancer.
Description
Technical field
The present invention relates to a kind of antibody and its preparation and application；More particularly to a kind of anti-HER2 bispecific antibodies and its
Preparation method and application, belong to biological technical field.
Background technology
Breast cancer is one of major malignant tumor for threatening women's health, and it is various that statistics show that its incidence of disease accounts for whole body
The 7%-10% of malignant tumour.The annual whole world there are about 1,200,000 breast cancer new cases, there are about 500,000 women and dies from breast cancer.
HER2 genes are the major pathogenicity-related genes of breast cancer, and its product has overexpression phenomenon, HER2 mistakes in 20%-30% patient
Degree expression prompting patient's prognosis mala, and resistance is produced to cytotoxic chemotherapeutics（Slamon DJ,Clark GM,Wong SG,
Levin WJ,Ullrich A,McGuire WL.Human breast cancer:correlation of relapse and
survival with amplification of the HER-2/neu oncogene.Science.1987Jan9;235
(4785):177-82）.
Human epidermal growth factor acceptor (human epidermal growth factor receptor) family has 4
Member：EGFR、HER2（ErbB-2）, ErbB3 and ErbB4, wide expression is in epithelium, interstitial and nerve
Organize, they participate in the cellular signal transduction pathways of activation a series of complex under physiological status, regulate and control the life of normal tissue cell
The important physiology courses such as long, division, differentiation.However, studies have shown that the generation of some tumours, development and malignant behaviors with
The unconventionality expression of EGFR families and activation are in close relations（Albanell J,Codony J,Rovira A,Mellado B,Gascó
n P.Mechanism of action of anti-HER2monoclonal antibodies:scientific update
on trastuzumab and2C4.Adv Exp Med Biol.2003;532:253-68）.HER2 does not have native ligand, in list
It is inactive under body state, tyrosine kinase activation can be made when forming dimer, MAPK and PI3K/Akt paths is activated, finally leads
Cause tumor cell proliferation and survival（Hudis CA.Trastuzumab-mechanism of action and use in
clinical practice.N Engl J Med.2007Jul5;357(1):39-51）.
Anti-HER 2 humanized monoclonal antibody trastuzumab（Trade name Herceptin, Herceptin, Trastuzumab）
It is the Humanized monoclonal antibodies for HER2 that Genentech companies of the U.S. develop, U.S. FDA approval was obtained in 1998 and is used
In the treatment of the high expression metastatic breast cancers of HER2.But Trastuzumab objective reactivity is only 30%（Vogel CL,
Cobleigh MA,Tripathy D,Gutheil JC,Harris LN,Fehrenbacher L,Slamon DJ,Murphy
M,Novotny WF,Burchmore M,Shak S,Stewart SJ,Press M.Efficacy and safety of
trastuzumab as a single agent in first-line treatment of HER2-overexpressing
metastatic breast cancer.J Clin Oncol.2002Feb 1;20(3):719-26；Nahta R,Yu D,
Hung MC,Hortobagyi GN,Esteva FJ.Mechanisms of disease:understanding
resistance to HER2-targeted therapy in human breast cancer.Nat Clin Pract
Oncol.2006May;3(5):269-80；Kümler I,Tuxen MK,Nielsen DL.A systematic review of
dual targeting in HER2-positive breast cancer.Cancer Treat Rev.2014;40(2):
259-70.）.And Trastuzumab is a humanized antibody, mouse source CDR region and a small amount of FR areas mouse source residue are remained, still
Fail to reach full humanization.Develop the active demand that more efficiently anti-HER2 therapeutic antibodies are clinical treatments.
Phage antibody library is the earliest antibody library for occurring and being most widely used at present.Phage display is that one kind will
The skill that foreign protein genes and the capsid protein gene of bacteriophage make exogenous protein expression be presented in phage surface after blending
Art.Phage antibody library is exactly that make use of principles above different specific antibody expressions is used in different phage surfaces
Antigen screens to them.Target cell for building phage antibody library can be hybridoma, immune human B cell
Or non-immunized human B cell.Non-immunized human B lymphocyte is to apply most target cells at present, and its storage capacity is big, reason
Contain all people source antibody gene on.The screening of phage antibody library is exactly the mistake for simulating internal antibody affinity maturation
Journey, with the phage library of immobilization Antigen adsorption surface expression specific antibody, free bacteriophage is then eluted, is inhaled with antigen
" absorption-elution-amplification " taken turns more again after attached phage-infect Host Strains propagation amplification is until screen special
Human antibody.
The content of the invention
It is an object of the invention to provide a kind of anti-HER2 bispecific antibodies and preparation method and application, the antibody is complete
Humanized antibody, there is stronger inhibitory action to breast cancer cell.
A kind of antibody provided by the invention, it is made up of two identical half molecules, wherein each half molecule is by passing through two sulphur
One light chain of key connection and a heavy chain form, and pass through disulfide bond between two heavy chains of described two half molecules；Institute
The amino acid sequence of heavy chain is stated as shown in SEQ ID No.19, the amino acid sequence of the light chain is as shown in SEQ ID No.20.
In above-mentioned antibody, in the coding gene sequence such as SEQ ID No.17 of the heavy chain the 73rd to from 5 ' ends
Shown in 1824 nucleotides；
In the coding gene sequence such as SEQ ID No.18 of the light chain from 5 ' ends the 82nd to the 1146th nucleosides
Shown in acid.
A kind of method for preparing any of the above-described antibody falls within protection scope of the present invention, comprises the following steps：Will
Contain the recombinant expression plasmid of DNA molecular shown in SEQ ID No.17 and the restructuring containing DNA molecular shown in SEQ ID No.18
Expression plasmid is imported in vitro mammalian cell, and screening obtains the transfectional cell of stable expression purpose antibody, then is expanded
Big culture, purifying obtain purpose antibody.
In the above method, the recombinant expression plasmid containing DNA molecular shown in SEQ ID No.17 is by SEQ ID
Obtained between Hind III and EcoR I site of DNA molecular insertion pcDNA3.1 (+) shown in No.17；
The recombinant expression plasmid containing DNA molecular shown in SEQ ID No.18 is by shown in SEQ ID No.18
Obtained between DNA molecular insertion pcDNA3.1 (+) Hind III and EcoR I site.
In any of the above-described described method, the mammalian cell is CHO-K1 cells；
Culture medium used is EX-CELL302 serum free mediums during the expansion culture, and the culture medium is purchased from SIGMA-
ALDRICH companies.
Application of any of the above-described described antibody in the product for preparing anti-human epidermal growth factor acceptor 2 falls within this
The protection domain of invention.
Application of any of the above-described described antibody in the product for suppressing breast cancer cell growth and/or propagation is prepared also belongs to
In protection scope of the present invention.
The breast cancer cell is specially human breast cancer cell；
The human breast cancer cell is specially human breast cancer cell BT-474, human breast cancer cell HCC-1569 or people's mammary gland
Cancer cell HCC-1419.
Any of the above-described described antibody suppresses breast cancer cell in preparation and forms answering in the product of tumour in animal body
With falling within protection scope of the present invention.
The breast cancer cell is specially human breast cancer cell；
The human breast cancer cell is specially human breast cancer cell BT-474 or human breast cancer cell HCC-1569.
In any of the above-described described application, the breast cancer cell is overexpression protection ErbB-2
Breast cancer cell.
Application of any of the above-described described antibody in anti-breast cancer medicines are prepared falls within protection scope of the present invention.
In above-mentioned application, the breast cancer is human breast carcinoma.
Anti- HER2 bispecific antibodies HER2-BsAb provided by the invention has the significant mammary gland for suppressing the high expression of HER2
Cancer cells in vitro proliferation activity and in vivo into function of tumor, shows that the antibody has wide application in the treatment of breast cancer
Prospect.
Brief description of the drawings
Fig. 1 is growth in vitro inhibitory activity of the anti-HER 2 monoclonal antibody to breast cancer cell BT-474.
Fig. 2 is growth in vitro inhibitory activity of the anti-HER 2 monoclonal antibody to breast cancer cell HCC-1569.
Fig. 3 is growth in vitro inhibitory activity of the anti-HER 2 monoclonal antibody to breast cancer cell HCC-1419.
Fig. 4 is anti-HER 2 monoclonal antibody to antitumor action inside BT-474 breast cancer.
Fig. 5 is anti-HER 2 monoclonal antibody to antitumor action inside HCC-1569 breast cancer.
Fig. 6 is growth in vitro inhibitory activity of the anti-HER2 bispecific antibodies to breast cancer cell BT-474.
Fig. 7 is growth in vitro inhibitory activity of the anti-HER2 bispecific antibodies to breast cancer cell HCC-1569.
Fig. 8 is growth in vitro inhibitory activity of the anti-HER2 bispecific antibodies to breast cancer cell HCC-1419.
Fig. 9 is anti-HER2 bispecific antibodies to antitumor action inside BT-474 breast cancer.
Figure 10 is anti-HER2 bispecific antibodies to antitumor action inside HCC-1569 breast cancer.
Embodiment
Only the present invention is further detailed for following examples, should not be construed as limiting the invention.
Embodiment does not include detailed descriptions of conventional methods, such as：Overlapping PCR react, and restricted digestion is anti-
Should, gene is inserted into plasmid vector, method that plasmid is introduced into host cell etc..Such method in this area for having
The personnel of ordinary skill be it is well known that and be all described in many publications, such as Sambrook, J.,
Fritsch, E.F.and Maniais, T. (1989) Molecular Cloning：A Laboratory Manual,
2ndedition, Cold spring Harbor Laboratory Press.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Human breast cancer cell BT-474 is purchased from American Type Culture Collection（ATCC）, catalogue
Number it is HTB-20.
Human breast cancer cell HCC-1569 is purchased from ATCC, catalog number CRL-2330.
Human breast cancer cell HCC-1419 is purchased from ATCC, catalog number CRL-2326.
PGEM-T carriers are purchased from Promega companies.
PcDNA3.1 (+) is purchased from Invitrogen companies.
CHO-K1 cells are purchased from ATCC, and catalog number is ATCC CRL-9618.
Lipofectamine2000Reagent is purchased from Invitrogen companies.
QIAGEN OneStep RT-PCR Kit are purchased from QIAGEN companies.
RProtein A Sepharose4Fast Flow are purchased from GE companies.
CellTiter96AQueous One Solution Cell Proliferation Assay (MTS) kit
Purchased from Promega companies, catalog number G3580.
Anti-HER 2 humanized antibody Pertuzumab（(Perjeta）Purchased from Roche companies.
Anti-HER 2 humanized antibody Trastuzumab（Herceptin）Purchased from Roche companies.
CD 20 antagonizing Chimeric antibody Rituximab（Rituxan）Purchased from Roche companies.
6 week old female BAl BIcs/c nude mices are purchased from Military Medical Science Institute's Experimental Animal Center.
The preparation of embodiment 1, full people source anti-HER 2 monoclonal antibody
First, human antibody light chain constant region, the clone of weight chain constant area gene
With lymphocyte separation medium (being purchased from Beijing Ding Guo biotechnologys Co., Ltd) separating health human lymphocyte,
Its total serum IgE is extracted with Trizol reagents (being purchased from Invitrogen companies), according to document " HieterPA, Max EE, Seidman
JG, Maizel JV Jr, Leder P.Cloned human and mouse kappa immunoglobulin constant
and J region genes conserve homology in functional segments.Cell.1980；22
(1Pt1):197-207 " and document " Ellison JW, Berson BJ, Hood LE.The nucleotide sequence of
a human immunoglobulin C gamma1gene.Nucleic Acids Res.1982Jul10；10(13)：4071-
It is permanent using the heavy chain and light chain of QIAGEN OneStep RT-PCR Kit amplification human antibodies that the sequence of 9 " reports separately designs primer
Determine area's gene, the heavy chain of human antibody and light chain constant region gene are connected respectively in pGEM-T carriers, obtain recombinant plasmid point
PGEM-T/C is not named as itHAnd pGEM-T/CL, confirm after sequence verification to obtain correct clone.
SEQ ID No.1 and SEQ ID No.2 respectively illustrate human antibody heavy chain's constant region (CH) nucleotide sequence and
Amino acid sequence.SEQ ID No.3 and SEQ ID No.4 respectively illustrate human antibody light chain constant region (CL) nucleotide sequence
And amino acid sequence.
2nd, the screening of phage antibody library
Antigen protein " people's HER2 film outskirts albumen " for elutriation is according to document " Carter P1, Presta L,
Gorman CM,Ridgway JB,Henner D,Wong WL,Rowland AM,Kotts C,Carver ME,
ShepardHM.Humanization of an anti-p185HER2antibody for human cancer
therapy.ProcNatl Acad Sci U S A.1992May15;89(10):It is prepared by 4285-9 " method.
Using people HER2 film outskirt albumen with full Large human naive scFv phage library（The antibody library document " Qiao Yuanyuan, Wang Yan,
Chen Xiaosui, Wang Yuxiao, the structure of change ice Large phage libraries and identification, Chinese microbiology and Journal of Immunology,
2004,24 (3), mistake disclosed in 194-197 "）According to document, " king is intended to Xiao Qiaoyuanyuan weeks beautiful king's a kind of jade from high-capacity antibody library
Screen anti-tnf-alpha people single-chain antibody cells and molecular immunology magazine .2008,24 (9):878-880 " method carries out take turns more
Screening, is finally obtained two plants of HER2 specific bacteriophage antibody clonings, 5A7Fv and 9C12Fv, its gene sequence is obtained after sequencing
Row.
Phage antibody clones 5A7Fv weight chain variable district VHNucleotide sequence as shown in SEQ ID No.5, its amino
Acid sequence is as shown in SEQ ID No.6；Light chain variable district VLNucleotide sequence as shown in SEQ ID No.7, its amino acid sequence
Row are as shown in SEQ ID No.8.
Phage antibody clones 9C12Fv weight chain variable district VHNucleotide sequence as shown in SEQ ID No.9, its ammonia
Base acid sequence is as shown in SEQ ID No.10；Light chain variable district VLNucleotide sequence as shown in SEQ ID No.11, its amino
Acid sequence is as shown in SEQ ID No.12.
3rd, anti-HER2 complete antibodies 5A7 preparation
（One）The structure of 5A7 heavy chain expression vector
With 5A7Fv antibody heavy chain variable region genes（SEQ ID No.5）And pGEM-T/CHFor template, design primer passes through
Overlapping PCR reactions obtain full people source anti-HER 2 monoclonal antibody 5A7 heavy chain gene, by this Overlapping
PCR primer is denoted as 5A7SH.The nucleotide sequence of 5A7SH genes is as shown in SEQ ID No.13.In SEQ ID No.13,
HindIII restriction enzyme sites are the signal peptide sequence in SEQ ID No.13 from 5 ' ends shown in the 1st to the 6th nucleotides
For in SEQ ID No.13 from 5 ' ends shown in the 16th to the 72nd nucleotides, 5A7 heavy chain of antibody 5A7VHCHGene order
For from 5 ' ends shown in the 73rd to the 1425th nucleotides, translation stop codon is SEQ ID in SEQ ID No.13
In No.13 from 5 ' ends shown in the 1426th to the 1428th nucleotides, restriction enzyme sites EcoR I is in SEQ ID No.13
From 5 ' ends shown in the 1429th to the 1434th nucleotides.5A7SH genes are inserted into pGEM-T carriers, obtain recombinating matter
Grain, is named as pGEM-T/5A7SH, is sent sequencing, as a result correctly.
Utilize restriction enzyme Hind III (being purchased from Promega companies) and EcoR I (being purchased from Promega companies) double digestion
PGEM-T/5A7SH, obtain genetic fragment；Using restriction enzyme Hind III and EcoR I double digestion pcDNA3.1 (+) plasmid,
Obtain carrier large fragment；Genetic fragment is connected with carrier large fragment, recombinant plasmid is obtained, is named as pcDNA3.1 (+)
(5A7SH), send pcDNA3.1 (+) (5A7SH) to sequencing, as a result correctly.
（Two）The structure of 5A7 light chain expression vector
With 5A7Fv antibody chain variable region genes（SEQ ID No.7）And pGEM-T/CLCarrier is template, is passed through
Overlapping PCR reactions obtain full people source anti-HER 2 monoclonal antibody 5A7 light chain gene, by this Overlapping
PCR primer is denoted as 5A7SL.The nucleotide sequence of 5A7SL genes is as shown in SEQ ID No.14.In SEQ ID No.14,
HindIII restriction enzyme sites are the signal peptide sequence in SEQ ID No.14 from 5 ' ends shown in the 1st to the 6th nucleotides
For in SEQ ID No.14 from 5 ' ends shown in the 16th to the 81st nucleotides, 5A7 heavy chain of antibody 5A7VHCHGene order
For from 5 ' ends shown in the 82nd to the 735th nucleotides, translation stop codon is SEQ ID in SEQ ID No.14
In No.14 from 5 ' ends shown in the 736th to the 738th nucleotides, restriction enzyme sites EcoR I be SEQ ID No.14 in from
Rise shown in the 739th to the 744th nucleotides 5 ' ends.5A7SL is inserted into pGEM-T carriers, recombinant plasmid is obtained, is named as
PGEM-T/5A7SL, sequencing is sent, as a result correctly.
Using restriction enzyme Hind III and the double digestion plasmid pGEM-T/5A7SL of EcoR I, genetic fragment is obtained；Utilize
Restriction enzyme Hind III and EcoR I double digestion pcDNA3.1 (+) plasmid, obtain carrier large fragment；By genetic fragment with carrying
Body large fragment connects, and obtains recombinant plasmid, is named as pcDNA3.1 (+) (5A7SL), pcDNA3.1 (+) (5A7SL) is sent
Sequencing, as a result correctly.
（Three）Full people source anti-HER 2 monoclonal antibody 5A7 expression and purification
5 × 10 are inoculated with 3.5cm tissue culture dishes5CHO-K1 cells, carried out when cell culture to 90%-95% is merged
Transfection, is comprised the following steps that：Take 5 μ g plasmid pcDNA3.1 (+) (5A7SH), 5 μ g plasmid pcDNA3.1 (+) (5A7SL) and 20 μ l
Lipofectamine2000Reagent, transfected by Lipofectamine2000Reagent kit specifications.Transfection
After carrying out 24h, cell culture medium is changed to the DMEM Screening of Media resistance clones containing 600 μ g/ml G418.
The CHO-K1 cell line serum free mediums of obtained stable expression antibody will be screened（EX-CELL302, it is purchased from
SIGMA-ALDRICH companies）Expand culture, obtained with rProtein A Sepharose4Fast Flow by specification affinity purifications
The antibody 5A7 arrived.The full people source anti-HER 2 monoclonal antibody 5A7 that purifying obtains is dialysed with PBS, finally uses UV absorption
Method determines the content of 5A7 antibody.
Antibody after purification detects its molecular weight under non-reduced and reducing condition with polyacrylamide gel electrophoresis respectively
Size.Electrophoresis result is shown：Under the reducing conditions, 5A7 antibody be rendered as molecular size range about 55KDa and 25KDa heavy chain and
Light chain bands.Under non reducing conditions, 5A7 antibody shows the single band that molecular weight is about 150KDa.
It can deduce full people source anti-HER 2 monoclonal antibody 5A7's according to the result of above polyacrylamide gel electrophoresis
Structure is as follows：The antibody is made up of two identical light chains and two identical heavy chains, and light chain is permanent by light chain variable district and light chain
District's groups are determined into heavy chain is made up of weight chain variable district and heavy chain constant region, wherein a light chain passes through two sulphur with a heavy chain respectively
Key connection, formation two near points is sub, and the antibody is formed by disulfide bond between two heavy chains of two near points；This is anti-
The amino acid sequence of the light chain variable district of body is as shown in SEQ ID No.8, and its coding gene sequence is as shown in SEQ ID No.7；
The amino acid sequence of weight chain variable district is as shown in SEQ ID No.6, and its coding gene sequence is as shown in SEQ ID No.5；Light chain
The amino acid sequence of constant region is as shown in SEQ ID No.4, and its coding gene sequence is as shown in SEQ ID No.3；Light chain constant
The amino acid sequence in area is as shown in SEQ ID No.2, and its coding gene sequence is as shown in SEQ ID No.1.
4th, anti-HER2 complete antibodies 9C12 preparation
（One）9C12 heavy chain expression vector structure
With 9C12Fv antibody heavy chain variable region genes（SEQ ID No.9）And pGEM-T/CHFor template, design primer passes through
Overlapping PCR reactions obtain full people source anti-HER 2 monoclonal antibody 9C12 heavy chain gene, by this Overlapping
PCR primer is denoted as 9C12SH, and the nucleotide sequence of 9C12SH genes is as shown in SEQ ID No.15.In SEQ ID No.15,
HindIII restriction enzyme sites are the signal peptide sequence in SEQ ID No.15 from 5 ' ends shown in the 1st to the 6th nucleotides
For in SEQ ID No.15 from 5 ' ends shown in the 16th to the 72nd nucleotides, 9C12 heavy chain of antibody 9C12VHCHGene sequence
It is classified as in SEQ ID No.15 from 5 ' ends shown in the 73rd to the 1422nd nucleotides, translation stop codon is SEQ ID
In No.15 from 5 ' ends shown in the 1423rd to the 1425th nucleotides, restriction enzyme sites EcoR I is in SEQ ID No.15
From 5 ' ends shown in the 1426th to the 1431st nucleotides.9C12SH is inserted into pGEM-T carriers, obtains recombinant plasmid, is ordered
Entitled pGEM-T/9C12SH, sequencing is sent, as a result correctly.
Using restriction enzyme Hind III and the double digestion pGEM-T/9C12SH of EcoR I, genetic fragment is obtained；Utilize limit
Double digestion pcDNA3.1 (+) plasmid of property restriction endonuclease Hind III and EcoR I processed, obtains carrier large fragment；By genetic fragment and carrier
Large fragment connects, and obtains recombinant plasmid, is named as pcDNA3.1 (+) (9C12SH), pcDNA3.1 (+) (9C12SH) is sent
Sequencing, as a result correctly.
（Two）9C12 light chain expression vector structure
With 9C12Fv antibody chain variable region genes（SEQ ID No.11）And pGEM-T/CLCarrier is template, is passed through
Overlapping PCR reactions obtain full people source anti-HER 2 monoclonal antibody 9C12 light chain gene, by this Overlapping
PCR primer is denoted as 9C12SL, and the nucleotide sequence of 9C12SL genes is as shown in SEQ ID No.16.In SEQ ID No.16,
HindIII restriction enzyme sites are the signal peptide sequence in SEQ ID No.16 from 5 ' ends shown in the 1st to the 6th nucleotides
For in SEQ ID No.16 from 5 ' ends shown in the 16th to the 81st nucleotides, 9C12 heavy chain of antibody 9C12VHCHGene sequence
It is classified as in SEQ ID No.16 from 5 ' ends shown in the 82nd to the 774th nucleotides, translation stop codon is SEQ ID
In No.16 from 5 ' ends shown in the 775th to the 777th nucleotides, restriction enzyme sites EcoR I be SEQ ID No.16 in from
Rise shown in the 778th to the 783rd nucleotides 5 ' ends.9C12SL is inserted into pGEM-T carriers, recombinant plasmid is obtained, is named as
PGEM-T/9C12SL, send pGEM-T/9C12SL to sequencing, as a result correctly.
Using restriction enzyme Hind III and the double digestion pGEM-T/9C12SL of EcoR I, genetic fragment is obtained；Utilize limit
Double digestion pcDNA3.1 (+) plasmid of property restriction endonuclease Hind III and EcoR I processed, obtains carrier large fragment；By genetic fragment and carrier
Large fragment connects, and obtains recombinant plasmid, is named as pcDNA3.1 (+) (9C12SL), pcDNA3.1 (+) (9C12SL) is sent
Sequencing, as a result correctly.
（Three）Full people source anti-HER 2 monoclonal antibody 9C12 expression and purification
5 × 10 are inoculated with 3.5cm tissue culture dishes5CHO-K1 cells, carried out when cell culture to 90%-95% is merged
Transfection, is comprised the following steps that：Take 5 μ g plasmid pcDNA3.1 (+) (9C12SH), 5 μ g plasmid pcDNA3.1 (+) (9C12SL) and 20
μ l Lipofectamine2000Reagent, are transfected by Lipofectamine2000Reagent kit specifications.Turn
After dye carries out 24h, cell culture medium is changed to the DMEM Screening of Media resistance clones containing 600 μ g/ml G418.
The CHO-K1 cell line serum free mediums of obtained stable expression antibody will be screened（EX-CELL302, it is purchased from
SIGMA-ALDRICH companies）Expand culture, obtained with rProtein A Sepharose4Fast Flow by specification affinity purifications
The antibody 9C12 arrived.The full people source anti-HER 2 monoclonal antibody 9C12 that purifying obtains is dialysed with PBS, finally with ultraviolet suction
Receipts method determines the content of 9C12 antibody.
Antibody after purification detects its molecular weight under non-reduced and reducing condition with polyacrylamide gel electrophoresis respectively
Size.Electrophoresis result is shown：Under the reducing conditions, 9C12 antibody be rendered as molecular size range about 55KDa and 25KDa heavy chain and
Light chain bands.Under non reducing conditions, 9C12 antibody shows the single band that molecular weight is about 150KDa.
Full people source anti-HER 2 monoclonal antibody 9C12 can be deduced according to the result of above polyacrylamide gel electrophoresis
Structure it is as follows：The antibody is made up of two identical light chains and two identical heavy chains, and light chain is by light chain variable district and light chain
Constant region is formed, and heavy chain is made up of weight chain variable district and heavy chain constant region, wherein a light chain passes through two with a heavy chain respectively
Sulfide linkage is connected, formation two near points, and the antibody is formed by disulfide bond between two heavy chains of two near points；Should
The amino acid sequence of the light chain variable district of antibody is as shown in SEQ ID No.12, its coding gene sequence such as SEQ ID No.11 institutes
Show；The amino acid sequence of weight chain variable district is as shown in SEQ ID No.10, and its coding gene sequence is as shown in SEQ ID No.9；
The amino acid sequence of constant region of light chain is as shown in SEQ ID No.4, and its coding gene sequence is as shown in SEQ ID No.3；Heavy chain
The amino acid sequence of constant region is as shown in SEQ ID No.2, and its coding gene sequence is as shown in SEQ ID No.1.
The anti tumor activity in vitro experiment of embodiment 2, full people source anti-HER 2 monoclonal antibody
Utilize CellTiter96AQueous One Solution Cell Proliferation Assay (MTS) are tried
Agent box detection HER2 antibodies on breast cancer cells BT-474, HCC-1569 and HCC-1419 growth in vitro inhibitory action.Specifically
Method is as follows：It is resuspended after human breast cancer cell BT-474, HCC-1569 and HCC-1419 pancreatin are digested with culture medium, makes its close
Spend for 5 × 104Individual/ml, it is inoculated according to every μ l of hole 100 in 96 orifice plates, next day discards old culture medium, and 100 μ are added in hole
L fresh cultures, wherein be 10 μ g/ml Rituximab, Pertuzumab containing concentration, Trastuzumab, 5A7 or
9C12, respectively as control group（Rituximab）, each HER2 antibody treatment group（Pertuzumab, Trastuzumab, 5A7 or
9C12）.37 DEG C, 5%CO2It is incubated.Detected after culture to the 5th day after dosing with MTS reagent box by operating procedure, ELIASA
Background absorbance value is read under 490nm wavelength.490nm Background absorbance value is corrected, method is as follows:3 repetitions
Blank well (acellular)（Cell culture medium and CellTiter96 containing volume same with experimental portAQueous One
Solution Reagent）, the absorbance that " acellular " blank well is subtracted from the light absorption value in the hole in addition to this hole in 490nm puts down
Average, you can obtain control group correction absorbance and HER2 antibody treatment group correction absorbance.Inhibitory rate of cell growth meter
Calculating formula is：Inhibiting rate（%）=(1-HER2 antibody treatment group correction absorbance ÷ control group corrections absorbance) × 100%.
HER2 antibodies on breast cancer cells BT-474 growth in vitro inhibitory action is as shown in Figure 1.
HER2 antibodies on breast cancer cells HCC-1569 growth in vitro inhibitory action is as shown in Figure 2.
HER2 antibodies on breast cancer cells HCC-1419 growth in vitro inhibitory action is as shown in Figure 3.
Fig. 1-Fig. 3 shows that Trastuzumab has obvious Inhibit proliferaton to make for breast cancer cell BT-474 cells
Be more than 50% with, growth inhibition ratio, and for remaining two plants of breast cancer cells HCC-1569 and HCC-1419 inhibitory action then
Unobvious, inhibiting rate is below 20%.Inhibitory action of the Pertuzumab for BT-474, HCC-1569 and HCC-1419 cell
Equal unobvious, inhibiting rate is below 20%.And full people source anti-HER 2 monoclonal antibody 5A7 and full people source anti-HER 2 monoclonal antibody
9C12 can effectively suppress the propagation of BT-474, HCC-1569 and HCC-1419 breast carcinoma cell strain, and growth inhibition ratio can
Reach more than 50%.
Fig. 1-Fig. 3 shows, full people source anti-HER 2 monoclonal antibody 5A7 and 9C12 show than Trastuzumab and
The effect for the suppression Cells Proliferation of Human Breast Cancer that Pertuzumab is significantly increased.
Anti-tumor experiment inside embodiment 3, full people source anti-HER 2 monoclonal antibody
6 week old female BAl BIcs/c nude mices difference subcutaneous vaccination quantity 3 × 106Breast cancer cell BT-474 or HCC-
1569, the length of knurl body is every other day measured with slide measure daily（mm）With width（mm）, tumor volume is calculated with equation below：
Gross tumor volume=length ×（It is wide）2/2.Treat that tumor volume reaches about 100mm3, obtain BT-474 breast cancer tumor-bearing mice groups and HCC-
Two kinds of tumor-bearing mices, are grouped by 1569 breast cancer tumor-bearing mice groups at random respectively, every group of 10 mouse.According to 20mg/kg body weight
Dosage to each HER2 antibody of each group mouse tail vein injection of each tumor-bearing mice（Pertuzumab,Trastuzumab,5A7,
9C12）Or control antibodies Rituximab, 2 times/week, continue 4 weeks., as the 0th day, to use slide measure during first time injection of antibodies
Every other day measure the length of knurl body（mm）With width（mm）, tumor volume is calculated with equation below：Gross tumor volume=length ×（It is wide）2/
2。
HER2 antibody is as shown in Figure 4 to antitumor action inside BT-474 breast cancer.
HER2 antibody is as shown in Figure 5 to antitumor action inside HCC-1569 breast cancer.
In Fig. 4 and Fig. 5, Control IgG represent control antibodies Rituximab.
Fig. 4 shows that, in BT-474 breast cancer tumor-bearing mice groups, full people source anti-HER 2 monoclonal antibody 5A7's and 9C12 is anti-
Function of tumor is similar to Trastuzumab, but much stronger than Pertuzumab.
Fig. 5 shows that, in HCC-1569 breast cancer tumor-bearing mice groups, Trastuzumab and Pertuzumab can not be effective
Suppress tumour growth, and full people source anti-HER 2 monoclonal antibody 5A7 and 9C12 show powerful antitumor activity, can be effective
Suppress the growth of tumour.
The preparation of embodiment 4, anti-HER2 bispecific antibodies HER2-BsAb
First, the heavy chain expression vector of bispecific antibody is built
Using Overlapping PCR method, by 5A7 weight chain variable district（SEQ ID No.5）C-terminal pass through
Linker small peptides " ASTKGPSVFPLAP " are connected with the N-terminal of 9C12 heavy chain of antibody variable region (SEQ ID No.9), then pass through
Overlapping PCR and heavy chain constant region CHThe N-terminal of (SEQ ID No.1) is connected, and obtains bispecific antibody HER2-
BsAb heavy chain gene, this Overlapping PCR primer is denoted as HER2-BsAbSH.The nucleotides of HER2-BsAbSH genes
Sequence is as shown in SEQ ID No.17.In SEQ ID No.17, HindIII restriction enzyme sites are SEQ ID No.17 last from 5 '
Hold shown in the 1st to the 6th nucleotides, signal peptide sequence is SEQ ID No.17 the 16th to the 72nd cores from 5 ' ends
Shown in thuja acid, HER2-BsAb heavy chain of antibody 5A7VH9C12VHCHGene order is SEQ ID No.17 the 73rd from 5 ' ends
Shown in the 1824th acid, translation stop codon 1825 to the 1827th nucleotides institutes from 5 ' ends for SEQ ID No.17
Show, restriction enzyme sites EcoR I is SEQ ID No.17 from 5 ' ends shown in the 1828th to the 1833rd nucleotides.Will
HER2-BsAbSH inserts pGEM-T carriers, obtains recombinant plasmid, is named as pGEM-T/HER2-BsAbSH, is sent sequencing, ties
Fruit is correct.
Using restriction enzyme Hind III and the double digestion pGEM-T/HER2-BsAbSH of EcoR I, genetic fragment is obtained；Profit
With restriction enzyme Hind III and EcoR I double digestion pcDNA3.1 (+) plasmid, carrier large fragment is obtained；By genetic fragment with
Carrier large fragment connects, and obtains recombinant plasmid, is named as pcDNA3.1 (+) (HER2-BsAbSH), by pcDNA3.1 (+)
(HER2-BsAbSH) sequencing is sent, as a result correctly.
2nd, the light chain expression vector of bispecific antibody is built
Using Overlapping PCR method, the C-terminal of 5A7 light chain variable district (SEQ ID No.7) is passed through
Linker small peptides " TVAAPSVFIFPP " are connected with the N-terminal of 9C12 antibody light chains variable region (SEQ ID No.11), then pass through
Overlapping PCR and antibody light chain constant region CLThe N-terminal of (SEQ ID No.3) is connected, and obtains bispecific antibody HER2-
BsAb light chain gene, this Overlapping PCR primer is denoted as HER2-BsAbSL.The nucleotides of HER2-BsAbSL genes
Sequence is as shown in SEQ ID No.18.In SEQ ID No.18, HindIII restriction enzyme sites are from 5 ' in SEQ ID No.18
End rise the 1st to the 6th nucleotides shown in, signal peptide sequence be SEQ ID No.18 in the 16th to the 81st from 5 ' ends
Shown in the nucleotides of position, HER2-BsAb heavy chain of antibody 5A7VL9C12VLCLGene order be SEQ ID No.18 in from 5 ' ends
Shown in 82nd to the 1146th nucleotides, translation stop codon be in SEQ ID No.18 the 1147th to the from 5 ' ends
Shown in 1149 nucleotides, restriction enzyme sites EcoR I be SEQ ID No.18 in the 1150th to the 1155th from 5 ' ends
Shown in nucleotides.HER2-BsAbSL is inserted into pGEM-T carriers, recombinant plasmid is obtained, is named as pGEM-T/HER2-BsAbSL,
Sequencing is sent, as a result correctly.
Using restriction enzyme Hind III and the double digestion plasmid pGEM-T/HER2-BsAbSL of EcoR I, gene piece is obtained
Section；Using restriction enzyme Hind III and EcoR I double digestion pcDNA3.1 (+) plasmid, carrier large fragment is obtained；By gene piece
Section is connected with carrier large fragment, is obtained recombinant plasmid, pcDNA3.1 (+) (HER2-BsAbSL) is named as, by pcDNA3.1
(+) (HER2-BsAbSL) send sequencing, as a result correctly.
3rd, expression of the bispecific antibody in eukaryotic
5 × 10 are inoculated with 3.5cm tissue culture dishes5CHO-K1 cells, carried out when cell culture to 90%-95% is merged
Transfection, is comprised the following steps that：
Take 5 μ g plasmid pcDNA3.1 (+) (HER2-BsAbSH), 5 μ g plasmid pcDNA3.1 (+) (HER2-BsAbSL) and 20
μ l Lipofectamine2000Reagent, are transfected by Lipofectamine2000Reagent kit specifications.Turn
After dye carries out 24h, cell culture medium is changed to the DMEM Screening of Media resistance clones containing 600 μ g/ml G418.
The CHO-K1 cell line serum free mediums of obtained stable expression antibody will be screened（EX-CELL302, it is purchased from
SIGMA-ALDRICH companies）Expand culture, with rProtein A Sepharose4Fast Flow by specification affinity purifications
HER2-BsAb antibody.The full people source anti-HER 2 monoclonal antibody HER2-BsAb that purifying obtains is dialysed with PBS, finally used
Ultraviolet absorption method determines the content of HER2-BsAb antibody.
Antibody after purification detects its molecular weight under reduction and non reducing conditions with polyacrylamide gel electrophoresis respectively
Size.Electrophoresis result is shown：Under the reducing conditions, HER2-BsAb antibody is rendered as molecular size range about 65KDa's and 35KDa
Heavy chain and light chain bands.Under non reducing conditions, HER2-BsAb antibody shows the single band that molecular weight is about 200KDa.
Anti- HER2 bispecific antibody HER2- can be deduced according to the result of above polyacrylamide gel electrophoresis
BsAb structure is as follows：The antibody is made up of two identical light chains and two identical heavy chains, wherein a light chain respectively with
One heavy chain forms two near points, passes through disulfide bond between two heavy chains of two near points by disulfide bond
Form the antibody；The amino acid sequence of heavy chain is as shown in SEQ ID No.19, encoding gene 5A7VH9C12VHCHSequence be
In SEQ ID No.17 from 5 ' ends shown in the 73rd to the 1824th nucleotides, the amino acid sequence such as SEQ ID of light chain
Shown in No.20, encoding gene 5A7VL9C12VLCLSequence be SEQ ID No.18 in the 82nd to the 1146th from 5 ' ends
Shown in the nucleotides of position.
Embodiment 5, anti-HER2 bispecific antibody HER2-BsAb anti tumor activity in vitro experiment
Utilize CellTiter96AQueous One Solution Cell Proliferation Assay (MTS) are tried
Agent box detection HER2 antibodies on breast cancer cells BT-474, HCC-1569 and HCC-1419 growth in vitro inhibitory action.Specifically
Method is as follows：It is resuspended after human breast cancer cell BT-474, HCC-1569 and HCC-1419 pancreatin are digested with culture medium, makes its close
Spend for 5 × 104Individual/ml, it is inoculated according to every μ l of hole 100 in 96 orifice plates, next day discards old culture medium, and 100 μ are added in hole
L fresh cultures, wherein be 10 μ g/ml Rituximab, Pertuzumab, Trastuzumab, 5A7,9C12 containing concentration,
5A7+9C12（Concentration is respectively 10 μ g/ml）Or HER2-BsAb, respectively as control group（Rituximab）, the processing of each HER2 antibody
Group（Pertuzumab, Trastuzumab, 5A7,9C12,5A7+9C12 or HER2-BsAb）, 37 DEG C, 5%CO2It is incubated.Wait to cultivate
Detected with MTS reagent box by operating procedure within the 5th day after to dosing, Background absorbance value is read under ELIASA 490nm wavelength.
490nm Background absorbance value is corrected, method is as follows:The blank well (acellular) of 3 repetitions（Containing same with experimental port
The cell culture medium and CellTiter96 of sample volumeAQueous One Solution Reagent）, from its in addition to this hole
Absorbance values of " acellular " blank well in 490nm are subtracted in the light absorption value in its hole, you can obtain control group correction extinction
Angle value and HER2 antibody treatment group correction absorbance.Inhibitory rate of cell growth calculation formula is：Inhibiting rate（%）=(1-HER2
Antibody treatment group correction absorbance ÷ control group corrections absorbance) × 100%.
HER2 antibodies on breast cancer cells BT-474 growth in vitro inhibitory action is as shown in Figure 6.
HER2 antibodies on breast cancer cells HCC-1569 growth in vitro inhibitory action is as shown in Figure 7.
HER2 antibodies on breast cancer cells HCC-1419 growth in vitro inhibitory action is as shown in Figure 8.
Fig. 6 shows that full people source anti-HER 2 monoclonal antibody 5A7 and 9C12 effectively suppress the life of BT-474 breast carcinoma cell strains
Long, inhibiting rate is respectively 58% and 55%.When 5A7 and 9C12 are used in combination, growth inhibition ratio further improves, and reaches 64%.And
Bispecific antibody HER2-BsAb growth inhibition ratio is up to 81%, far above 5A7,9C12, and 5A7+9C12.
Fig. 7 and Fig. 8 show that each HER2 antibody obtains and BT-474 cells in HCC-1569 and HCC-1419 cell lines
The similar result of strain, i.e., when 5A7 and 9C12 is used alone compared with, when 5A7 and 9C12 are used in combination, the life to breast cancer cell
Long inhibiting rate further improves, and bispecific antibody HER2-BsAb growth inhibition ratio reaches and is far above 5A7,9C12 and 5A7+
9C12。
Embodiment 6, anti-HER2 bispecific antibody HER2-BsAb inside anti-tumor experiment
6 week old female BAl BIcs/c nude mices difference subcutaneous vaccination quantity 3 × 106Breast cancer cell BT-474 or HCC-
1569, the length of knurl body is every other day measured with slide measure daily（mm）With width（mm）, tumor volume is calculated with equation below：
Gross tumor volume=length ×（It is wide）2/2.Treat that tumor volume reaches about 100mm3, obtain BT-474 breast cancer tumor-bearing mice groups and HCC-
Two kinds of tumor-bearing mices, are grouped by 1569 breast cancer tumor-bearing mice groups at random respectively, every group of 10 mouse.To each of each tumor-bearing mice
Group mouse tail vein injects 5A7 respectively（20mg/kg body weight）,9C12（20mg/kg body weight）,5A7+9C12（It is respectively 20mg/kg bodies
Weight））,HER2-BsAb（20mg/kg body weight）Or the control antibodies Rituximab of 20mg/kg body weight, 2 times/week, continue 4 weeks.With
It is the 0th day during first time injection of antibodies, the length of knurl body is every other day measured with slide measure（mm）With width（mm）, with following public affairs
Formula calculates tumor volume：Gross tumor volume=length ×（It is wide）2/2。
HER2 antibody is as shown in Figure 9 to antitumor action inside BT-474 breast cancer.
HER2 antibody is as shown in Figure 10 to antitumor action inside HCC-1569 breast cancer.
In Fig. 9 and Figure 10, Control IgG represent control antibodies Rituximab.
Fig. 9 shows that, in BT-474 breast cancer tumor-bearing mice groups, 5A7 and 9C12 can suppress tumour growth.As 5A7 and
When 9C12 is combined, its antitumor activity further improves.And bispecific antibody HER2-BsAb antitumor action is much stronger than 5A7,
9C12 and 5A7+9C12.Figure 10 shows, HCC-1569 breast cancer mice with tumor groups acquired with BT-474 breast cancer lotus knurls
The similar result of mouse group.
Claims (9)
1. a kind of antibody, it is made up of two identical half molecules, wherein each half molecule is by passing through the one light of disulfide bond
Chain and a heavy chain form, and pass through disulfide bond between two heavy chains of described two half molecules；The amino acid of the heavy chain
Sequence is as shown in SEQ ID No.19, and the amino acid sequence of the light chain is as shown in SEQ ID No.20.
2. antibody according to claim 1, it is characterised in that：The coding gene sequence of the heavy chain such as SEQ ID No.17
In from 5 ' ends shown in the 73rd to the 1824th nucleotides；
In the coding gene sequence of the light chain such as SEQ ID No.18 from 5 ' ends the 82nd to the 1146th nucleotides institute
Show.
3. a kind of method for preparing the antibody of claim 1 or 2, comprises the following steps：It will contain shown in SEQ ID No.17
The recombinant expression plasmid of DNA molecular and recombinant expression plasmid containing DNA molecular shown in SEQ ID No.18 import the in vitro food in one's mouth
In newborn zooblast, screening obtains the transfectional cell of stable expression purpose antibody, then is expanded culture, and purifying obtains purpose and resisted
Body.
4. according to the method for claim 3, it is characterised in that：The weight containing DNA molecular shown in SEQ ID No.17
Group expression plasmid is that the DNA molecular shown in SEQ ID No.17 is inserted into pcDNA3.1's (+)HinThe Hes of d IIIEcoBetween the sites of R I
Arrive；
The recombinant expression plasmid containing DNA molecular shown in SEQ ID No.18 is by the DNA shown in SEQ ID No.18 points
Son insertion pcDNA3.1's (+)HinThe Hes of d IIIEcoR I is obtained between site.
5. the method according to claim 3 or 4, it is characterised in that：The mammalian cell is CHO-K1 cells.
6. application of the antibody in the product for preparing anti-human epidermal growth factor acceptor 2 described in claim 1 or 2.
7. application of the antibody in the product for suppressing breast cancer cell growth and/or propagation is prepared described in claim 1 or 2.
8. the antibody described in claim 1 or 2 suppresses breast cancer cell in preparation and formed in animal body in the product of tumour
Using.
9. application of the antibody described in claim 1 or 2 in anti-breast cancer medicines are prepared.
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| CN103509117A (en) * | 2013-05-06 | 2014-01-15 | 江苏匡亚生物医药科技有限公司 | Bispecific antibody capable of resisting human epidermal growth factor receptor 2 (HER2) and human insulin-like growth factor-IR (IGF-IR), and preparation method and applications thereof |
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| CN103509117A (en) * | 2013-05-06 | 2014-01-15 | 江苏匡亚生物医药科技有限公司 | Bispecific antibody capable of resisting human epidermal growth factor receptor 2 (HER2) and human insulin-like growth factor-IR (IGF-IR), and preparation method and applications thereof |
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