CN104974259B - Anti-vegf/PIGF bispecific antibody, preparation method and the usage - Google Patents
Anti-vegf/PIGF bispecific antibody, preparation method and the usage Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and more specifically, the invention discloses anti-vegf/PIGF bispecific antibodies, preparation method and the usage.Anti-vegf of the invention/PIGF bispecific antibody can combine in conjunction with VEGF and with PIGF, and intratumoral vasculature can be effectively suppressed and formed, and then more effectively inhibit tumour growth.
Description
Technical field
The invention belongs to field of biotechnology, more specifically, the invention discloses a kind of energy while target spots different from two kinds
Bispecific antibody, preparation method and its application in oncotherapy combined.
Background technique
New vessels generation plays highly important effect during the occurrence and development of tumour.When the volume of tumour is super
Cross 1mm3When, tumour can not be supported to continue to develop the demand for nutrition by tissue infiltration nutrition obtained.Therefore, it swells
Tumor tissue needs to obtain various nutrients required for it develops by the generation of new vessels.In anoxic and nutritional deficiency
The tumour cell of state can secrete the factors that a variety of rush new vessels generate, such as vascular endothelial growth factor (VEGF), at fiber
Porcine HGF (FGF), Human plactnta growth factor (PIGF), tumor necrosis factor-alpha (TNF-α) etc..In these growth factors
Under the action of, by the process of a series of complex, the migration of degradation, endothelial cell including blood vessel endothelium matrix, endothelial cell
Proliferation, pipeline branch formed vascular circle and new basilar memebrane and etc. form new blood vessel.Since new vessels generate
Importance in tumor development inhibits the growth of tumour to have become now by inhibiting the new vessels of tumour to generate
The important means of clinical therapy of tumor.
In the growth factor that the various rush new vessels of tumor cell secretion generate, VEGF is generally acknowledged thin for endothelium
Born of the same parents' specificity highest, the strongest mitogen of effect of promoting vascular growth.VEGF and two kinds of height on vascular endothelial cell are affine
After force receptor VEGFR1 and VEGFR2 are combined, vascular endothelial cell proliferation is directly stimulated, and it is induced to migrate and formed lumen sample
Structure；Microvascular permeability can also be increased simultaneously, plasma protein (mainly fibrinogen) is caused to exosmose, and by between induction
Matter generates and internal new vessels is promoted to generate.VEGF plays the regulating and controlling effect of central in angiogenesis and forming process,
It is that crucial new vessels generate stimulating factor.
Currently, the monoclonal antibody specific Bevacizumab(Avastin for VEGF of Genetech company exploitation)
Good effect is had been achieved in clinical treatment, is approved by the FDA in the United States it with chemotherapy drugs in combination for metastatic colon cancer, lung
The treatment of the tumor diseases such as cancer and glioma.But the multiplicity of the angiogenesis promoting factor as secreted by tumour cell
Property and the relevant various factors functionally there is certain substitutability, therefore by inhibit single angiogenesis promoting because
The activity of son such as VEGF can not block the generation of the new vessels in tumour completely.Preclinical animal studies show tumor group
It is woven in after Bevacizumab is handled, other angiogenesis promoting factors such as PIGF in tissue, Ang-1
(angiopoietin-1), the expression quantity of FGF significantly rises, (Casanovas O, Hicklin DJ, Bergers G,
Hanahan D.Drug resistance by evasion of antiangiogenic targeting of VEGF
signaling in late-stage pancreatic islet tumors.Cancer Cell2005;8:299-309.).
In addition, clinical test also further demonstrates that, and in colorectal cancer patients, after the treatment of anti-vegf, the PIGF water of patient's body
Head up display, which writes, increases (Willett CG, Boucher Y, Duda DG, di Tomaso E, Munn LL, et al.Surrogate
markers for antiangiogenic therapy and dose-limiting toxicities for
bevacizumab with radiation and chemotherapy:continued experience of phase I
trial in rectal cancer patients.J Clin Oncol2005;23:8136-9).
Unlike VEGF, PIGF selectively with VEGFR1 and its accessory receptor neuropilin-1
(neuropilin-1) and neuropilin -2(neuropilin-2) combine, and in macrophage, bone marrow precursor
The a large amount of VEGFR1 of surface expression.Functionally, PIGF is other than capableing of the proliferation of stimulating endothelial cell, migrating, additionally it is possible to logical
It crosses chemotaxis recruitment macrophage and myeloid precursor enters tumor tissues, form one in tumor tissues and be conducive to blood
The microenvironment of pipe new life.The macrophage and related myeloid cell that dependence PIGF is recruited are generated in tumor tissues new vessels
During played an important role.Moisten the macrophage for entering tumor tissues quilt in related microenvironment studies have shown that invading
Induce into M2 type macrophage.Mainly play a part of to activate body specific immune response different from M1 type macrophage, M2 type
Macrophage can largely synthesize and discharge various anti-inflammatory cytokines such as interleukin-10 (IL-10), tumor growth factor-β
(TGF-β) etc., immunosuppressive factor and a variety of cell factors for promoting tumour growth, to inhibit inflammatory reaction and promote swollen
Tumor cell growth and transfer.Profit is invaded by clodronate liposome (clodronate liposomes) or the removal of anti-PIGF antibody
After the macrophage of tumor tissues, the growth of angiogenesis and tumour in tissue is suppressed significantly.In addition, preclinical animal body
It is interior it is demonstrated experimentally that in the Transplanted tumor model being resistant to anti-VEGF antibody, anti-PIGF antibody and anti-VEGF antibody combination can be shown
Write enhancing anti-VEGF antibody Anticancer effect in vivo (Fischer C, Jonckx B, Mazzone M, Zacchigna S,
Loges S,et al.Anti-PIGF inhibits growth of VEGF(R)-inhibitor-resistant tumors
without affecting healthy vessels.Cell2007;131:463-75).But in clinical practice, a side
Face, since two kinds of antibody costs of use in conjunction are too high, and the antibody Limited Number that can be used for being treated in combination, it is on the other hand, clinical
Upper two kinds different targeting antibodies drugs carry out combination therapy, and there is all multi-risk Systems in terms of safety.
Therefore, the bispecific antibody that can be combined simultaneously with VEGF and PIGF is constructed, and then is more effectively pressed down
Tumour growth processed is always those skilled in the art's problem anxious to be resolved.
Summary of the invention
To solve the above-mentioned problems, the present inventor has carried out a large number of experiments, and having obtained one can block simultaneously
The bispecific antibody of two target spots of VEGF and PIGF, i.e., it is " recombination anti-vegf/PIGF bispecific antibody " of the present invention,
So as to complete the present invention.
Specifically, the invention discloses:
1, a kind of anti-vegf/PIGF bispecific antibody, which is characterized in that the antibody can in conjunction with VEGF and
It is combined with PIGF.
2, above-mentioned 1 anti-vegf/PIGF bispecific antibody, which is characterized in that the bispecific antibody amino acid
Sequence includes the variable region of Anti-X activity and the variable region sequences of anti-PIGF monoclonal antibody.
3, above-mentioned 1 or 2 anti-vegf/PIGF bispecific antibody, which is characterized in that the bispecific antibody light chain
Variable region amino acid sequence is the bispecific antibody heavy chain variable region amino shown in SEQ ID NO:6 and SEQ ID NO:8
Acid sequence is shown in SEQ ID NO:10 and SEQ ID NO:12.Preferably,
The bispecific antibody further includes constant region, and its chain constant region amino acid sequence is SEQ ID NO:2 institute
Show, light chain constant region amino acid sequence is shown in SEQ ID NO:4.
4, a kind of isolated nucleic acid molecules encode any anti-vegf/PIGF bispecific antibody of above-mentioned 1-3.
Preferably, the nucleotide sequence of bispecific antibody light chain variable region described in the nucleic acid molecule encoding such as SEQ ID NO:5 and
Shown in SEQ ID NO:7, the nucleotides sequence for encoding the bispecific antibody heavy chain variable region is classified as SEQ ID NO:9 and SEQ
Shown in ID NO:11.
5, a kind of expression vector is connected containing nucleic acid molecules described in above-mentioned 4 with the series of operations of the nucleic acid molecules
Expression regulation sequence；Preferably, the carrier can be pCHO1.0, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO
(+) or pDHFR.
6. a kind of host cell contains carrier described in above-mentioned 5.Preferably, the host cell is eukaryocyte.It is preferred that
, the host cell is mammalian cell.Most preferably, the host cell is Chinese hamster ovary celI.7, a kind of to prepare above-mentioned 1-
3 any anti-vegf/PIGF bispecific antibody methods, the method includes the steps:
A) under expression condition, host cell described in culture above-mentioned 6, so that it is anti-to express anti-vegf/PIGF bispecific
Body,
B) anti-vegf described in isolated or purified/PIGF bispecific antibody.
8, a kind of composition contains above-mentioned the 1-3 any antibody and pharmaceutically acceptable carrier.
9, composition described in any anti-vegf/PIGF bispecific antibody or above-mentioned 8 of above-mentioned 1-3 is anti-in preparation
Purposes in tumour medicine.
10, above-mentioned 9 purposes further includes and other anti-tumor drugs is used in combination.
In the present invention, it is any can simultaneously and the amino acid sequence that combines of VEGF, PIGF specificity and the corresponding ammonia of coding
The DNA sequence dna of base acid sequence is adapted to the present invention.
In the present invention, any suitable carrier can be used in the building of anti-vegf/PIGF bispecific antibody expression vector,
Can be pCHO1.0, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO (+), pDHFR etc. include being connected with conjunction in expression vector
Suitable transcription and translation adjusts the DNA sequence dna of the corresponding amino acid sequence of coding of sequence.
Mammal or insect host cell culture systems can be used for the present invention, such as COS, CHO, NS0, sf9 and sf21
Etc. may be applicable to the present invention.
Available host cell may be the prokaryotic cell containing above-mentioned carrier, can be DH5a, BL21 (DE3), TG1
One of.
Anti-vegf disclosed in the present invention/PIGF bispecific antibody preparation method is under expression condition, in culture
The host cell stated, to express anti-vegf/PIGF bispecific antibody, anti-vegf/PIGF described in isolated or purified is bis- special
Heterogenetic antibody.Using the above method, anti-vegf/PIGF bispecific antibody can be purified as substantially uniform substance, such as
It is single band on irreducibility SDS-PAGE electrophoresis.
The method that can use affinity chromatography separate to anti-vegf disclosed by the invention/PIGF bispecific antibody pure
Change, according to the characteristic of the affinity column utilized, the methods of conventional method such as high-salt buffer, change PH elution can be used
Anti-vegf/PIGF the bispecific antibody being incorporated on affinity column.
The structure of above-mentioned anti-vegf/PIGF bispecific antibody disclosed by the invention can be expressed simply as: light chain
VL1-linker1- VL2-CL, heavy chain VH1-linker2-VH2-CH.Wherein VL1, VH1 are respectively anti-vegf/PIGF bispecific
First Variable domain of antibody light and heavy chain；VL2, VH2 are respectively that anti-vegf/PIGF bispecific antibody light and heavy chain is
Two Variable domains；CL, CH are respectively anti-vegf/PIGF bispecific antibody light and heavy chain constant region；linker1,
linker2Respectively be connect anti-vegf/PIGF bispecific antibody light chain, heavy chain the first and second Variable domains company
Connect polypeptide.Above-mentioned bispecific antibody obtains by the following method: light chain VL1 (comes from anti-VEGF antibody, nucleotide sequence
SEQ ID No.7), VL2 (comes from anti-PIGF antibody, nucleotide sequence SEQ ID No.5), and heavy chain VH1 is (anti-from anti-vegf
Body, nucleotide sequence SEQ ID No.11), VH2 (coming from anti-PIGF antibody, nucleotide sequence SEQ ID No.9) is given birth to by Shanghai
The synthesis of object engineering services Co., Ltd's full genome.Light chain VL1 and VL2 pass through linker1(QPKAAPSVTLFPP) after connecting
Rear clone is connect with human antibody light chain constant region gene (SEQ ID No.1) with overlapPCR (to be purchased to pCHO1.0 carrier
Life Technology company) first restricted multienzyme enzyme site, construct carrier for expression of eukaryon pCHO-VEGF/PIGF
DVD-Ig-L, equally, heavy chain VH1 and VH2 pass through linker2(ASTKGPSVFPLAP) connect after with human antibody heavy chain's constant region
Gene (SEQ ID No.3) connection is cloned into the pCHO-VEGF/PIGF DVD-Ig-L containing bispecific antibody light chain
Expression vector second restricted multienzyme enzyme site, construct complete bispecific expression plasmid.Above-mentioned plasmid is used
FreeStyleTMMAX Reagent transfection enters CHO-S cell, and with containing 50 μ g/ml puromycins and 1 μM of methopterin
The single cell clone that Selective agar medium screens stable VEGF expression/PIGF bispecific antibody passes through parent using Protein A column
Anti-vegf/PIGF bispecific antibody is purified from the supernatant of cell culture with chromatography.
Applicant of the present invention carries out the affinity with VEGF, PIGF to above-mentioned anti-vegf/PIGF bispecific antibody
The experiments such as detection, inhibition HUVEC cell Proliferation, internal tumor suppression.The experimental results showed that anti-vegf/PIGF disclosed by the invention is bis-
Specific antibody can be very good in conjunction with VEGF and PIGF.It can blocking VEGF participate in the vascular endothelial cell of regulation
Proliferation, migration and activation, at the same it also PIGF can be blocked to be mediated tumor tissues in macrophage and myeloid cell invade
Profit, to block oxygen during the generation of new vessels in tumor tissues, cutting tumor development more significantly and support
Point supply, inhibit the growth of tumour, due to simultaneously inhibit participate in new vessels generate various kinds of cell (endothelial cell, it is huge
Phagocyte, myeloid cell etc.) activity, therefore, anti-vegf of the present invention/PIGF bispecific antibody can reduce tumor tissues is resistance to
The generation of pharmacological property.
The present invention discloses above-mentioned anti-vegf/PIGF bispecific antibody, can be with pharmaceutically acceptable auxiliary material together
For composition pharmaceutical preparations composition to more stably play curative effect, these preparations can guarantee fusion receptors ammonia disclosed by the invention
The conformation integrality of base acid core sequence, while the polyfunctional group of protected protein matter also being wanted to prevent its degradation (including but not limited to
Cohesion, deamination or oxidation).Under normal conditions, it for liquid preparation, can usually be saved under the conditions of 2 DEG C -8 DEG C at least stable
1 year, for lyophilized preparation, stablize in 30 DEG C of holdings at least six months.Herein preparation can commonly be suspended for pharmaceutical field,
The preparations such as water needle, freeze-drying, preferably water needle or lyophilized preparation, for disclosed by the invention above-mentioned, anti-vegf/PIGF bispecific is anti-
The water needle or lyophilized preparation of body, pharmaceutically acceptable auxiliary material include but is not limited to: surfactant, solution stabilizer, etc.
Seep one or a combination set of regulator and buffer.Wherein surfactant includes but is not limited to: nonionic surface active agent is such as
Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80)；Poloxamer(such as poloxamer188)；Triton；Dodecyl
Sodium sulphate (SDS)；Sodium Laurylsulfate；Myristyl, sub- oil base or octadecyl sarcosine；Pluronics；MONAQUATTMDeng,
Its additional amount should make anti-vegf/PIGF bispecific antibody granulating trend minimum.Solution stabilizer includes but is not limited to: sugar
Class can be reducing sugar and nonreducing sugar；Amino acids can be monosodium glutamate or histidine；Alcohols can be three
First alcohol, advanced sugar alcohol, propylene glycol, polyethylene glycol etc., the additional amount of solution stabilizer should make the preparation eventually formed at this
The technical staff in field thinks to reach stable time interior holding stable state.Isotonic regulator can be sodium chloride, mannitol
One of；Buffer can be one of TRIS, histidine buffering liquid, phosphate buffer.
Above-mentioned preparation is comprising above-mentioned anti-vegf/PIGF bispecific antibody composition, to including people
After animal administration, antitumous effect is obvious.Specifically, effectively to the prevention of tumour and/or treatment, it can be used as antineoplastic
Object uses.
Signified tumour of the invention, including but not limited to: gland cancer, leukaemia, lymthoma, melanoma, sarcoma, tumor group
The source knitted include but is not limited to adrenal gland, gall-bladder, bone, marrow, brain, mammary gland, bile duct, gastrointestinal tract, heart, kidney, liver,
Lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, skin, salivary gland, spleen, testis, thymus gland, thyroid gland and son
Palace.Other than above-mentioned tumour, it may also be used for tumour of central nervous system such as spongiocyte diversity tumor, astrocytoma etc.,
Furthermore the tumour of eye includes basal-cell carcinoma, squamous cell carcinoma, melanoma etc., further includes endocrine disrupting effects, in nerve
Excretory system tumour, gastrointestinal tract pancreatic endocrine system tumor, genital system and head and neck neoplasm etc..Here no longer one by one
It enumerates.
The so-called anti-tumor drug of the present invention, refers to the drug for inhibiting and/or treating tumour, may include with tumour
The delay of related symptoms development and/or the reduction of these severity of symptom are grown, it further comprises already present tumour
It grows the mitigation of simultaneous phenomenon and prevents the appearance of other symptoms, still reduce or prevent transfer.
Anti-vegf disclosed by the invention/PIGF bispecific antibody and combinations thereof can also be with other antineoplastic connection
Close administration, for the treatment of tumour, these antineoplastics include but is not limited to: 1, cytotoxic drug (1) acts on DNA chemistry
The drug of structure: alkylating agent such as nitrogen mustards, nitrous urinate class, methane sulfonic acid esters；Platinum-like compounds such as cis-platinum, carboplatin and oxalic acid platinum
Deng；Mitomycin (MMC)；(2) influence nucleic acid synthesis drug: dihydrofolate reductase inhibitor such as methopterin (MTX) and
Alimta etc.；Thymus nucleoside synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine) etc.；Purine nucleosides closes
At enzyme inhibitor such as Ismipur (6-MP) and 6-TG etc.；Ribonucleotide reductase inhibitor such as hydroxycarbamide (HU) etc.；DNA
Poly enzyme inhibitor such as cytarabine (Ara-C) and gemzar (Gemz) etc.；(3) act on the drug of transcribed nucleic acid: selectivity is made
For DNA profiling, inhibit DNA dependenc RNA polymerase, so that the drug for inhibiting RNA to synthesize is such as: actinomycin D, daunorubicin,
Adriamycin, Epi-ADM, aclacinomycin, mithramycin etc.；(4) mainly act on tubulin synthesis drug: taxol,
Taxotere, vinblastine, vinorelbine, Podophyllum emodi var chinense alkali class, homoharringtonine；(5) other Cytotoxic drugs: L-Asparaginasum is main
Inhibit the synthesis of protein；2, steroids antiestrogenic: tamoxifen, Droloxifene, Exemestane etc.；Aromatizing enzyme inhibits
Agent: aminoglutethimide, Lactel be grand, Letrozole, auspicious Ningde etc.；Antiandrogen: its ammonia RH-LH agonist/antagonist of fluorine: Zoladex,
Enatone etc.；3, biological response modifiers: main that tumour interferon is inhibited by body's immunity；Interleukin 2；Chest
Gland peptides；4, monoclonal antibody: Mabthera (MabThera)；Cetuximab(C225)；Trastuzumab (Trastuzumab),
Bevacizumab(Avastin)；5, some other current mechanism is unknown and needs the drug further studied；Cell differentiation lures
Lead agent such as retinoids；Cell death inducer.Anti-vegf disclosed by the invention/PIGF bispecific antibody and combinations thereof can be with
With one or a combination set of above-mentioned anti-tumor drug drug combination.
Detailed description of the invention
Fig. 1 .pCHO1.0 expression vector structural schematic diagram,
Fig. 2 anti-vegf/PIGF bispecific antibody structural schematic diagram；
Fig. 3 anti-vegf/PIGF bispecific antibody inhibits HUVEC cell-proliferation activity experimental result picture；
Fig. 4 anti-vegf/PIGF bispecific antibody is to colon cancer HT-29 transplantable tumor Anticancer effect in vivo experimental result
Figure；
Fig. 5 anti-vegf/PIGF bispecific antibody is to colon cancer Lovo transplantable tumor Anticancer effect in vivo experimental result
Figure；
Specific embodiment
Following embodiment, experimental example are that the present invention is further detailed, and be should not be understood as to limit of the invention
System.Embodiment does not include detailed descriptions of conventional methods, such as the method that those are used to construct antibody expressing plasmid, will encode egg
White gene be inserted into corresponding carrier and plasmid method or by plasmid introduce host cell method as method for
Person having ordinary skill in the art is well-known, and is all described in many publications, including
Sambrook, J., Fritsch, E.F.and Maniais, T.(1989) Molecular Cloning:A Laboratory
Manual, 2ndEdition, Cold spring Harbor Laboratory Press.
The clone of 1. human antibody light and heavy chain constant region gene of embodiment
With lymphocyte separation medium separating health human lymphocyte, mentioned with Trizol reagent (Invitrogen Products)
Take total serum IgE, according to document (Cell, 1980,22:197-207) and document (Nucleic Acids Research, 1982,10:
Report 4071-4079) separately designs primer amplification heavy chain of antibody and light chain constant region gene.PCR reaction is all made of heat and opens
It is dynamic, reaction condition: 94 DEG C of minutes；94 DEG C 45 seconds, 60 DEG C 45 seconds, 72 DEG C 1 point 10 seconds, 30 circulation；72 DEG C 10 minutes.PCR is produced
Object is through agarose gel electrophoresis purification and recovery and is cloned into pGEM-T carrier (purchased from Life Technology company, the same below)
In, confirmation obtains correct clone after sequence verification.SEQ ID NO:1 and SEQ ID NO:2 respectively illustrates chain constant
The nucleotide and amino acid sequence in area (CL).SEQ ID NO:3 and SEQ ID NO:4 respectively illustrates heavy chain constant region (CH)
Nucleotide and amino acid sequence.Correct clone in this example is denoted as pGEM-T/CL and pGEM-T/CH.
The building of 2. anti-vegf of embodiment/PIGF bispecific antibody expression vector
Light chain VL1 (coming from anti-VEGF antibody, nucleotide sequence SEQ ID No.7), VL2 (come from anti-PIGF antibody, nucleosides
Acid sequence SEQ ID No.5), heavy chain VH1 (coming from anti-VEGF antibody, nucleotide sequence SEQ ID No.11), VH2 are (from anti-
PIGF antibody, nucleotide sequence SEQ ID No.9) nucleotide sequence by the full base of Shanghai biotechnology Services Co., Ltd
Because of synthesis.Light chain VL1 and VL2 nucleotide sequence is attached by the method and Linker1 of overlapPCR with cascade
(Linker1 nucleotide sequence is shown in SEQ ID No.13), and it is cloned into pGEM-T carrier.Then the area light chain Shuan Bian gene
(VL1-VL2) nucleotide sequence is connect with overlapPCR with human antibody light chain constant region nucleotide sequence and is cloned into pGEM-T
In carrier, positive colony is selected, sequencing is correct.With AvrII and BstZ17I digestion, obtained through agarose gel electrophoresis purification and recovery
The digestion segment obtained is purchased from Life Technology company with the plasmid pCHO1.0(with digestion, the same below) it is connected with T4DNA
Enzyme is attached, and is built into light chain expression vector pCHO (VEGF/PIGF DVD-IgG-L).Heavy chain VH1 and VH2 nucleotide sequence
It is attached with cascade that (Linker2 nucleotide sequence is shown in SEQ ID by the method and Linker2 of overlapPCR
No.15), and it is cloned into pGEM-T carrier.Then the area heavy chain Shuan Bian gene (VH1-VH2) nucleotide sequence and human antibody
IgG1 light chain constant region nucleotide sequence is cloned into pGEM-T carrier with overlapPCR connection, selects positive colony, is surveyed
Sequence is correct.With EcoRV and Pacl digestion, the digestion segment obtained through agarose gel electrophoresis purification and recovery and the light chain with digestion
Expression vector pCHO (VEGF/PIGF DVD-IgG-L) is attached with T4DNA ligase, and it is bis- to construct complete VEGF/PIGF
Specific antibody expression vector pCHO (VEGF/PIGF DVD-IgG).Attached drawing 1 illustrates pCHO1.0 expression vector structural representation
Figure.Wherein, PPGK is phosphoglycerate kinase；PEF2/CMV is EF2/CMV hybrid promoter, and PCMV/EF1 is CMV/
EF1 hybrid promoter, SV40pA indicate SV40 polyadenylation signal；CMVpA indicates that CMV polyadenylation signal, DHFR indicate two
Hydrogen reduction of folates enzyme gene；Pac is puromycin resistance gene, and pMB1ori is Plasmid replication origins.Fig. 2 illustrates acquisition
Anti-vegf/PIGF bispecific antibody structural schematic diagram, anti-vegf/PIGF bispecific antibody light chain VL1-linker1-VL2-
CL, heavy chain VH1-linker2Wherein VL1, VH1 are respectively anti-vegf/PIGF bispecific antibody light and heavy chain to-VH2-CH
One Variable domain；VL2, VH2 are respectively that anti-vegf/PIGF bispecific antibody light and heavy chain is the second Variable domain；
CL, CH are respectively anti-vegf/PIGF bispecific antibody light and heavy chain constant region；linker1, linker2It is that connection is anti-respectively
VEGF/PIGF bispecific antibody light chain, heavy chain the first and second Variable domains connecting peptides, light chain VL1 (comes from
Anti-VEGF antibody, nucleotide sequence SEQ ID No.7, amino acid sequence SEQ ID No.8), VL2 (comes from anti-PIGF antibody, core
Nucleotide sequence SEQ ID No.5, amino acid sequence SEQ ID No.6), heavy chain VH1 (comes from anti-VEGF antibody, nucleotide sequence
SEQ ID No.11, amino acid sequence SEQ ID No.12), VH2 (comes from anti-PIGF antibody, nucleotide sequence SEQ ID
No.9, amino acid sequence SEQ ID No.10), light chain CL(nucleotide sequence SEQ ID No.1, amino acid sequence SEQ ID
No.2), heavy chain CH(nucleotide sequence SEQ ID No.3, amino acid sequence SEQ ID No.4).
5 × 10 are inoculated in 125ml shaking flask5/ ml CHO-S cell 30ml, cell culture are transfected after 24 hours: being taken
PCHO (VEGF/PIGF DVD-IgG) plasmid 50 μ g and 50 μ l FreeStyleTMMAX Reagent(Invitrogen company produces
Product) it is dissolved in 1.45ml OptiPRO respectivelyTMSFM is stored at room temperature 5 minutes, and above 2 kinds of liquid is mixed, and is incubated at room temperature 10 minutes
Make to form DNA- liposome complex, DNA- liposome complex be added in shaking flask, then by the CHO-S cell after transfection in
37 DEG C, 8%CO2130-150rpm continues to cultivate in shaking table culture case.Transfection is mould with 50 μ g/ml purine are contained after carrying out 48 hours
Expression anti-vegf/PIGF bispecific antibody single cell clone is stablized in the screening of the Selective agar medium of element and 1 μM of methopterin
Cell.
Take cells and supernatant to detect screening high-expression clone with ELISA: mouse anti human IgG1 (CH2) is coated in ELISA
Plate, 4 DEG C overnight, is closed 2 hours with 2%BSA-PBS in 37 DEG C, culture supernatant to be measured and standard items (Human is added
Myeloma IgG1, κ), 37 DEG C are incubated for 2 hours, and HRP- mouse anti human IgG (CH3) is added and is combined reaction, 37 DEG C are incubated for 1
Hour, TMB is added and is acted on 5 minutes in 37 DEG C, finally uses H2SO4Reaction is terminated, OD is surveyed450Value.The Gao Biaodake that screening is obtained
Grand expanded with serum free medium is cultivated, and it is bis- special to isolate and purify VEGF/PIGF with Protein A affinity column (GE Products)
Property antibody, be sequenced it is consistent with SEQ ID NO:8, SEQ ID NO:10 sequence, by the bispecific antibody of purifying with PBS into
Row dialysis, finally with UV absorption standard measure.Fig. 2 is bispecific antibody structure chart.
Experimental example
Experimental example 1.Biacore method detects anti-vegf/PIGF bispecific antibody and VEGF affinity is tested
Anti-vegf/PIGF bispecific antibody is purchased from VEGF(: R&D company) affinity be according to BIAcore-
2000TMThe association rate constant and dissociation rate constant that surface plasmon resonance system measures are calculated.With hydrochloric acid N- ethyl-
N '-(3- dimethylaminopropyl) carbodiimides (EDC) and HOSu NHS activate biologic sensor chip CM5,
In order to VEGF covalent coupling.The buffer of VEGF is changed to 20mM sodium acetate (PH4.8), is then diluted to about 50 micrograms/milli
It rises.One aliquots (35 microlitres) are injected with 2 mul/min of flow velocity, to reach about 700-1400 resonance units (RU)
Coupling protein is then injected into 1M ethanol amine as sealer.Anti-vegf/PIGF bispecific antibody and VEGF affinity are examined
It surveys, anti-vegf/PIGF bispecific antibody or anti-vegf-antibody (voluntarily preparing according to 2 method of embodiment, the same below) is existed
PBS/TWEEN TMBuffer (contains 0.05%TWEEN20TMPhosphate buffer) in 2 times of serial dilutions, with 10 microlitres/
The speed injection of minute, association and dissociation rate standard method (Karlsson et al., J. Immunol. Methods
(J.Immun.Methods) 145:229-240(1991)) it calculates, equilibrium dissociation constant Kd (survey by surface plasmon resonance (SPR)
The K of magnituded) press Koff/KonIt is calculated.Data are shown in Table 1.As shown in table 1, anti-vegf/PIGF bispecific antibody and anti-vegf
Antibody is compared, and has quite similar kinetic constant, and the combination balance dissociation constant speed Kd value of VEGF is 7 × 10-10M is left
It is right.
2. anti-vegf of experimental example/PIGF bispecific antibody and PIGF affinity test experience
Anti-vegf/PIGF bispecific antibody and PIGF(are purchased from R&D company) affinity be according to BIAcore-
2000TMThe association rate constant and dissociation rate constant that surface plasmon resonance system measures are calculated.With hydrochloric acid N- ethyl-
N '-(3- dimethylaminopropyl) carbodiimides (EDC) and HOSu NHS activate biologic sensor chip CM5,
In order to PIGF covalent coupling.The buffer of PIGF is changed to 20mM sodium acetate (PH4.8), is then diluted to about 60 micrograms/milli
It rises.One aliquots (35 microlitres) are injected with 2 mul/min of flow velocity, to reach about 700-1400 resonance units (RU)
Coupling protein is then injected into 1M ethanol amine as sealer.Anti-vegf/PIGF bispecific antibody and PIGF affinity are examined
It surveys, by anti-vegf/PIGF bispecific antibody or anti-PIGF- antibody (being prepared according to 2 method of embodiment) in PBS/TWEENTMBuffer (contains 0.05%TWEEN20TMPhosphate buffer) in 2 times of serial dilutions, with 10 mul/min of speed
Injection, association and dissociation rate standard method (Karlsson et al., J. Immunol. Methods (J.Immun.Methods)
It 145:229-240(1991)) calculates, the equilibrium dissociation constant Kd (K of surface plasmon resonance (SPR) measured valued) press Koff/Kon
It is calculated.Data are shown in Table 2.Anti-vegf/PIGF bispecific antibody and anti-PIGF antibody have quite similar dynamics normal
The binding force of number, anti-vegf/PIGF bispecific antibody and PIGF are slightly below anti-PIGF antibody, dissociate constant speed Kd value in conjunction with balance
It is 8 × 10-9M or so.
3. anti-vegf of experimental example/PIGF bispecific antibody inhibits HUVEC cell proliferation experiment
Experimental procedure: take the good HUVEC cell of growth conditions (purchased from Chinese Academy of Sciences's type culture classical collection committee
Cell bank), adjustment cell concentration is 2.5 × 104/ ml is inoculated in 96 porocyte culture plates, 100 holes μ l/, in 37 DEG C, 5%C02
It is cultivated in incubator and changes serum free medium afterwards for 24 hours, continued culture for 24 hours, be separately added into the VEGF/PIGF of various concentration gradient
DVD-IgG, anti-VEGF antibody, anti-PIGF antibody are control with PBS, and each concentration takes 3 parallel holes, and 37 DEG C of incubation 1h are added
The VEGF-C of the VEGF-A or 100ng/ml of final concentration of 50ng/ml continue after cultivating 72h, and l00 μ l/ hole CellTiter is added
(Promega Products) reagent, after incubation at room temperature 15 minutes, the fluorescent value of 96 orifice plates carries out reading detection in SpectraMax
The proliferative conditions of HUVEC cell calculate the related inhibiting rate of different disposal group.As shown in Figure 3, the results showed that VEGF/PIGF
DVD-IgG, anti-VEGF antibody, anti-PIGF antibody can inhibit HUVEC cell Proliferation caused by VEGF-A, and VEGF/PIGF simultaneously
DVD-IgG inhibits the activity of HUVEC cell Proliferation caused by VEGF-A strong compared with anti-VEGF antibody and anti-PIGF antibody.
Inhibit the experiment of HT-29 growth of transplanted human in 4. anti-vegf of experimental example/PIGF bispecific antibody body
Experimental procedure: for the Anticancer effect in vivo for evaluating VEGF/PIGF bispecific antibody, human colon cancer cell HT-
29(5×106Cell/only) (be purchased from Chinese Academy of Sciences's type culture classical collection committee cell bank, the same below) be inoculated in Balb/
The right side of ca nude mouse (being purchased from Shanghai Slac Experimental Animal Co., Ltd., the same below) is subcutaneous, the subcutaneous tumor to nude mouse
Grow to 100-200mm3When, lotus knurl nude mouse is grouped at random, be divided into four groups: blank control group, anti-VEGF antibody are controlled
Treatment group, anti-PIGF Antybody therapy group, anti-vegf/PIGF bispecific antibody treatment group.Tail vein injection administration is carried out every three days
Once, continued administration surrounding.The length and width for measuring tumour weekly twice, calculate relative tumour volume, as a result as shown in Figure 4.As a result
Show: in colon cancer HT-29 Transplanted tumor model, compared with anti-VEGF antibody treatment group and anti-PIGF Antybody therapy group, resisting
The tumour growth of VEGF/PIGF bispecific antibody treatment group receives to be inhibited more significantly, and gross tumor volume is significantly less than anti-
VEGF Antybody therapy group and anti-PIGF Antybody therapy group, and compared with the primary tumor volume treated, the volume of tumour is aobvious
It writes and reduces.
Inhibit the experiment of LoVo growth of transplanted human in experimental example 5.VEGF/PIGF bispecific antibody body
Experimental procedure: to evaluate anti-vegf/PIGF bispecific antibody Anticancer effect in vivo, human colon cancer cell
Lovo(5×106Cell/only) be inoculated in Balb/CA nude mouse right side it is subcutaneous, the subcutaneous tumor to nude mouse grows to 100-
200mm3When, lotus knurl nude mouse is grouped at random, be divided into four groups: blank control group, anti-VEGF antibody treatment group resist
PIGF Antybody therapy group, anti-vegf/PIGF bispecific antibody treatment group.Tail vein injection is carried out every three days to be administered once, and is held
Continuous administration surrounding.The length and width for measuring tumour weekly twice, calculate relative tumour volume, as a result as shown in Figure 5.The result shows that:
In colon cancer Lovo Transplanted tumor model, compared with anti-VEGF antibody treatment group and anti-PIGF Antybody therapy group, anti-vegf/PIGF is bis-
The tumour growth of specific antibody therapy group receives to be inhibited more significantly, and gross tumor volume is significantly less than anti-VEGF antibody treatment
Group and anti-PIGF Antybody therapy group.
Claims (9)
1. a kind of anti-vegf/PIGF bispecific antibody can combine in conjunction with VEGF and with PIGF, which is characterized in that
The bispecific antibody light chain construct be VL1-linker1-VL2-CL, heavy chain structure VH1-linker2-VH2-CH,
Middle bispecific antibody light chain variable region VL1 amino acid sequence is that SEQ ID NO:8, VL2 amino acid sequence is SEQ ID NO:
The amino acid sequence of 6, link peptide linker1 are SEQ ID NO:14, bispecific antibody heavy chain variable region VH1 amino acid sequence
It is SEQ ID NO:10 for SEQ ID NO:12, VH2 amino acid sequence, the amino acid sequence of link peptide linker2 is SEQ ID
NO:16。
2. anti-vegf described in claim 1/PIGF bispecific antibody, constant region of light chain CL amino acid sequence is SEQ ID
Shown in NO:2, heavy chain constant region CH amino acid sequence is shown in SEQ ID NO:4.
3. a kind of isolated nucleic acid molecules, any anti-vegf/PIGF bispecific antibody of coding claim 1-2.
4. a kind of expression vector, containing nucleic acid molecules as claimed in claim 3 and with the series of operations phase of the nucleic acid molecules
Expression regulation sequence even；Its described carrier is pCHO1.0, pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+) or pDHFR.
5. a kind of host cell contains carrier as claimed in claim 4.
6. a kind of prepare any anti-vegf/PIGF bispecific antibody method of claim 1-2, the method includes
Step:
A) under expression condition, host cell described in claim 5 is cultivated, so that it is anti-to express anti-vegf/PIGF bispecific
Body,
B) separate and purify a) described in anti-vegf/PIGF bispecific antibody.
7. a kind of composition contains claim the 1-2 any antibody and pharmaceutically acceptable carrier.
8. any anti-vegf/PIGF bispecific antibody of claim 1-2 or composition as claimed in claim 7 are being made
Purposes in standby anti-tumor drug.
9. any anti-vegf/PIGF bispecific antibody of claim 1-2 or composition as claimed in claim 7 are being made
Purposes in standby anti-tumor drug, the purposes further includes and other anti-tumor drugs are used in combination.
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| WO2012061558A2 (en) * | 2010-11-04 | 2012-05-10 | Abbott Laboratories | Dual variable domain immunoglobulins and uses thereof |
| WO2013181087A2 (en) * | 2012-06-01 | 2013-12-05 | Ibc Pharmaceuticals, Inc. | Multimeric complexes with improved in vivo stability, pharmacokinetics and efficacy |
| 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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| WO2012061558A2 (en) * | 2010-11-04 | 2012-05-10 | Abbott Laboratories | Dual variable domain immunoglobulins and uses thereof |
| WO2013181087A2 (en) * | 2012-06-01 | 2013-12-05 | Ibc Pharmaceuticals, Inc. | Multimeric complexes with improved in vivo stability, pharmacokinetics and efficacy |
| 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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