CN105481981B - Target VEGF bispecific antibody and application thereof - Google Patents
Target VEGF bispecific antibody and application thereof Download PDFInfo
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Abstract
The present invention is in Bevacizumab and full human monoclonal antibody B5E3On the basis of building one be directed to two epitopes of VEGF bispecific antibody Bev-B5E3BsAb, experiment show its not with Bevacizumab competitive binding VEGF, thus it is speculated that its epitope is the new epitope different from Bevacizumab.Meanwhile compared with its two parental monoclonal antibody, VEGF can be more effectively combined.In addition, showing the ability for more strongly inhibiting human umbilical vein endothelial cell (HUVEC) proliferation in experiment in vitro；Animal experiments show that its growth that can significantly inhibit tumour.Therefore, which is expected to develop into for tumour and treatment of eye disorders or diagnosis novel antibodies preparation.
Description
Technical field
The invention belongs to field of biotechnology, more specifically, the invention discloses a kind of anti-human target vascular therapy endothelial growths
The factor (VEGF) bispecific antibody and its reagent preparing anti-tumor drug and preparing diagnosis or treatment ophthalmology disease
Or the purposes in drug.
Background technique
Malignant tumour is the disease that the world today seriously endangers human health, its disease incidence becomes in obvious rise in recent years
Gesture.Therapeutic effect of malignant tumour is poor, and the advanced stage rate of transform is high, and often prognosis is bad.Clinically used three big routines are controlled at present
Treatment method: although chemotherapy, radiotherapy and operative treatment largely alleviate slight illness, extending life span, these sides
There is certain limitation in method, curative effect is difficult to further increase.
Growth and transfer important role of the angiogenesis to tumour, there are two visibly different ranks for the growth of tumour
Section, i.e., be changed into the fast breeding stage for having blood vessel from avascular slow growth phase, to promote tumor proliferation, invasion.
The growths of new vessels and maturation are a complicated multiple-factor and multi signal conductive process as a result, being directed to many blood
Pipe generate the factor, wherein most importantly vascular endothelial growth factor (vascular endothelial growth factor,
VEGF).VEGF can be with vascular endothelial growth factor receptor (the vascular endothelial growth of cell surface
factor receptor；VEGFR it) combines, which includes the tyrosine kinase receptor of 3 kinds of high-affinities, i.e. FLT1/
VEGFR1、FLK1/KDR/VEGFR2And FLT4/VEGFR3, can be with by activating tyrosine kinase signal transduction pathway to function
Promote endothelial cell proliferation, migration, induction of vascular is formed, and promotes tumour continued propagation, and improve vasopermeability, causes surrounding
Tissue fibrin is calm, promotes monocyte, fibroblast and endothelial cell infiltration, is conducive to tumor stroma and is formed and swollen
Oncocyte enters new vessels, promotes metastases.
Targeting VEGF antibody has following advantage in antineoplaston: target spot is directly exposed in blood, straight convenient for drug
Connect effect；It can inhibit the transfer of tumour；Target gene expression is stablized, and drug resistance is not likely to produce；There is downstream enlarge-effect；Without examining
Consider the histological characteristic of tumour；Adverse reaction is relatively fewer.Large result card, from VEGF is during promoting Tumor Angiongesis
Important function, blocking VEGF are considered as presently most one of effective antitumour treatments.Such as the shellfish of Genentech company
Cut down humanized antibody (the Cancer Res 1997 that monoclonal antibody (Avastin, bevacizumab) is anti-human VEGF；57:4593-9),
It was approved listing by U.S. FDA in 2 months 2004, is used for first-line treatment metastatic colorectal carcinoma.It is by inhibiting VEGF's
Activity, including endothelial cell enhancing vasopermeability activity, mitogenic activity and other Angiogensis activity, to press down
The formation of new vessels processed reduces the blood supply of tumour, oxygen supplies and the supply of other nutriments and inhibits tumour growth.
Retinal vasculature and choroidal artery system are to form the important component of retina.Wound or disease and
Caused wall structures or the abnormal change of function are the main reason for leading to visual impairment or visual loss.For example, glycosuria
Sick retinopathy be due to diabetes caused by retinal vessel proliferation, in turn result in retinal detachment, be patient's vision
The main reason for forfeiture.The life of intraretinal neovascularization may also be caused in eye injury or postoperative recovery process
It is long.This kind of retinal vessel proliferations are also major reason (the Nature 438:932- for causing visual impairment or blindness
938,2005).Age-dependent macular degeneration (AMD) is a kind of cell or tissue degeneration and blood vessel increasing by foveal region of retina area
Disease caused by life.Stemness and two kinds moist can be divided into.Wherein moist AMD is the most common shape that choroidal neovascularization is formed
Formula is the main reason for causing patient's eye to be blinded.
Much research shows that once the photosensory cell of eye retina starts atrophy (referred to as " ischemic due to subalimentation
Property atrophy "), VEGF just starts to increase in intraretinal concentration, to promote neovascularization growth.This process is known as " new
The formation of angiogenic ".Within the eye, different from normal blood vessels on newborn vascular morphology, official jargon is irregular, and tube wall is mostly to leak.
The paraplasm of this high-permeability or the blood vessel of leakage, which often results in, generates scar on retina, and can further fall off from
And influence eyesight.
Research shows that there is high-caliber vegf expression in the tela chorioidea of moist patient AMD
(Invest.Opthal.Vis.Sci 37:855-868,1996；Microvascular Res.64:162-169,2002).By
In vegf expression level and the correlation between moist AMD, VEGF is used as the biochemical indicator of diagnosis AMD
(Br.J.Opthalmol.88:809-815,2004).Diabetic macular edema is a kind of diabetes correlation ocular complication, meeting
Lead to fuzzy patient's vision, serious vision loss or even blinds.Diabetes are the main reason for causing U.S. adults to be blinded, in beauty
State's diabetic macular edema influences about 560,000 diabetics, and the diabetic's number for having report to report visual problems
Up to nearly 4,000,000 people.And VEGF improves the albumen of vascular permeability, causes the thin vessel leakage of diabetic's ocular hair and macula lutea water
It is swollen.Thunder Buddhist nun's strain monoclonal antibody (Lucentis, ranibizumab) is the monoclonal of a targeting VEGF of Genentech company exploitation
Antibody, it has been ratified in June, 2006 by Food and Drug Adminstration of the US (FDA) related yellow for treating the neovascular age
Spot denaturation.And in August, 2012, U.S. FDA has approved one New indication of Lei Nizhu monoclonal antibody, i.e., with monthly (about 28d) 1 glass
The Diabetic Macular of glass internal injection 0.3mg (preparation specification is 6mg/ml, takes its 0.05ml) Regimen Chemotherapy diabetic
Inpairment of vision caused by oedema.The existing standard treatments of diabetic macular edema are laser surgey, though this can slow down patient
Visual loss and help stabilizing vision, can hardly but restore the eyesight lost.Lei Nizhu monoclonal antibody category antibody fragment class
VEGF inhibitor, undoubtedly Lei Nizhu monoclonal antibody is that such patient brings hope.
Into after the nineties in last century, the appearance of Antibody library makes the preparation of humanized antibody reach one completely newly
Level, it has bypassed required hybridoma approach in previous monoclonal antibody development process, in this embodiment it is not even necessary to even more important by immune
, it makes people it is expected that the dream for obtaining therapeutic human antibody becomes reality for a long time, thus shows powerful
Vitality.Phage antibody library is the earliest antibody library for occurring and being most widely used at present.Phage display is by Smith
(Science, 1985,228 (4705): 1315-1317) are initially set up a kind of by foreign protein genes and bacteriophage
Capsid protein gene makes exogenous protein expression be presented in the technology of phage surface after blending.Phage antibody library is exactly to utilize
Principles above screens the antibody expression of not homospecificity with antigen to them in different phage surfaces
(Science,1989,246(4935):1275-1281；Proc Natl Acad Sci USA,1991,88(18):7978-
7982；Human Antibodies,1997,8(4):155-168).Target cell for constructing phage antibody library can be miscellaneous
Hand over oncocyte, immune human B cell or non-immunized human B cell.Non-immunized human B lymphocyte is that current application is most
Target cell, its storage capacity is big, theoretically contain all people source antibody gene.
The screening of phage antibody library is exactly the process for simulating internal antibody affinity maturation, with immobilization Antigen adsorption table
The phage library of face expression specificity antibody, then elutes free bacteriophage, with the phage-infect host strain of Antigen adsorption
" absorption-elution-amplification " more taken turns again after proliferation amplification is until screen special human antibody.High-capacity antibody library
Foundation be the key that obtain high-affinity source of people antibody, if building phage antibody storage capacity be greater than 1010, that may
Therefrom screening obtains high-affinity (>=109M-1) specific antibody.
Have the report of two kinds of monoclonal preclinical laboratories of conjunctive use, but this use in conjunction Dan Ke in clinical test
But having for grand antibody is many restricted.For example, the safety of two kinds of monoclonal antibodies of use in conjunction and effect need to go respectively
Evaluation, resulting long period, high development cost limit its development process, and bispecific antibody solves this just
One bottleneck.In addition, part bispecific antibody can also obtain some new characteristics not available for its parental monoclonal antibody: such as past
Toward with dissociation speed more lower than former parental antibody, stronger sealing process etc..
Summary of the invention
The first purpose of this invention is to provide a kind of target vascular therapy endothelial growth factors bispecific antibody or antibody
Segment；Second is designed to provide the gene order for encoding these antibody or antibody fragment and can express gene
Carrier；Third purpose is to excavate the purposes of these antibody or antibody fragment.
In order to realize the first purpose of this invention, the present invention provides a kind of bispecific antibody Bev-B5E3BsAb,
With including hypervariable region CDR1、CDR2、CDR3、CDR4、CDR5、CDR6Heavy-chain variable domains and include hypervariable region CDR1',
CDR2', CDR3', CDR4', CDR5', CDR6The light variable domains of '.Wherein, CDR1Amino acid sequence are as follows: Gly-Tyr-
Thr-Phe-Thr-Asn-Tyr-Gly；CDR2Amino acid sequence are as follows: Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro；
CDR3Amino acid sequence are as follows: Ala-Lys-Tyr-Pro-His-Tyr-Tyr-Gly-Ser-Ser-His-Trp-Tyr-Phe-
Asp-Val；CDR4Amino acid sequence are as follows: Gly-Gly-Thr-Phe-Ser-Ser-Tyr-Ala；CDR5Amino acid sequence are as follows:
Ile-Ile-Pro-Ile-Phe-Gly-Thr-Ala；CDR6Amino acid sequence are as follows: Ala-Arg-His-Trp-Gly-Tyr-
Asp-Ala-Phe-Asp-Ile；CDR1The amino acid sequence of ' are as follows: Gln-Asp-Ile-Ser-Asn-Tyr；CDR2The amino acid of '
Sequence are as follows: Phe-Thr-Ser；CDR3The amino acid sequence of ' are as follows: Gln-Gln-Tyr-Ser-Thr-Val-Pro-Trp-Thr；
CDR4The amino acid sequence of ' are as follows: Gln-Ser-Val-Ser-Ser-Ser-Tyr；CDR5The amino acid sequence of ' are as follows: Gly-Ala-
Ser；CDR6The amino acid sequence of ' are as follows: Gln-Gln-Tyr-Gly-Ser-Ser-Pro-Tyr-Thr.
Bispecific antibody Bev-B5E3BsAb is selected from the bispecific antibody and the bispecific antibody of anti-vegf
Any one in segment, bispecific antibody fragment are the Fab segment or F (ab ') of bispecific antibody2Segment.
Bispecific antibody Bev-B5E3The above-mentioned bispecific antibody Bev-B of the antibody fragment of BsAb5E3The Fab piece of BsAb
Section or F (ab ')2Segment.
Above-mentioned humanized antibody is obtained by display technique of bacteriophage.Humanization anti-VEGF antibody of the present invention and its
Functional fragment is in addition to having the advantageous feature of above-mentioned anti-VEGF antibody and its functional fragment, also with high-affinity combination people
Vegf protein.
Under the premise of not substantial effect antibody activity, those skilled in the art can to sequence of the invention replace,
One or more (such as 1,2,3,4,5,6,7,8,9 or 10 or more) amino acid are added and/or lack, to obtain
State the variant of antibody or the sequence of its functional fragment.They are considered as including within the protection scope of the present invention.Such as can
Become area to be replaced the amino acid with similarity.The sequence of variant of the present invention can have at least with its derived sequences
There is 95%, 96%, 97%, 98% or 99% consistency.It is soft that sequence analysis can be used in sequence identity of the present invention
Part measurement.Such as the computer program BLAST using default parameter, especially BLASTP or TBLASTN.
Antibody of the invention can be (for example, IgG1 or IgG4 antibody) of overall length or can only include antigen-binding portion thereof
(for example, Fab, F (ab ')2Or scFv segment), or can be modified to influence function.In some applications, it is modified to remove
It goes undesirable glycosylation site can be useful, or fucose moiety is not present on oligonucleotide chain.In other applications,
It can carry out galactosylation modification.
Term as used herein " functional fragment " refers in particular to antibody fragment such as Fv, scFv (sc refers to single-stranded), Fab, F
(ab ') 2, Fab ', scFv-Fc segment or double antibody (diabody) pass through chemical modification or pass through in incorporation liposome
Any segment of half-life period should be able to be increased, poly- (alkylidene) glycol such as polyethylene glycol (" poly- second is for example added in the chemical modification
It is diolation, PEGylated ") (the referred to as polyethylene glycol of Fv-PEG, scFv-PEG, Fab-PEG, F (ab ') 2-PEG or Fab '-PEG
Change segment) (" PEG " is polyethylene glycol), there is the segment VEGF to combine activity.Preferably, the function fragment will by its Lai
The heavy chain of source antibody or the partial sequence of light chain variable region constitute or comprising them, and the partial sequence is enough to retain to be come with it
The identical binding specificity of source antibody and sufficient affinity, for VEGF, preferably at least equal to its derived antibodies affinity
1/100, at least equal to 1/10 in more preferable mode.This function fragment will include minimum 5 amino acid, preferably its source
10,15,25,50 and 100 continuous amino acids of antibody sequence.
In order to realize second object of the present invention, the present inventor additionally provides encodes weight as shown in SEQ ID NO.9
Chain variable region nucleotide sequence, the coding light chain variable region nucleotide sequence as shown in SEQ ID NO.11.
Those skilled in the art can will encode the cloned dna molecule of anti-VEGF antibody of the present invention into carrier, into
And convert host cell.Therefore, the present invention also provides a kind of recombinant DNA carriers, contain coding anti-vegf of the present invention
The DNA molecular of antibody.Preferably, the recombinant DNA carrier is a kind of expression vector, and those skilled in the art are by the antibody
Cloned dna molecule converts host cell, obtains antibody by inducing expression into expression vector.Expression vector of the invention contains
There are the heavy chain variable region of coding anti-VEGF antibody, the DNA sequence dna of light chain variable region and/or constant region.However, it is also possible to difference structure
Two kinds of expression vectors are built, one kind containing heavy chain variable region and constant region, and another kind contains light chain variable region and constant region, turns together
Contaminate mammal.In a preferred embodiment, the expression vector further contains promoter and encoding secretion signals
The DNA sequence dna of peptide and at least one drug resistant gene for screening.
Host cell of the present invention can be prokaryotic host cell, eukaryotic host cell or bacteriophage.The protokaryon place
Chief cell can be Escherichia coli, hay bacillus, streptomycete or proteus mirabilis etc..The eukaryotic host cell, can be for such as
The fungies such as pichia pastoris yeast, saccharomyces cerevisiae, fission yeast, trichoderma, such as meadow mythimna separata insect cell, such as tobacco plant
Cell, such as bhk cell, Chinese hamster ovary celI, COS cell, myeloma cell's mammalian cell.In some embodiments, this hair
The bright host cell is preferably mammalian cell, more preferable bhk cell, Chinese hamster ovary celI, NSO cell or COS cell.
In order to realize third object of the present invention, antibody provided by the present invention or its functional fragment have following spy
At least one of property: can be with the interaction of high-affinity blocking VEGF and KDR；Antineoplaston；For ophthalmology disease
Treatment.Therefore, bispecific antibody Bev-B5E3BsAb or its antibody fragment can be used in preparing anti-tumor drug and preparation
The reagent or drug of diagnosis or treatment ophthalmology disease, these drugs or diagnostic reagent also include pharmaceutically acceptable excipient, diluent with
Any one in carrier.
Wherein, term " pharmaceutical composition " expression combine with realize at least one drug of certain specific purpose with
And the combination of optionally pharmaceutical acceptable carrier or auxiliary material.In certain embodiments, described pharmaceutical composition be included in the time and/or
Spatially separated combination, if its can collective effect to achieve the object of the present invention.For example, institute in described pharmaceutical composition
The ingredient (such as the combination of antibody according to the present invention, nucleic acid molecules, nucleic acid molecules and/or conjugate) contained can be with whole application
In object or separate administration in object.It is described when the ingredient contained in the described pharmaceutical composition is dividually applied to object
Ingredient can simultaneously or sequentially be applied to object.Preferably, described pharmaceutical acceptable carrier is water, buffered aqueous solution, isotonic salting liquid
Such as PBS (phosphate buffered saline), glucose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose, carbon
Sour magnesium, 0.3% glycerol, hyaluronic acid, ethyl alcohol or polyalkylene glycol such as polypropylene glycol, triglycerides etc..
The type of pharmaceutical acceptable carrier used depend particularly on composition according to the present invention whether be formulated as taking orally,
Nose, intradermal, subcutaneous, intramuscular or intravenous administration.Composition according to the present invention may include wetting agent, emulsifier or buffer substances
As additive.
Caused by terms used herein " anti-tumor drug " are intended to indicate that by vegf expression or using vegf expression as disease
Shape/feature disease or illness.In some embodiments, the tumour includes but is not limited to lung cancer (lung cancer), non-
Small Cell Lung Cancer (non small cell lungcancer (NSCL)), bronchial alveolar cells lung cancer
(bronchioloalviolar cell lung cancer), osteocarcinoma (bone cancer), cancer of pancreas (pancreatic
Cancer), cutaneum carcinoma (skin cancer), head or neck cancer (cancer of the head or neck), skin or intraocular black
Plain tumor (cutaneous or intraocular melanoma), uterine cancer (uterine cancer), oophoroma (ovarian
Cancer), the carcinoma of the rectum (rectal cancer), cancer of the anal region (cancer of the anal region), gastric cancer (stomach
Cancer), gastric cancer (gastric cancer), colon cancer (colon cancer), breast cancer (breast cancer), uterus
Cancer (uterine cancer), carcinoma of fallopian tube (carcinoma of the fallopian tubes), carcinoma of endometrium
(carcinoma of the endometrium), cervix cancer (carcinoma of the cervix), carcinoma of vagina
(carcinoma of the vagina), carcinoma of vulva (carcinoma of the vulva), Hodgkin's disease (Hodgkin ' s
Disease), the cancer of the esophagus (cancer of the esophagus), carcinoma of small intestine (cancer of the small
Intestine), internal system cancer (cancer of the endocrine system), thyroid cancer (cancer of
The thyroid gland), parathyroid carcinoma (cancer of the parathyroid gland), adrenal
(cancer of the adrenal gland), soft tissue sarcoma (sarcoma of soft tissue), carcinoma of urethra
(cancer of the urethra), carcinoma of penis (cancer of the penis), prostate cancer (prostate
Cancer), bladder cancer (cancer of the bladder), kidney or carcinoma of ureter (cancer of the kidney or
Ureter), clear-cell carcinoma (renal cell carcinoma), carcinoma of renal pelvis (carcinoma of the renal pelvis),
Celiothelioma (mesothelioma), hepatocellular carcinoma (hepatocellular cancer), cholangiocarcinoma (biliary cancer),
Central nervous system (CNS) tumour (neoplasms of the central nervous system (CNS)), vertebra axis are swollen
Tumor (spinal axis tumors), brain stem glioma (brain stem glioma), glioblastoma multiforme
(glioblastoma multiforme), astrocytoma (astrocytomas), neurinoma (schwanomas), room pipe
Film tumor (ependymonas), medulloblastoma (medulloblastomas), meningioma (meningiomas), squamous cell carcinoma
(squamous cell carcinomas), pituitary adenoma (pituitary adenoma) and Evans sarcoma (Ewing ' s
Sarcoma), the combination including any intractable form of above-mentioned cancer or one or more above-mentioned cancers.
Caused by terms used herein " treatment of ophthalmology disease " are intended to indicate that by vegf expression or with vegf expression
For symptom/feature disease or illness.In some embodiments, the ophthalmology disease includes but is not limited to that intraocular new blood vessel is comprehensive
Simulator sickness (intraocular neovascular syndrome), proliferating retinopathy (proliferative
Retinopathies), age-dependent macular degeneration (age-related macular degeneration (AMD)), glycosuria
Sick retinopathy (diabetic retinopathy).
It is enough to show it for the dosage of institute's subject benefit in addition, " therapeutically effective amount " used herein refers to.
The actual amount of application, and the velocity and time process of application can be depending on the own situation and severity of institute curer.It controls
The prescription (such as decision etc. to dosage) for the treatment of is finally the responsibility of general practitioner and other doctors and relies on it and make a decision, usually
The case where considering treated disease, individual patients, site of delivery, method of administration and for doctor it is known it is other because
Element.
Unless otherwise defined, all scientific and technical terminologies used herein there are those of ordinary skill in the art to be understood identical
Meaning.Definition and term about this field, professional specifically refer to Current Protocols in Molecular
Biology Ausubel).The abbreviation of amino acid residue is the mark that one of 20 common l-amino acids are referred to used in this field
3 letter of standard and/or 1 alphanumeric codes.
Invention action and effect
The present invention is in above-mentioned Bevacizumab and full human monoclonal antibody B5E3On the basis of building one be directed to VEGF two
The bispecific antibody Bev-B of a epitope5E3BsAb, experiment show its not with Bevacizumab competitive binding VEGF, thus it is speculated that its
Epitope is the new epitope different from Bevacizumab.It, can be more effectively meanwhile compared with its two parental monoclonal antibody
In conjunction with VEGF.In addition, showing the energy for more strongly inhibiting human umbilical vein endothelial cell (HUVEC) proliferation in experiment in vitro
Power；Animal experiments show that its growth that can significantly inhibit tumour.Therefore, which is expected to develop into for tumour
And treatment of eye disorders or diagnosis novel antibodies preparation.
Detailed description of the invention
Fig. 1 is VEGF antibody Activity determination result figure in conjunction with VEGF；
Fig. 2 is the test result figure of the combination of VEGF antibody blocking VEGF and KDR；
Fig. 3 is bispecific antibody Bev-B5E3The result figure of BsAb inhibition HUVEC cell proliferation experiment；
Fig. 4 is bispecific antibody Bev-B5E3Inhibit the experimental result picture of tumour growth in BsAb body.
Specific embodiment
The present invention is further detailed with reference to embodiments, but should not be construed as limiting the invention.
Embodiment does not include detailed descriptions of conventional methods, such as: Overlapping PCR reaction, restricted digestion are anti-
It answers, gene is inserted into plasmid vector, plasmid is introduced to the method etc. of host cell.Such method in this field for having
The personnel of ordinary skill are well-known, and are all described in many publications, such as Sambrook, J.,
Fritsch, E.F.and Maniais, T. (1989) Mole-cular Cloning:A Laboratory Manual,
2ndedition, Cold spring Harbor Laboratory Press.And material as used in the following examples, reagent
Deng being commercially available unless otherwise specified.
Embodiment 1, the targeting full human monoclonal antibody B of VEGF5E3Preparation
(1) clone of human antibody light and heavy chain constant region gene
With lymphocyte separation medium (GE Products) separating health human lymphocyte, with Trizol reagent
(Invitrogen Products) extract total serum IgE, according to document (Cell, 1980,22:197-207) and document (Nucleic
Acids Research, 1982,10:4071-4079) sequence of report separately designs primer, anti-using RT-PCR reaction amplification
Weight chain and light chain constant region gene.PCR product is through agarose gel electrophoresis purification and recovery and is cloned into pGEM-T carrier
In (Promega Products), confirmation obtains correct clone after sequence verification.By the heavy chain and constant region of light chain of human antibody
Gene is connected respectively in expression vector constructs carrier IgG-AbVec plasmid (for containing heavy chain constant region nucleosides respectively
The carrier of acid, is constructed and is saved by this room) and Ig κ-AbVec plasmid (for the carrier containing antibody light chain constant region nucleotide, by
This room constructs and saves), confirmation obtains correct clone after sequence verification.Wherein, the nucleotides sequence of heavy chain constant region (CH)
Column and amino acid sequence show such as SEQ ID NO:1 and SEQ ID NO:2 respectively, the nucleotide sequence of constant region of light chain (CL) with
Amino acid sequence is respectively as SEQ ID NO:3 and SEQ ID NO:4 is shown.
(2) preparation of cDNA
Each 20ml of peripheral blood of 50 Healthy Peoples is collected, is mixed, with lymphocyte separation medium (Sigma-aldrich company)
Separate mononuclearcell.Cell is extracted from isolated human peripheral lymphocyte with Trizol reagent (Invitrogen company)
Total serum IgE.Go out cDNA with cDNA reverse transcription reagent box (Invitrogen company) reverse transcription.Above step is provided according to producer
Specification carries out.
(3) design of primers
It is variable that bibliography (Immunotechnology, 1998,3:271-278) designs and synthesizes clone human antibody heavy chain
VHBack, VHFor, VLBack and the VLFor primer in area (VH) and light chain variable region (VL) gene.VHBack, VHFor,
The sequence of VLBack and VLFor is see (Immunotechnology, 1998,3:271-278).Wherein the 5 ' of VHBack primer
End adds the sequence atg gcc cag ccg gcc atg gcc containing Sfi I site, adds sequence at 5 ' ends of VHFor primer
Gcc aga acc acc gcc gcc gga gcc acc acc gcc is arranged, adds sequence tcc at 5 ' ends of VLBack primer
Ggc ggc ggt ggt tct ggc gga ggc gga tct is added at 5 ' ends of VLFor primer and is contained Not I site
Sequence atg cgg ccg c.
(4) building and screening of phage antibody library
Using the primer in the cDNA and step (3) in step (2), recombinant Phage antibody is utilized
System kit (Amersham Biosciences company) constructs phage antibody library, then with specific antigen pair
Library carries out elutriation.Antibody library building and Panning methods are referring to recombinant Phage antibody system kit
Specification carries out, and the specific antigen " rhVEGF165 albumen " for elutriation is purchased from R&D company.Through multiple elutriation antibody
One plant of anti-human VEGF single-chain antibody B is obtained behind library5E3ScFv obtains its gene order after sequencing.SEQ ID NO:5 and SEQ
ID NO:6 respectively illustrates B5E3The nucleotide sequence and amino acid sequence of ScFv heavy chain variable region VH.SEQ ID NO:7 and SEQ
ID NO:8 respectively illustrates the nucleotide sequence and amino acid sequence of B5E3ScFv light chain variable region VL.
(5) human antibody B5E3The building of expression vector in eukaryocyte
With B5E3ScFv gene is template, synthesizes human antibody heavy chain variable region gene, reaction condition are as follows: 95 by PCR
DEG C 15 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 50 seconds, 30 circulation；72 DEG C 10 minutes.And make this human antibody heavy chain
Restriction enzyme sites AgeI and signal peptide gene sequence are contained in the end 5' of variable region gene, and 3' contains at end restriction enzyme sites SalI.Letter
Number peptide gene sequence is (ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTC).Finally
Agarose gel electrophoresis separates pcr amplification product, recycles purpose band and is cloned into IgG-AbVec carrier, and positive gram of screening
Grand sequencing.Selecting sequencing, correctly clone is full source of people heavy chain carrier for expression of eukaryon IgG-AbVec-B5E3.
With B5E3ScFv gene is template, synthesizes full humanized antibody light chain's variable region gene, reaction condition by PCR are as follows:
95 DEG C 15 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 50 seconds, 30 circulation；72 DEG C 10 minutes, obtain PCR product B5E3VLCL,
Restricted enzyme site AgeI and signal peptide gene sequence are contained in its end 5', and 3' contains at end restriction enzyme sites BsiwI.Signal peptide gene
Sequence is (ATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCA).Through agarose gel electrophoresis purification and recovery purpose
Band is simultaneously cloned into Ig κ-AbVec carrier, screening positive clone sequencing.Selecting sequencing, correctly clone is full source of people heavy chain
Carrier for expression of eukaryon Ig κ-AbVec-B5E3。
(6) human antibody B5E3Expression in eukaryocyte
3 × 10 are inoculated in 3.5cm tissue culture dishes5CHO-K1Cell (ATCC CRL-9618), cell culture to 90%-
It is transfected when 95% fusion: taking 10 μ g (plasmid IgG-AbVec-B of plasmid5E34 μ g, plasmid Ig κ-AbVec-B5E36 μ g) and 20
2000 Reagent of μ L Lipofectamine (Invitrogen Products) presses Lipofectamine2000 Reagent
Kit specification is transfected.After transfection carries out for 24 hours cell change containing 600 μ g/ml G418 (Invitrogen Products) and
The DMEM Screening of Media resistance clone of 250 μ g/ml Zeocin (Invitrogen Products).Cells and supernatant is taken to use
ELISA detection screening high-expression clone: Goat Anti Human IgG (Fc) (KPL company) is coated in elisa plate, 4 DEG C of mistakes
Resistance clone culture supernatant to be measured or standard items Human myeloma is added with 2%BSA-PBS in 37 DEG C of closing 2h in night
Goat Anti Human IgG-HRP ((Southern Biotechnology is added in IgG1 (Sigma), 37 DEG C of incubation 2h
Associates company) it is combined reaction, 37 DEG C of incubation 1h are added TMB developing solution in 37 DEG C of effect 5min, finally use HCL
Reaction is terminated, microplate reader is in wavelength 450nm readings.The high-expression clone that screening is obtained is expanded with serum free medium to be cultivated,
Human antibody B is isolated and purified with Protein A affinity column (GE Products)5E3.Antibody purification is dialysed with PBS, most
Afterwards with UV absorption standard measure.
Embodiment 2, the targeting full source of people bispecific antibody Bev-B of VEGF5E3The preparation of BsAb
(1), bispecific antibody heavy chain variable region, light-chain variable sequence acquisition and the building of recombinant expression carrier
Using the method for Overlapping PCR, the C-terminal of Bevacizumab heavy chain variable region is passed through into Linker small peptide
" ASTKGPSVFPLAP (nucleotide sequence are as follows: GCT AGC ACC AAG GGA CCT TCT GTG TTC CCT CTG GCC
) " and B CCT5E3The N-terminal of antibody heavy chain variable region is connected, and introduces restriction enzyme A ge I and restriction enzyme Sal I
Point constitutes Bev-B5E3The heavy chain variable region of BsAb bispecific antibody, nucleotide sequence and amino acid sequence are respectively SEQ
ID NO:9 and SEQ ID NO:10, by Bev-B5E3BsAb heavy chain variable region is cloned into IgG-AbVec carrier and constitutes its heavy chain table
Up to carrier IgG-AbVec-Bev-B5E3BsAb.Same method, the C-terminal of Bevacizumab light chain variable region is short by Linker
Peptide " TVAAPSVFIFPP (nucleotide sequence are as follows: ACC GTG GCT GCC CCT AGC GTG TTC ATC TTC CCT
) " and B CCT5E3The N-terminal of antibody's light chain variable region is connected, and introduces restriction enzyme A ge I and restriction enzyme Bsiw I
Site constitutes Bev-B5E3The light chain variable region of BsAb bispecific antibody, nucleotide sequence and amino acid sequence are respectively
SEQ ID NO:11 and SEQ ID NO:12, by Bev-B5E3BsAb light chain variable region is cloned into Ig κ-AbVec carrier and constitutes it gently
Chain expression vector Ig κ-AbVec-Bev-B5E3BsAb。
(2) bispecific antibody Bev-B5E3BsAb expression and purifying
5 × 10 are inoculated in 3.5cm Tissue Culture Dish5CHO-K1Cell, by cell culture to 90%-95% merge when into
Row transfection, the specific steps are as follows: take 5 μ g plasmid IgG-AbVec-Bev-B5E3BsAb, 5 μ g plasmid Ig κ-AbVec-Bev-
B5E32000 Reagent of BsAb and 20 μ LLipofectamine is said by 2000 Reagent kit of Lipofectamine
Bright book is transfected.After transfection carries out for 24 hours, cell culture medium is changed to containing 600 μ g/mL G418DMEM Screening of Media resistance
Clone.
The CHO-K for stablizing expression antibody that screening is obtained1Cell strain is expanded with serum free medium EX-CELL302 to be trained
It supports, with 4 Fast Flow purification system of Protein A Sepharose, by specification affinity purification obtains bispecific antibody
Bev-B5E3BsAb.The bispecific antibody that purifying obtains is dialysed with PBS, finally measures Bev- with ultraviolet absorption method
B5E3The content of BsAb antibody.
Using polyacrylamide gel electrophoresis method, the structure of antibody is identified.Antibody after purification respectively non-reduced and
Its molecular size range is detected with polyacrylamide gel electrophoresis under reducing condition.Electrophoresis result is shown: under the reducing conditions, Bev-
B5E3BsAb antibody is rendered as the heavy chain and light chain bands of molecular size range about 65KDa and 35KDa.Under non reducing conditions, Bev-
B5E3BsAb antibody shows the single band that molecular weight is about 200KDa.
It thereby it is assumed that out the bispecific antibody Bev-B for VEGF5E3The structure of BsAb: the antibody is by two phases
Same light chain and two identical heavy chains compositions, light chain are made of light chain variable region and constant region of light chain, and heavy chain is by weight chain variable
Area and heavy chain constant region composition, wherein a light chain is connect with a heavy chain by disulfide bond respectively, formation two near points, two
It connects to form the antibody by disulfide bond between two heavy chains of a half molecule.So far, we construct and give expression to complete
Bispecific antibody Bev-B5E3BsAb。
The combination Activity determination of embodiment 3, VEGF antibody and VEGF
VEGF (R&D Products) is diluted to 0.05mmol/L sodium carbonate sodium bicarbonate buffer liquid (pH 9.6)
The hole 2ug/ml, 50ul/, 4 DEG C of coatings are overnight.10% defatted milk room temperature close 2h after, be added various concentration control IgG,
Bevacizumab (Roche Products), B5E3And D2F6, the hole 50u l/, each concentration takes 3 parallel holes, is incubated at room temperature 2h.
Supernatant is abandoned, PBS is washed 3 times, goat anti-human igg's monoclonal antibody (KPL Products) of the HRP label diluted by potency is added,
The hole 50u1/ is incubated at room temperature 45min.After PBS is sufficiently washed, after TMD colour developing, microplate reader (the automatic enzyme mark colour comparatour of Elx type, beauty are set
BIO-TEK company, state) in measure 450nm absorbance (OD450) value.Experimental result is as shown in Figure 1, the results showed that full people
Source antibody B5E3And D2F6Can be in conjunction with vegf protein, but binding ability is slightly weaker than and Bevacizumab.
The combination test of embodiment 4, VEGF antibody blocking VEGF and KDR
Vascular endothelial growth factor receptor KDR (VEGFR2) is mainly distributed on vascular endothelial cell, is also distributed about Hematopoietic Stem
The cell surfaces such as cell, macrophage, effect are the vascular endothelial proliferation for mediating VEGF, chemotactic endothelial cell and increase
The functions such as vasopermeability are the major function receptors of VEGF.Vascular endothelial growth factor receptor KDR (VEGFR2) albumen is used
0.05mmol/L sodium carbonate sodium bicarbonate buffer liquid (pH 9.6) is diluted to 2ug/ml, and the hole 100ul/, 4 DEG C of coatings are overnight.10%
After skimmed milk power room temperature closes 2h, the antibody of various concentration and the VEGF mixed liquor of 400ng/ml, the hole 100ul/, Mei Genong is added
Degree takes 3 parallel holes, is incubated at room temperature 2h.Supernatant is abandoned, PBS is washed 5 times, and the Biotinylated diluted by potency is added
HVEGF purified Goat lgG (R&D, BAF293), 100 holes μ L/ are incubated at room temperature 2h.After PBS is sufficiently washed, addition is pressed
The streptavidin-HRP (R&D, DY998) that potency has diluted sets microplate reader after 37 DEG C are protected from light incubation 45min, TMD colour developing
Middle measurement 450nm absorbance (A450) value.Experimental result is as shown in Figure 2, the results showed that: Bevacizumab can be blocked effectively
The combination of VEGF and KDR, but B5E3And D2F6Antibody cannot effectively blocking VEGF and KDR combination.
Embodiment 5, bispecific antibody Bev-B5E3BsAb inhibits HUVEC cell proliferation experiment
We utilize Bev-B5E3BsAb antibody on human huve cell HUVEC cell growth inhibition assay is further
Verify bispecific antibody Bev-B5E3The function of BsAb antibody.The good HUVEC cell of growth conditions (purchased from ATCC) is taken, is adjusted
Whole cell concentration is 5 × 104/ ml is inoculated in 96 porocyte culture plates, 100 holes μ L/, in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator
After change serum free medium, continue cultivate 48h, be added various concentration gradient antibody and final concentration be respectively 200ng/ml VEGF
Mixed liquor continues culture for 24 hours, and cck-8 reagent is added, and continues to be incubated for 3h, sets measurement 450nm absorbance (A450) in microplate reader
Value.
Fig. 3 is bispecific antibody Bev-B5E3The result figure of BsAb inhibition HUVEC cell proliferation experiment.Such as Fig. 3-A institute
Show, Bevacizumab can effectively inhibit HUVEC cell Proliferation compared with compareing IgG, the full source of people compared with Bevacizumab
Antibody B5E3Inhibit HUVEC ability of cell proliferation very weak, Bevacizumab and B5E3Antibody combination is living to the Proliferation Ability of HUVEC
Property is similar to Bevacizumab.It is surprising that bispecific antibody Bev-B5E3The work of BsAb inhibition HUVEC cell Proliferation
Property be significantly stronger than Bevacizumab (* P < 0.01, t examine).As shown in Fig. 3-B, Bevacizumab can compared with compareing IgG
Effectively inhibit HUVEC cell Proliferation, the human antibody D compared with Bevacizumab2F6Inhibit HUVEC ability of cell proliferation very
It is weak.Bevacizumab and D2F6Antibody combination is similar to Bevacizumab to the increment inhibitory activity of HUVEC, and bispecific is anti-
Body Bev-D2F6BsAb inhibits the activity of HUVEC cell Proliferation also similar to Bevacizumab.
Inhibit tumour growth experiment in embodiment 6, VEGF antibody body
For antitumor activity in detection VEGF antibody body, LM3 cell (human liver cancer cell) is used first, is inoculated in female SCID
Mouse (The 2nd Army Medical College Experimental Animal Center) right flank side is subcutaneous, give on the day of inoculated tumour each VEGF antibody 25mg/kg and
Then homotype irrelevant protein control is subcutaneously injected for 4 weeks every other day.After 6 weeks, the length and width of every 3 days measurement tumours calculate tumour body
Product.Similar to Vitro Experimental Results, as shown in Fig. 4-A, Bevacizumab can effectively inhibit LM compared with compareing IgG3Cell
Knurl growth, the human antibody B compared with Bevacizumab5E3Inhibit LM3Cell knurl growth ability is very weak,
Bevacizumab and B5E3Antibody combination is similar to Bevacizumab to knurl inhibitory activity.However, bispecific antibody Bev-
B5E3BsAb inhibits LM3 cell knurl growth activity to be significantly stronger than Bevacizumab (* P < 0.01, t inspection).As shown in Fig. 4-B,
Bevacizumab can effectively inhibit LM compared with compareing IgG3The growth of cell knurl, full source of people is anti-compared with Bevacizumab
Body D2F6Inhibit LM3Cell knurl growth ability is very weak.Bevacizumab and D2F6Antibody combination is to LM3The growth suppression of cell knurl
Make activity, bispecific antibody Bev-D similar to Bevacizumab2F6BsAb inhibit HUVEC cell Proliferation activity also with
Bevacizumab is similar.B5E3And D2F6Inhibit LM3Cell knurl growth activity it is similar, but they respectively with Bevacizumab
But there were significant differences for the inhibitory activity of the bispecific antibody of composition, it is presumed that this may be due to B5E3And D2F6The two are anti-
The epitope of body is different, and difference occurs in the function for the bispecific antibody for causing each and Bevacizumab to form.
Bev-B5E3BsAb has the activity than the Bevacizumab inhibition HUVEC being remarkably reinforced, it is expected to develop into use
In the new antibody preparation for the treatment of of solid tumors.
The action and effect of embodiment
Above-described embodiment specifically describes the bispecific antibody Bev-B for two epitopes of VEGF5E3The building of BsAb
Journey, and it is shown not with Bevacizumab competitive binding VEGF by specific experiment, and then deducing its epitope is to be different from
The conclusion of the new epitope of Bevacizumab.Meanwhile compared with its two parental monoclonal antibody, bispecific antibody Bev-
B5E3BsAb can more effectively combine VEGF.More strongly inhibit in human umbilical vein in addition, its in experiment in vitro is shown
The ability of chrotoplast (HUVEC) proliferation, zoopery also indicate that bispecific antibody Bev-B5E3BsAb can significantly inhibit tumour
Growth.Therefore, which is expected to develop into for tumour and treatment of eye disorders or diagnosis novel antibodies system
Agent.
Claims (6)
1. a kind of targeting VEGF bispecific antibody Bev-B5E3BsAb comprising:
Heavy-chain variable domains include hypervariable region CDR1、CDR2、CDR3 、CDR4、CDR5、CDR6；And
Light variable domains include hypervariable region CDR1', CDR2', CDR3', CDR4', CDR5', CDR6',
Wherein, the CDR1Amino acid sequence are as follows: Gly-Tyr-Thr-Phe-Thr-Asn-Tyr- Gly；
The CDR2Amino acid sequence are as follows: Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro；
The CDR3Amino acid sequence are as follows: Ala-Lys-Tyr-Pro-His-Tyr-Tyr-Gly-Ser-Ser-His-Trp-
Tyr-Phe-Asp-Val；
The CDR4Amino acid sequence are as follows: Gly-Gly-Thr-Phe-Ser-Ser-Tyr-Ala；
The CDR5Amino acid sequence are as follows: Ile-Ile-Pro-Ile-Phe-Gly-Thr-Ala；
The CDR6Amino acid sequence are as follows: Ala-Arg-His-Trp-Gly-Tyr-Asp-Ala- Phe-Asp-Ile；
The CDR1The amino acid sequence of ' are as follows: Gln-Asp-Ile-Ser-Asn-Tyr；
The CDR2The amino acid sequence of ' are as follows: Phe-Thr-Ser；
The CDR3The amino acid sequence of ' are as follows: Gln-Gln-Tyr-Ser-Thr-Val-Pro-Trp-Thr；
The CDR4The amino acid sequence of ' are as follows: Gln-Ser-Val-Ser-Ser-Ser-Tyr；
The CDR5The amino acid sequence of ' are as follows: Gly-Ala-Ser；
The CDR6The amino acid sequence of ' are as follows: Gln-Gln-Tyr-Gly-Ser-Ser-Pro-Tyr- Thr,
The heavy chain variable amino acid sequence is as shown in SEQ ID NO.10, the chain variable region amino acid sequence such as SEQ
Shown in ID NO.12.
2. a kind of antibody fragment for VEGF, it is characterised in that:
The antibody fragment is bispecific antibody Bev-B described in claim 15E3 The Fab segment or F (ab ') of BsAb2Piece
Section.
3. a kind of bispecific antibody Bev-B for encoding targeting VEGF described in claim 15E3The gene of BsAb, feature
It is:
The nucleotide sequence of the heavy chain variable region is encoded as shown in SEQ ID NO.9, encodes the nucleosides of the light chain variable region
Acid sequence is as shown in SEQ ID NO.11.
4. a kind of expression vector, it is characterised in that:
The expression vector contains the gene as claimed in claim 3 of at least one copy.
5. the bispecific antibody Bev-B of targeting VEGF described in claim 15E3BsAb prepare anti-tumor drug and
The reagent or the purposes in drug of preparation diagnosis or treatment ophthalmology disease.
6. the antibody fragment of targeting VEGF as claimed in claim 2 is preparing anti-tumor drug and is diagnosing or treat eye in preparation
The reagent of section's disease or the purposes in drug.
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| CN1259961A (en) * | 1997-04-07 | 2000-07-12 | 基因技术股份有限公司 | Humanized antibodies and method for forming same |
| CN102167740A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and preparation method as well as application thereof |
| CN104592390A (en) * | 2013-10-30 | 2015-05-06 | 上海张江生物技术有限公司 | Bispecific recombinant anti-HBsAg antibody, and preparation method and application thereof |
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| US20020103345A1 (en) * | 2000-05-24 | 2002-08-01 | Zhenping Zhu | Bispecific immunoglobulin-like antigen binding proteins and method of production |
| WO2006020258A2 (en) * | 2004-07-17 | 2006-02-23 | Imclone Systems Incorporated | Novel tetravalent bispecific antibody |
| EP1928506A4 (en) * | 2005-08-19 | 2009-10-21 | Abbott Lab | Dual variable domain immunoglobin and uses thereof |
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| CN1259961A (en) * | 1997-04-07 | 2000-07-12 | 基因技术股份有限公司 | Humanized antibodies and method for forming same |
| CN102167740A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and preparation method as well as application thereof |
| CN104592390A (en) * | 2013-10-30 | 2015-05-06 | 上海张江生物技术有限公司 | Bispecific recombinant anti-HBsAg antibody, and preparation method and application thereof |
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