CN105085677B - Anti-vegf R2 source of people nano antibody NTV1 and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of anti-vegf R2 source of people nano antibody, including framework region and complementary determining region, the nano antibody has amino acid sequence shown in SEQ ID NO:1.The present invention also provides a kind of preparation methods for preparing anti-vegf R2 nano antibody drug, comprising the following steps: a. screens the nano antibody in conjunction with VEGFR2 using display technique of bacteriophage；B. the expression and purification of nano antibody；C. using the interaction between AlphaScreen technology measurement antigen and antibody, the highest antibody of the affinity of further screening.The present invention obtains the source of people nano antibody NTV1 with more high-affinity and good anti-tumor activity.The present invention expresses and optimizes the VEGFR2 nano antibody albumen for obtaining affinity height and stable uniform, successfully researches and develops source of people anti-vegf R2 nano antibody drug, opens the frontier of the medicament research and development of targeting VEGFR2 antibody.

Description

Anti-vegf R2 source of people nano antibody NTV1 and its preparation method and application

Technical field

The present invention relates to field of biological pharmacy.More particularly it relates to a kind of VEGFR2 nano antibody, the antibody The purposes of preparation method and the antibody in preparation anticancer medicine.

Background technique

Malignant tumour is that current serious influences one of human health, the principal disease for threatening human life, ends 2012, The whole world has more than 8,200,000 people and dies of cancer, and the World Health Organization and hygiene department of national governments are all classified as one cancer is captured Item top priority.But the treatment of tumour is still the problem of current medical field, researcher always searches for oncotherapy New direction.By research tumour discovery, angiogenesis is the necessary condition of tumorigenesis, and vascular endothelial growth factor Receptor (vascular endothelial growth factor receptor, VEGFR) is used as angiogenesis key factor The receptor of VEGF also becomes the research hotspot of the targeting of anti-tumor drug.

Tumor vessel provides sufficient nutrition supply for the growth of tumour, while also promoting the diffusion of cancer cell and turning It moves.VEGF is the growth factor for mainly acting on vascular endothelial cell, has and promotes endothelial cell proliferation, increase capilary penetrating Property, induction of vascular generate etc. multiple functions.Existing research confirms the formation and its transfer, wind of the high expression and entity tumor of VEGF The diseases close relation such as wet arthritis, psoriasis, diabetic retinopathy, artery sclerosis.VEGF by with blood Specific receptor VEGFR on endothelial cell film is conducted in conjunction with activation signal is carried out.The member of VEGFR family include: VEGFR1, VEGFR2 and VEGFR3.They are transmembrane protein, by the functional domain of 7 extracellular immunoglobulin-likes, tyrosine intracellular Kinase domain and one section of intermediate cross-film sequence composition.Wherein VEGFR2 is positioned at people 4q11-12, by 1356 amino acid structures At, can promote vascular endothelial cell proliferation, migration and increase capillary permeability.It studies and has confirmed, tumor vascular endothelium The mitosis of cell and the permeability of tumor-microvessel are mainly to be occurred by the interaction of VEGF and VEGFR2. VEGFR2 is in the very low at expression in human body of health or even does not express；And in many tumours, such as breast cancer, lung In the tumor vascular endothelial cell of cancer, bladder cancer, colon cancer, cervical carcinoma, hepatocellular carcinoma, hemangioblastoma, glioma etc. It is highly expressed.In addition, VEGFR2 extracellular part deletion experiments confirm: VEGF mainly passes through the I- extracellular with VEGFR2 The area III specifically binds signal transduction occurs.

In decades since Kohler and Milstein invention hybridoma technology prepares monoclonal antibody, antibody is It is widely used in the diagnosing and treating of clinical disease.Therapeutic monoclonal antibodies drug, which has become in bio-pharmaceuticals, at present increases Long most fast field, the advantages such as monoclonal antibody medicine is strong with targeting, curative effect is clear, Small side effects are in autoimmune disease, cancer Equal fields are widely used.Wherein, Antineoplastic angiogenesis antibody has the characteristics such as targeting, specificity, specificity, can specificity Tumor vascular occurrence and development are blocked, the lethal effect to its hetero-organization is reduced.Currently used for clinical Antineoplastic angiogenesis Antibody drug bevacizumab (bevacizumab, Avastin) be approved by the fda in the United States for non-small cell lung cancer in 2004 Treatment, and good curative effect is shown in the treatment of other tumours.However, due to its also having to VEGFR1 and VEGFR3 Inhibiting effect, the associated signal paths for having blocked VEGF to mediate comprehensively, causes patient hypertension, nosebleed, albuminuria etc. occur Serious adverse reaction.The heavy chain antibody of naturally occurring missing light chain in camel body, variable region is the smallest functional antigen knot Close segment.In general, the relative molecular mass of this kind of antibody is 15KDa, and only the 1/10 of conventional antibody, numberator height is 4.8nm, diameter 2.2nm, therefore referred to as nano antibody (nanobody).Nano antibody is as the smallest functional antigen bonding pad Section, unique biological structure feature impart their many advantages, as stability is strong, solubility is good, immunogenicity is low, easy Expression etc..Nano antibody contains 3 complementary determining regions (complementarity determining regions, CDRs). It is found from the stereochemical structure of nano antibody, longer CDR3 connect cyclization by disulfide bond to stablize structure with adjacent CDR1 or CDR2 As.The conventional antibodies Fab segment and antigen binding domain shape of single-chain antibody ScFv antibody is dished or planar structure, can only identify Positioned at the site of antigenic surface, and the CDR3 longer of heavy chain antibody, stable big bulge loop structure can be formed, deeply has crack recessed It falls into inside the antigen of structure, such as active site, virus and host cell binding site of enzyme etc..Therefore nano antibody has more Add extensive antigen binding power.Secondly, there are cysteine residues (Cys), Ke Yihe for the area CDR1 and skeleton area of nano antibody Cys residue in the area CDR3 forms disulphide bridges, increases the stability and structure change of variable region.In addition, nano antibody has Molecular weight is small, penetration power is relatively strong and the features such as being prepared into multipurpose antibody or carry chemical drug, so that this kind of antibody exists To solid tumor and need that there is significant advantage in the treatment of blood-brain barrier disease.In addition to this, random mutation and bacteriophage The introducing of the technologies such as displaying is but also the building in nano antibody library and the screening process of specific nano antibody become more just It is prompt, efficient.VEGFR2 is one of most important drug target, has wide Prospect of R & D and important realistic meaning.However, The technical bottleneck of conventional antibodies affinity maturation, protein expression and purification and biological effect is serious to limit VEGFR2 antibody drug Research and development.

Summary of the invention

In order to solve the above-mentioned technical problem, an object of the present invention is to provide a kind of with high-affinity and good anti-swollen Tumor activity, efficiently, toxic side effect is low and the anti-vegf R2 source of people nano antibody that is readily produced.

The purpose of another aspect of the present invention is to provide a kind of method for preparing above-mentioned antibody.

The purpose of another aspect of the invention is to provide above-mentioned antibody in preparation for treating the purposes in anti-tumor drug.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme that:

The present invention provides a kind of anti-vegf R2 source of people nano antibody, including framework region and complementary determining region, which is characterized in that The nano antibody has amino acid sequence shown in SEQ ID NO:1.

The present invention also provides a kind of preparation methods for preparing anti-vegf R2 nano antibody, comprising the following steps:

A. the nano antibody in conjunction with VEGFR2 is filtered out using display technique of bacteriophage, and is compared and is analyzed by gene, Pick out the highest sequence of repetition；

B. nano antibody previous step filtered out is rebuild in expression vector, and uses procaryotic cell expression system Expression obtains high purity protein；And

C. most using the affinity of AlphaScreen technology further screening from the nano antibody that previous step filters out High antibody.

Above-mentioned steps are described in more detail below.

A. the nano antibody in conjunction with VEGFR2 is screened using display technique of bacteriophage

Preferably, positive bacteriophage is screened using biopanning technique first, and positive colony is expanded in host strain TG1 Increase enrichment；Then positive colony determined dna sequence, gene comparison analysis are carried out, the highest sequence of repetition is selected.

It is highly preferred that antigen is fixed on magnetic bead surfaces using liquid phase panning technique first, it is added anti-containing source of people nanometer In the combination liquid in body library, positive bacteriophage is screened, and positive colony is expanded to enrichment in host strain TG1, it is preferable that the expansion Promote row 4 to take turns；Then it selects positive colony at random in last two-wheeled elutriation and carries out gene sequencing and gene comparison analysis, therefrom Pick out the highest sequence of number of repetition.

B. the expression and purification of nano antibody

Preferably, sequence application Not I and BamH the I restriction enzyme and DNA recombinant technique that will be singled out are by digestion In the pET carrier for the linearization for enzyme restriction that pcr amplification product insertion afterwards carries 6His-sumo label, expression plasmid is constructed；By matter Grain conversion is cultivated into host strain to logarithmic phase, inducing expression；Bacterial precipitation is collected, ultrasonic method sufficiently cracks bacterium, takes supernatant, For thick leach protein；Thick leach protein passes through nickel affinity column and molecular sieve column, obtains purified protein samples；With electrophoresis testing goal albumen Expression, to the higher clone of the frequency of occurrences carry out prokaryotic system expression and purification.

C. the screening of nano antibody affinity preferably, is measured mutual between antigen and antibody using AlphaScreen technology Effect, the highest antibody of the affinity of further screening.

The present invention also provides above-described anti-vegf R2 source of people nano antibody in preparation for the use in anticancer medicine On the way.

Wherein, the cancer includes breast cancer, lung cancer, bladder cancer, colon cancer, cervical carcinoma, hepatocellular carcinoma, angioblast Tumor, glioma.

The present inventor passes through further investigation VEGFR2 or even the identification of entire VEGFRs receptor family ligand and receptor activation Mechanism filters out enrichment humanization nano antibody using display technique of bacteriophage for the first time and carries out gene sequencing and albumen table to it Up to purifying；The nano antibody having with antigen compared with high-bond is further gone out by AlphaScreen technology screening；Had There is the source of people nano antibody NTV1 of more high-affinity and good anti-tumor activity.Pass through surface plasma resonance (SPR) technology table Levy the dynamic behavior of the antibody；Cell experiment demonstrates it and inhibits vascular endothelial cell proliferation ability and angiogenesis inhibiting living Property.The present invention expresses and optimizes the VEGFR2 nano antibody albumen for obtaining affinity height and stable uniform, and it is anti-successfully to research and develop source of people VEGFR2 nano antibody drug opens the frontier of the medicament research and development of targeting VEGFR2 antibody, has far-reaching social effect With wide potential applicability in clinical practice.Also, the present invention is that relevant subsequent medicament research and development provides the foundation, and can research and develop carrying accordingly Chemical drug merges the antibody of Fc to increase curative effect.

Detailed description of the invention

Fig. 1: display NTV (1-4) four kinds of nano antibody amino acid alignments.The gene order of antibody is translated into amino Acid sequence carries out Multiple Sequence Alignment.Unlabelled is complementary determining region, and label is framework region.

Fig. 2: the expression and purification of display NTV (1-4) albumen.10% polyacrylamide gel electrophoresis detects purity of protein, Standard molecular weight is marked on the left of gel.It is from left to right respectively NTV1, NTV2, NTV3, NTV4.

Reaction between Fig. 3: AlphaScreen detection VEGFR2D3 and NTV (1-4).(A) AlphaScreen reaction is shown It is intended to, the AlphaScreen testing result that (B-E) NTV1, NTV2, NTV3, NTV4 is reacted with VEGFR2D3.Fig. 4: SPR analysis NTV1 and VEGFR2D3 kinetics.

Fig. 5: NTV1 to the effect of human umbilical vein cell Proliferation.

Fig. 6: NTV1 inhibits HUVEC vascularization effect in vitro.

Specific embodiment

Laboratory apparatus used in following embodiment and experimental example and experimental material information are as follows:

Reagent and kit

Small hundred Tyke of extraction reagent kit of plasmid

Hundred Tyke of PCR product QIAquick Gel Extraction Kit

Hundred Tyke of Ago-Gel QIAquick Gel Extraction Kit

Agarose gives birth to work

Peptone sigma

Yeast extract sigma

Tris-base sigma

Biotin sigma

Imidazoles sigma

Restriction endonuclease BamH1NEB

Restriction endonuclease Not1 NEB

T4 DNA ligase Fermentas

Q5-High Fidelity DNA polymerase NEB

Maltose sigma

Phage display library HuSdLTMCreative BioLabs

Helper phage M13K07 helper phage GE Healthcare

Magnetic beadMyOne^TM Streptavidin C1 life technologies

IPTG gives birth to work

PEG8000 sigma

SDS sigma

Tween-20 traditional Chinese medicines

Biotin CAPture Kit Series S GE Healthcare

Alphascreen Histidine Detection Kit Perkin Elmer

Instrument and equipment

AKTA FPLC system (GE)

NanoVue spectrophotometer(GE)

centrifuges 5415R(Eppendorf)

Sorvall RC 12BP centrifuge(Thermo)

avanti J-26xp(BECKMAN COULTER)

Incubator/shaker for2L bacterial cultures(Thermo)

Chromatography freezer (Beijing moral day is helped)

PCR machines(Eppendorf)

High pressure cell cracker

Ultraviolet specrophotometer (BECKMAN COULTER)

Biorad imager(BIA-Rad)

Pure water meter (Milli-Q)

Superclean bench (AIRTECH)

High-pressure steam sterilizing pan (MIURA)

Liquid-transfering gun (device) (Eppendorf)

Biacore T200(GE Healthcare)

Envision microplate reader (PerkinElmer)

CO2 incubator (Thermo)

Flat tissue culture plate (Nunclon Denmark)

Crystallography instrument (FORMULATRIX)

Art Robbins Instruments(PHOENIX)

Crystal incubator (Molecular Dimensions)

Microscope (OLYMPUS)

Constant-temperature table (her Fu Sen Bioisystech Co., Ltd)

Prepare embodiment

The present embodiment provides a kind of preparation methods for preparing anti-vegf R2 nano antibody, comprising the following steps:

A. display technique of bacteriophage screening antibodies are used

Biotin-VEGFR2 fused antigen is coated on magnetic bead surfaces, 4 DEG C are incubated for 2 hours.After PBS washes 3 times, with containing 2% The PBS of skimmed milk power is closed 1 hour in 37 DEG C.It is 1 × 10 by capacity¹²The source of people nano antibody phage antibody library of clone's number adds In immune pipe after to closing, 37 DEG C are combined 1 hour.It is washed 10 times with PBST, PBS is washed 5 times.1mL glycine elution buffering is added Liquid (pH2.2), room temperature shake 10min, and eluent is sucked out, is neutralized to pH7.4 with Tris.10 times of volumes of eluent are sucked out TG1 room temperature infects 10min, measures titre and is transferred to 37 DEG C plus helper phage M13KO7 shaking overnight incubations.Above procedure into Row four-wheel.To filter out the nano antibody for having affinity to VEGFR2.It is chosen from the culture plate after the screening of last two-wheeled at random 300 clones are selected, gene sequencing is carried out and gene compares.Pick out the higher clone standby of sequence repetition number.Experimental result Display: being obtained 8 may compare through gene and find with the nano antibody in conjunction with VEGFR2, and NTV1 and NTV2 goes out in 8 clones Existing frequency highest, and each sequence complementary determining region conservative is not high (as shown in table 1 and Fig. 1).Prompt phage display skill Art can filter out the nano antibody in conjunction with VEGFR2.

B. the expression and purification of nano antibody

Sequence application Not I and BamH the I restriction enzyme and DNA recombinant technique that will be singled out are by the PCR after digestion In the pET carrier for the linearization for enzyme restriction that amplified production insertion carries 6His-sumo label, expression plasmid is constructed.With what is filtered out Sequence gene (i.e. NTV1-4) is template, introduces BamH1, Not1 restriction enzyme site, and target gene is introduced and carries 6His-sumo mark In the Pet-duet expression vector of label, expression plasmid is constructed.In molar ratio by the NTV1-4 genetic fragment of gel extraction and carrier 5:1 establishes 10 μ l linked systems, 25 DEG C of connection 10min of T4DNA ligase.Connection product is transferred to competent cell TOP10, is taken Bacterium solution after 150 μ l conversion is coated on the LB solid medium tablets containing 50 μ g/ml Amp, and 37 DEG C of inversion stationary cultures are stayed overnight. Expression plasmid is transferred to e. coli bl21 competent cell referring to above step by picked clones, and secondary morning receives LB plate.It will The 100ml LB liquid medium that sterilizes is packed into 250ml shaking flask, and the BL21 bacterial strain monoclonal on picking LB plate is to wherein, and 37 DEG C, 180rpm/min shake culture is stayed overnight；The bacterium solution OD value being incubated overnight is surveyed, is seeded in 4 × 2L LB (OD value 0.15) shaking flask, 24 DEG C, 180rpm/min shake culture；Its OD600 is surveyed after about 4 hours, when it is between 0.6~0.8, shaking table temperature is adjusted to 16 DEG C, cooling down and continuing culture to OD value is between 0.8~1.2；It is added IPTG (0.5M), until ultimate density is 50~100 μM, 16 DEG C of overnight inductions (16~18h), precipitating (4000rpm/min, 30 minutes) that thalline were collected by centrifugation；Every 2L bacterium solution 30ml His Buffer A is transferred in 50ml centrifuge tube after being sufficiently suspended, ice bath.With high pressure cracker smudge cells.16000rpm/min, High speed centrifugation 20 minutes, collecting supernatant was thick leach protein.Thick leach protein is crossed into nickel column.Nickel column is loaded into FPLC system, is set Elution program carries out albumen wash-out.By the protein concentration being collected into volume appropriate (be slightly larger than 10ml), over-molecular sieve column into One step purifies destination protein, obtains purified protein samples.With the expression of 12%SDS-PAGE electrophoresis detection destination protein.To appearance The higher clone NTV1-4 of frequency carries out prokaryotic system expression and purification, as shown in Fig. 2, result prompt is available using this method The albumen of stable homogeneous.

C. it is further screened using AlphaScreen and and nano antibody and VEGFR2 is measured using SPR technique Affinity

Due to the positive colony being enriched to by biopanning technique be likely to occur false positive as a result, therefore apply AlphaScreen technology further screening and highest nanometer of VEGFR2 affinity from the antibody that previous step filters out is anti- Body.It takes 1mg luminous particle and is separately added into centrifuge tube containing antigen or antibody response liquid, room temperature, which is protected from light, is incubated for 1h.It is micro- by two kinds (biotin-NTV (1-4) and His8-VEGFR2D3 equal proportion of concentration 20nM, 50nM, 100nM, 200nM are mixed for grain mixing Close), optical signal is detected in nanometer homogeneous phase time discrimination fluorescence detector after being incubated for 15min, each concentration is in triplicate. AlphaScreen testing result is as shown in Figure 3, it is seen that using AlphaScreen technology further screening and VEGFR2 parent With the highest nano antibody NYV1 of power.

Using BIAcore T200 system research nano antibody and VEGFR2 based on Applications of surface plasmon resonance Binding ability.2 channels are set in CAP sensor core on piece, as sense channel, another is not fixed a coupling VEGFR2 VEGFR2 is as blank reference channel.Using HBS solution as working solution, flow velocity is 2 μ L/min；Activation and closing chip；Again with 2 Respectively with gradient concentration sample introduction nano antibody, each concentration rank detects 2 times the flow velocity of μ L/min2；It obtains and combines dynamic map, Parameter calculating is carried out with software module after process of fitting treatment.As shown in figure 4, injection concentration is 10 μM of biotinylated NTV1, make letter Number reach 3500RU or so, by concentration be 20nM, 60nM, 180nM, 550nM, 1.6 μM, 5 μM of VEGFR2D3 flow through chip list Kinetic curve is drawn according to signal intensity in face.By Biacore T200 assessment software calculations incorporated constant (ka) and dissociate Constant (kd).Dissociation equilibrium constant (K is obtained by kd/ka_D).SPR technique confirms the binding ability of NTV1 and VEGFR2, solution From constant (K_D) it is 4.9nM, prompt nano antibody NTV1 and VEGFR2 that there is very high affinity, thus it is speculated that it may have anti-swollen Tumor function.

Experimental example: nano antibody biological activity determination

This experimental example measures inhibition Human umbilical vein endothelial cells (HUVECs) cell Proliferation of nano antibody using MTT experiment Ability forms influence of the measuring nano antibody to HUVECs angiogenesis using micro-pipe.

MTT experiment: with the dedicated culture medium of HUVECs cell (5%FBS-ECM) at 37 DEG C after incubation HUVECs24 hours The nano antibody of respective concentration is added, continues that MTT dyestuff is added in tissue culture plate after being incubated for 72 hours, uses enzyme after 4 hours Absorbance is read under mark instrument 570nm.

It is 5 × 10 that the NTV1 that concentration is 1nM, 10nM, 100nM and 1000nM, which is added to HUVECs concentration,³The 96 of cells/well It in orifice plate, is read at 570nm wavelength after 72 hours, blank control is set.Each concentration is repeated 3 times, and calculates standard deviation, is carried out The statistical analysis of P < 0.05, as a result as shown in Figure 5.As seen from Figure 5, compared with the control group, NTV1 are in concentration There is significant inhibiting effect to HUVECs proliferation when 10nM, 100nM, 1000nM, and dose-dependent relationship is presented.

Micro- tube formation assay: by 4.5 × 10³HUVECs is inoculated into paving in advance and is incubated by 37 DEG C in 96 orifice plates of Geltrix It educates overnight.Next day takes culture plate to observe each hole micro-pipe under inverted microscope and generates situation.

It is 4.5 × 10 that the NTV1 that concentration is 0nM, 10nM, 100nM, 1000nM, which is added to HUVEC concentration,³The 96 of cells/well It being incubated overnight in orifice plate, forms photographic analysis with inverted microscope Human Umbilical Vein Endothelial Cells official jargon, 100 times of amplifications carry out artificial counting, Not plus NTV1 as a control group, be calculated as 100%.Each concentration is repeated 3 times, and calculates standard deviation, carries out the statistics of P < 0.05 Analysis.As a result as shown in fig. 6, compared with blank control (0nMNTV1), NTV1, can be significant under 100nM, 1000nM concentration Inhibit the formation of endothelial cell official jargon, and with the raising of concentration, which has the tendency that becoming larger.

Above-mentioned experiment in vitro shows that HUVECs cell Proliferation and Angiogenesis can be effectively suppressed in NTV1.

Above embodiments are enumerated only as the example of embodiment of the present invention, do not constitute any limit to the present invention System, it will be appreciated by those skilled in the art that modification in the range of without departing from essence and design of the invention each falls within the present invention Protection scope.

Claims (3)

1. a kind of anti-vegf R2 source of people nano antibody, including framework region and complementary determining region, which is characterized in that the nano antibody With amino acid sequence shown in SEQ ID NO:1.

2. purposes of the antibody described in claim 1 in preparation anticancer medicine.

3. purposes according to claim 2, which is characterized in that the cancer includes breast cancer, lung cancer, bladder cancer, colon Cancer, cervical carcinoma, hepatocellular carcinoma, hemangioblastoma and glioma.