CN102190729A - Anti-human CD80 bivalent dimer and application thereof - Google Patents

Abstract

The invention discloses an anti-human CD80 bivalent dimer, which consists of a heavy chain variable region and a light chain variable region, and is characterized in that: the heavy chain variable region and the light chain variable region are connected through connecting peptide Gly4Ser; the nucleotide sequence of the heavy chain variable region is shown as SEQIDNO.1; the nucleotide sequence of the light chain variable region is shown as SEQIDNO.2; and the nucleotide sequence of the bivalent dimer is shown as SEQIDNO.3. The bivalent dimer has high capacity of combining antigen, has obvious inhibition effect on Raji cells, can be used for tumor imaging, and also can be applied to preparing medicines for inhibiting tumor cell proliferation.

Description

Anti-people CD80 bivalent antibody and application thereof

Technical field

The present invention relates to a kind of bivalent antibody, be specifically related to a kind of anti-people CD80 bivalent antibody.

Background technology

B7 family costimulatory molecules belongs to immunoglobulin superfamily (Immunoglobulin superfamily, IgSF) member comprises CD80(B7-1), CD86(B7-2), the ICOS equimolecular.Wherein, the CD80 molecule is again member important in the B7 family.The part of CD80 molecule is CD28 and the CTLA-4 that is expressed in the T cell surface, combine the costimulatory signal of transduction T lymphocyte activation with CD28, can promote secretion, the inductive effect T cytodifferentiation of cytokines such as specific T-cells propagation, IL-2 and provide to prevent and treat the signal etc. that too early apoptosis takes place the T cell; Can produce the inhibition signal and combine, stop the T cell activation, regulate the intensity of T cellullar immunologic response with CTLA-4.If lack this costimulatory signal, not only first signal of antigen-specific can not effectively activate specific T-cells, cause T cell incapability on the contrary, perhaps tolerance [4].Studies show that the signal path of CD80 mediation not only plays a significant role in T cell activation process, but also participate in the generation development and the prognosis situation of numerous disease.Given this, can use the specific antibody of CD80 to regulate the immune response strength of T cell or blocking-up signal that CD80 mediated to suppress the progress of disease.

The basic form of single-chain antibody: single-chain antibody (single-chain variable fragment, ScFv) comprise variable region of heavy chain (the variable heavy chain, VH) and variable region of light chain (the variable light chain, VL), size is 26-28kDa, it has complete antigen binding site, is the functional VH-VL zone of an antibody molecule minimum.There are two kinds of form: VH-linker-VL and VL – linker-VH in the variable region, and the former is more general.Link to each other by one section connection peptides between VH and the VL, 10-25 amino acid is arranged on its length usually, that the most general is (Gly ₄Ser) ₃Pentadecapeptide.To be Huston etc. succeed in developing in 1988 with Bird etc. first ScFv molecule simultaneously and can combine with target antigen with the avidity similar with parental antibody.

The derivative form of single-chain antibody: the ScFv major part is single aggressiveness, is with V by 12 or more amino acid connecting peptides _HAnd V _LLink to each other and get.Can not be folded into a functional Fv zone but have in the ScFv chain of 3 ~ 12 amino acid connecting peptides, on the contrary can and another ScFv molecular chain between to be cross-linked to form a dimer (diabody, ~ 60 kDa) be bivalent antibody.In addition, reduce connection peptides to 3 amino acid or ScFv is linked to each other form the tripolymer (triabodies, ~ 90 kDa) or the tetramer (tetrabodies, ~ 120 kDa).

Recombinant antibodies is widely used in the diagnosis and treatment of various diseases such as tumour, and the annual market sales revenue can reach 1,000,000,000 dollars and the trend that increases is arranged gradually.Therefore, exploitation high specificity, recombinant antibodies that avidity is high are subjected to the favor of each country.Single-chain antibody ScFv is by variable region of heavy chain (the variable heavy chain of 12 connection peptides more than the amino acid with antibody molecule, VH) and variable region of light chain (the variable light chain, VL) connect, it has complete antigen binding site.Compare with complete IgG molecule, ScFv shows the tumour-specific of obvious raising and the perviousness between tumour cell.Yet aspect tumor imaging, because their clearance rates in blood are very fast, and the avidity that single binding site causes is relatively low, causes the local level of this antibody-like higher, and imaging results is poor.Bivalent antibody (diabody) contains two antigen binding sites, and affine activity significantly improves, and obviously improves aspect tumor imaging.Connection peptides between single-chain antibody VH and the VL is at least 12 amino acid.When connection peptides was reduced to 3-10 amino acid, intramolecular VH and VL can not be combined into the effective antigens binding site usually, but this has promoted the pairing of intermolecular VH and VL, formed to have active diabody.

Diabody is an a kind of pair of valency dimer, promptly adopts the genetic engineering technique transformation to form on the basis of single-chain antibody.Diabody is divided into poly-type and different poly-type.The Diabody molecular weight generally between 50-60kDa, demonstrates favorable tissue penetration power, cancer target and serum clearance rate, becomes the carrier of the tool potentiality of immunotherapy of tumors most probably.At present, the anti-CEA-diabody approved of a strain enters clinical application, is mainly used in radioimmunodiagnosis.In addition, in addition the anti-CEA-diabody of two strains, anti-CD20-diabodies, anti-PSCA-diabodies (prostate stem cell antigen, PSCA) and anti-HER-2-diabodies through after the isotopic labeling in the middle of the research of preclinical phase and show good tumor imaging ability.

Therefore, need a kind of anti-people CD80 bivalent antibody of development with independent intellectual property right.

Summary of the invention

Goal of the invention of the present invention provides a kind of anti-people CD80 bivalent antibody, described bivalent antibody can specificity in conjunction with the CD80 molecule; Another object of the present invention provides the cell strain that produces anti-people CD80 bivalent antibody.

To achieve the above object of the invention, the technical solution used in the present invention is: a kind of anti-people CD80 bivalent antibody, described bivalent antibody is by variable region of heavy chain (the variable heavy chain, VH) and variable region of light chain (the variable light chain, VL) form, between the two by connection peptides Gly ₄Ser links to each other, and described variable region of heavy chain VH nucleotide sequence is shown in SEQ ID NO.1; Described variable region of light chain VL nucleotide sequence is shown in SEQ ID NO.2; The nucleotide sequence of bivalent antibody is shown in SEQ ID NO.3.

The cell strain of the above-mentioned anti-people CD80 bivalent antibody of the present invention's claimed generation simultaneously, the preservation information of described cell strain is: depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on 01 24th, 2011; Deposit number: CGMCCNo. 4571; Classification name: the cell strain of secreting anti-people CD80 molecule bivalent antibody.

The present invention is the application of claimed described anti-people CD80 bivalent antibody in tumor imaging simultaneously.

The present invention's application of claimed described anti-people CD80 bivalent antibody in the medicine of preparation inhibition tumor cell proliferation simultaneously.

Because the technique scheme utilization, the present invention compared with prior art has following advantage:

1. the ability that cell strain of the present invention is secreted anti-people CD80 bivalent antibody is strong, stable performance; Anti-people CD80 bivalent antibody of the present invention and antigenic binding ability are strong, and parental antibody 4E5 compares with the mouse source, have higher avidity;

2. anti-people CD80 bivalent antibody of the present invention can obviously suppress Raji cells in vitro propagation and be dose-dependently, the obvious suppression effect can occur when final concentration is 25 μ g/ml, reaches maximum value at 50 μ g/ml; Restraining effect obviously is better than the single-chain antibody of control group.

3. anti-people CD80 bivalent antibody of the present invention can successfully be blocked the signal path of CD80 mediation.

Description of drawings

Fig. 1 is the figure as a result that the double digestion of anti-CD80 bivalent antibody, variable region of heavy chain and chain variable region gene and pIRES2-EGFP/diabody among the embodiment is identified, wherein, and M:DL2 000 DNA marker; 1: heavy chain variable region gene; 2; Chain variable region gene; 3: anti-CD80 bivalent antibody gene; 4: carrier for expression of eukaryon pIRES2-EGFP/diabody is through by EcoR I and Sal I double digestion; 5: carrier for expression of eukaryon pIRES2-EGFP/diabody; 6: empty carrier pIRES2-EGFP;

Fig. 2 is Chinese hamster ovary celI (400 *) the light microscopic microgram of transfection 72h among the embodiment;

Fig. 3 is transfection 72h Chinese hamster ovary celI culture supernatant activity identification figure as a result among the embodiment;

Fig. 4 is the figure as a result that combines of anti-CD80 bivalent antibody supernatant and L929-CD80 cell among the embodiment;

Fig. 5 is the evaluation result figure that tires of anti-CD80 bivalent antibody among the embodiment;

Fig. 6 is the bivalent antibody sex change (A) of Ni post affinitive layer purification among the embodiment and non-sex change (B) polyacrylamide gel electrophoresis figure as a result;

Fig. 7 is the figure as a result that combines of anti-CD80 bivalent antibody and people B lymphoma cell strain Raji and Daudi among the embodiment;

Fig. 8 is that anti-CD80 bivalent antibody combines figure as a result to the anti-people CD80 of mouse source property antibody 4E5 with the competition of L929-CD80 cell membranous type CD80 molecule among the embodiment, wherein, and A:a: blank group; B: positive controls (4E5); C: anti-CD80 bivalent antibody+4E5; The folded figure of d:b and c; B:a: negative control group; B: anti-CD80 bivalent antibody and 4E5 adds simultaneously; C: anti-CD80 single-chain antibody and 4E5 adds simultaneously;

Fig. 9 be among the embodiment anti-CD80 bivalent antibody to Raji cells in vitro inhibition of proliferation exercising result figure, wherein, a: P＜0.05vs negative control; B: P＜0.01vs negative control; C: P＜0.05vs negative control; D: PThe anti-CD80 single-chain antibody of＞0.05vs (50 μ g/ml);

Figure 10 be among the embodiment anti-CD80 bivalent antibody to the blocking effect of APC surface membranous type CD80 molecule figure as a result, wherein, a:P＜0.05; B:P＞0.05.

Embodiment

Below in conjunction with drawings and Examples the present invention is further described:

Embodiment one: make up the pIRES2-EGFP/diabody expression vector.

1) recovery contains pIRES2-EGFP/ScFv intestinal bacteria Top10: that draws-80 ℃ of frozen guarantors kinds contains pIRES2-EGFP/ScFv intestinal bacteria Top10 50 μ l, joins and contains 4ml Kan ⁺In the Fahrenheit pipe of LB substratum, 37 ℃, 200rpm shakes bacterium 12h, and next day is standby.

2) extract the pIRES2-EGFP/ScFv plasmid, operate according to plasmid DNA small volume of reagent box specification sheets, concrete steps are as follows:

A. get the bacterium liquid of the above-mentioned overnight incubation of 4 ml, centrifugal 1 min of 12,000 * g abandons most supernatant.

B. add 250 μ l Buffer S1(and contain RNase A) the suspension bacterial precipitation, suspending needs evenly should not leave little bacterium piece.

C. add 250 μ l Buffer S2, gentleness also spins upside down fully to mix for 10-15 time and makes the abundant cracking of thalline, until forming bright solution.

D. add 350 μ l Buffer S3, gentle also spinning upside down fully mixed centrifugal 15 min of 12,000 * g 6-8 time.

E. draw and go up centrifugal supernatant of step and transfer to preparation pipe (2 ml centrifuge tubes (providing in the test kit) are provided), the centrifugal 1min of 12,000 * g abandons filtrate.

F. will prepare pipe and put back centrifuge tube, and add 500 μ l Buffer W1, centrifugal 1 min of 12,000 * g abandons filtrate.

G. will prepare pipe and put back centrifuge tube, and add 700 μ l Buffer W2(and added dehydrated alcohol), the centrifugal 1min of 12,000 * g abandons filtrate; With same method more once with 700 μ l Buffer W2 washing.Abandon filtrate.

H. will prepare pipe and put back in the 2ml centrifuge tube, centrifugal 1 min of 12,000 * g.

I. will prepare pipe and move in the 1.5 new ml centrifuge tubes, central authorities add 60 μ l Eluent at the preparation periosteum, and room temperature leaves standstill 1 min.Centrifugal 1 min of 12,000 * g.

Preserve in-80 ℃ extracting good plasmid, standby.

3) extract anti-CD80-diabody weight chain gene: with the above-mentioned pIRES2-EGFP/ScFv plasmid that obtains is template, is primer with P1/P2 and P3/P4 respectively, PCR reaction amplification heavy and light chain gene,

Reaction system is as follows:

Wherein, F, R represent forward and reverse primer, if obtain the VH gene, then F is P1, and R is P2; If obtain the VL gene, then F is P3, and R is P4.

The cycling condition parameter is: heavy chain: 94 ℃ of pre-sex change 5min, 94 ℃ of 1min, 60 ℃ of 45s, 35 circulations of 72 ℃ of 50s; Extend 10min in 72 ℃ again.Light chain: 94 ℃ of pre-sex change 5min, 94 ℃ of 1min, 61 ℃ of 45s, 35 circulations of 72 ℃ of 50s; Extend 10min in 72 ℃ again.

Get above-mentioned PCR product in 1.5% agarose gel electrophoresis analysis, the results are shown in Figure 1: visible V clearly _HAnd V _L, stripe size is about 440bp and 360bp respectively.Reclaim test kit purifying VH, VL gene through glue, standby, concrete steps are as follows:

A. under ultraviolet lamp, downcut the sepharose that contains target DNA, exhaust gel surface liquid and chopping with paper handkerchief.Calculated for gel weight (writing down 1.5ml centrifuge tube weight in advance), this weight is as a gel volume (as 100mg=100 μ l)

B. the Buffer DE-A that adds 3 gel volumes mixes the back in 75 ℃ of heating (the low melting-point agarose gel is in 40 ℃ of heating), is interrupted and mixes (every 2-3 min), melts (about 6-8min) fully until gel piece

C. add the Buffer DE-B of 0.5 Buffer DE-A volume, mix; When separated DNA fragment during, add the Virahol of 1 gel volume less than 400bp

D. draw the mixed solution of going up in the step, transfer to DNA preparation pipe (placing in the 2 ml(test kits provides) centrifuge tube), centrifugal 1 min of 12000 * g.Abandon filtrate

E. will prepare pipe and put back the 2ml centrifuge tube, and add 500 μ l Buffer W1, centrifugal 30 s of 12000 * g abandon filtrate

F. will prepare pipe and put back the 2ml centrifuge tube, and add 700 μ l Buffer W2, centrifugal 30 s of 12000 * g abandon filtrate, wash the centrifugal 1min of 12000 * g with 700 μ l Buffer W2 again with same method

G. will prepare pipe and put back in the 2ml centrifuge tube, the centrifugal 1min of 12,000 * g

H. will prepare pipe 1.5 clean ml centrifuge tubes (providing in the test kit) are provided, central authorities add 25-30 μ l Eluent or deionized water at the preparation film, and room temperature leaves standstill 1min, the centrifugal 1min eluted dna of 12000 * g.

4) make up recombinant expression vector: adopt overlapping extension splicing PCR method (splicing by overlapping extension PCR, SOE-PCR) (as seen electrophoresis is about the band of 800bp the weight chain gene to be connected structure diabody gene, see Fig. 1), detailed process is as follows: at first with the weight chain gene mixing behind the purifying, add in nothing under the situation of primer, primer and template each other, PCR reacts (94 ℃ of 40s, 55 ℃ of 45s, 72 ℃ of 50s) 10 circulations of amplification, in reaction system, add P1 and P4 subsequently, same 35 circulations of PCR reaction conditions amplification.After the PCR product reclaims, make up cloning vector pMD19-T/diabody and connect, transformed competence colibacillus Top10, picking positive colony enzyme cut the order-checking of checking back.PMD19-T/diabody and plasmid pIRES2-EGFP that order-checking is correct use EcoRI and SalI is carried out double digestion (double digestion the results are shown in Figure 1) respectively, and the T4 dna ligase connects, and makes up the pIRES2-EGFP/diabody expression vector.

Embodiment two: the anti-CD80-diabody cell strain of stably express build strain and screening

1) recovery of CHO: at first bath temperature is adjusted to 40 ℃, in Bechtop, draws the RPMI1640 nutrient solution that contains 10% calf serum and place centrifuge tube, take out freeze-stored cell, its lower end is inserted in the water-bath, shake short its thawing gently from liquid nitrogen.Ice cube in waiting to manage takes out when melting to the soya bean size, places off-the-shelf centrifuge tube with suction pipe sucking-off cell, shakes up, and 800rpm * 5min is centrifugal, abandon supernatant, with after an amount of substratum washing, re-suspended cell moves into culturing bottle again, microscopically is put 37 ℃, 5%CO after observing ₂Cultivate in the incubator.After treating that cell state is adjusted, a little cell is moved in 6 orifice plates, treat that cell grows at 80% o'clock in the hole, can supply transfection.

2) anti-CD80-diabody cell strain build strain and screening: adopt liposome method with the Chinese hamster ovary celI (operating) of growing in pIRES2-EGFP/diabody transfection 6 orifice plates by the test kit specification sheets, 6h changes perfect medium into, 1:6 dilution next day, day by day observe after the transfection, as seen green fluorescence is arranged under the fluorescent microscope, and transfection efficiency can reach 60%(and see Fig. 2); Collect transfection 72h cells and supernatant to do active the detection: connect immunofluorescence technique 72h cells and supernatant qualitative experiment is shown, anti-CD80-diabody active good (Fig. 3).With more than 1mg/ml G418 pressurization two weeks of screening, continue to cultivate behind the cell clone of acquisition stable growth during 72h.Collect well-grown people CD80 gene transfecting cell strain L929-CD80 as antigen, cell concn to 1 * 10 are adjusted in 2%FCS-PBS washing 2 times ⁷/ ml, get the culture supernatant reaction of 50mL cell suspension and transfection goal gene cell, behind 4 ℃ of 30min, 2%FCS-PBS washing 2 times, add PE mark mouse-anti-His antibody 2mL, reaction is after flow cytometry analysis, and with L929-mock as the negative control cell strain, the analytical results of FCM shows that the positive combination rate of gene transfecting cell culture supernatant and L929-CD80 is that 96.5%(sees Fig. 4).Therefore, obtain plant height secretion strain, called after SQA-8.SQA-8 is through continuous passage culture in vitro, well-grown behind the cryopreservation resuscitation repeatedly, the stable performance of secretory antibody.

Embodiment three: the expression of anti-CD80-diabody and purifying

With the above-mentioned cell strain large scale culturing that filters out, treat that cell is covering with at the bottom of the culturing bottle and during the substratum flavescence, collect culture supernatant, through high speed centrifugation and 0.22 μ m filter membrane ultrafiltration and concentrated, enriched material pack into dialysis tubing after the PBS dialysed overnight, adjust PH to 8.0, the scheme by bio-red company provides adopts Profinity ^TMIMAC nickel prepacked column manually carries out separation and purification, the lowry standard measure, and pure product filtration sterilization is sub-packed in-20 ℃ of preservations.

The result shows: the SQA-8 cells and supernatant is through Profinity ^TMAfter the IMAC Cartridges separation and purification, the lowry standard measure, anti-CD80-ScFv productive rate is about 15mg/L.

Embodiment four: the SDS-PAGE of the two valencys of antibody identifies

1) electrophoresis under the sex change environment may further comprise the steps:

A., the vertical electrophoresis instrument is installed.

B. dispose 12%SDS-PAGE separation gel (5ml): deionized water (1.6ml), 30% acrylamide (2.0ml), 1.5M Tris pH8.8(1.3ml), 10%SDS(50 μ l), 10% ammonium persulphate (50 μ l), TEMED(4 μ l).

C. mixing is poured between two sheet glass rapidly, reserves perfusion and concentrates the glue requisite space.Add water seal at once, parked 30min, the complete polymerization of glue to be separated.

D. configuration concentrates glue (2ml): deionized water (1.4ml), 30% acrylamide (0.33ml), 1.0M Tris pH6.8(0.25ml), 10%SDS(20 μ l), 10% ammonium persulphate (20 μ l), TEMED(4 μ l).Discard the water that is used to seal, and blot with thieving paper, mixing pouring into concentrated glue on the polymeric separation gel, inserts comb rapidly.

E. after concentrated glue polymerization is finished, put into electrophoresis apparatus, add electrophoretic buffer.

F. anti-people CD80-diabody and anti-people CD80-ScFv be 40 μ l and the mixing of 8 μ l, 6 * sex change sample loading buffer respectively, 100 ℃ of sex change 5min, and sample is gone up in ice bath, centrifugal back.

G. connect power supply, concentrate glue voltage 80V, separation gel voltage 120V treats to finish when tetrabromophenol sulfonphthalein arrives the bottom.

The dismounting gel, gel is put into the Xylene Brilliant Cyanine G solution 4h that dyes, and finishes after the water flushing, puts into destainer and decolours that (methyl alcohol: acetate: water=4.5:1:4.5) is also observed.

2) electrophoresis under the non-sex change environment: except sample-loading buffer and electrophoretic buffer, do not add in the system outside the SDS, other conditions are identical with electrophoresis under the sex change environment.Non-in addition sex change electrophoresis should be tried one's best and be carried out at low temperatures.

Sex change SDS-PAGE result shows that a band is only arranged after the SQA-8 sex change, and Mr is about 27kDa, and is consistent with albumen size after its sex change; Non-sex change SDS-PAGE result shows that anti-CD80-diabody is with single band migration, and the SA-II also shows single band.The electrophoresis result under sex change and non-sex change condition according to SQA-8 and SA-II is not difficult to infer that the SQA-8 size about 53kDa, sees Fig. 6.

Embodiment five: the analysis of tiring of anti-CD80-diabody

Collect well-grown L929-CD80 cell, with 5 * 10 ⁵/ 10 μ l/test add the anti-CD80-diabody antibody of 0.5 μ g, 1.0 μ g, 1.5 μ g, 2.0 μ g and 2.5 μ g more respectively, hatch 45min altogether in 4 ℃, and it is anti-to add mouse-anti His-PE two after the PBS washing, and 4 ℃ of reaction 30min are behind the thorough washing.Add 0.5mL PBS re-suspended cell, through flow cytometry.Mouse source property parental antibody 4E5 is set to compare.

The flow cytometry analysis result shows, 6 * 10 ⁵Among the individual L929-CD80, add single-chain antibody 0.1,0.5,1.0,1.5 and 2.0 μ g respectively, positive combination rate is followed successively by 35.6%, 60.7%, 80.2%, 99.7% and 99.8%.Positive combination rate (99.9%) with mouse source property parental antibody 4E5 is reference, and the relative potency of anti-CD80-ScFv antibody is 1.5 μ g/5 * 10 ⁵Individual cell.See Fig. 5.

Embodiment six: anti-CD80-diabody is in conjunction with activation analysis

The people B that collects natural high expression level CD80 molecule respectively is lymphoma cell strain Raji and Daudi, and people T is lymphoma cell strain Jurkat, with 5 * 10 ⁵/ pipe is sub-packed in the streaming pipe, adds anti-CD80-diabody 2 μ g, 4 ℃ of reactions down, and behind the 30min, 2%FCS-PBS washing 2 times adds anti--His antibody 3mL that PE connects, and reaction is after flow cytometry analysis.

The result that FCM analyzes shows that anti-CD80-diabody can combine with the people's Malignant B lymphoma cell strain Raji and the Daudi of natural high expression level CD80 molecule, and positive rate is respectively 98.1% and 93.0%, sees Fig. 7.Point out anti-CD80-diabody can discern the CD80 molecule.

Embodiment seven: anti-CD80-diabody suppresses to combine with parental antibody 4E5 competition

Collect well-grown L929-CD80,4 * 105/ pipes.Experiment is divided into: 1. add anti-CD80-diabody 3mg in advance, hatch 40min for 4 ℃, washing adds the anti-CD80 antibody of mouse source parent 4E5 1mg/50mL again; 2. add 4E51mg separately; 3. will resist each 3mg of CD80-diabody and ScFv and 4E5 1mg to add simultaneously in the streaming pipe; Above-mentioned cell is hatched 40min in 4 ℃.After the washing, cell is resuspended among the 2%FCS-PBS, adds 10ml sheep anti-mouse igg-PE two and resist, hatch 40min for 4 ℃, the 2%FCS-PBS washing, flow cytometer detects.To 4E5 competition inhibiting rate=(1. 4E5 control group A value-experimental group is worth))/4E5 control group A value * 100%.

The analytical results of flow cytometry shows that the L929-CD80 cell adds parental antibody 4E5 in advance again after SQA-8 is hatched, and positive combination rate only is 2.8%; And the positive combination rate of independent and 4E5 is respectively 97.0%, and the competition inhibiting rate is 97.11%.SQA-8 and SA-II with amount add streaming Guan Zhongyu L929-CD80 reaction simultaneously with 4E5 respectively, the result shows that SQA-8 can be with higher avidity and antigen-reactive, make that the positive combination rate of L929-CD80 and 4E5 only is 17.4%, and SA-II group, positive combination rate reaches 32.5%, and prompting SQA-8 has higher affine activity.More than the results are shown in Figure 8.

Embodiment eight: anti-CD80-diabody is to the influence of Raji in-vitro multiplication

It is 1 * 10 that Raji is adjusted cell density with the RPMI1640 that contains 20%FCS ⁶/ ml, 100 μ l/holes place 96 well culture plates, adding final concentration respectively is the anti-CD80-diabody of 5,25,50,75,100 μ g/ml, 37 ℃, 5% CO2 are cultivated 72h, and every hole adds MTT (5 g/L) 20 μ l, continue to cultivate 4h, abandon supernatant after centrifugal and add 100 μ l DMSO again, leave standstill 10min, the 570nm place measures the A value, draws growth curve.Every group is provided with 3 multiple holes, the 4E5 control group is set simultaneously, and calculates inhibiting rate.Inhibiting rate=(control group A value-experimental group A value)/control group A value * 100%.

After anti-CD80-diabody and the Raji co-culture of cells, observe day by day, the microscopically visible cell is assembled agglomerating, occurs black particle in the kytoplasm, and refractivity and stereoscopic sensation weaken.Be cultured to 72 h, the MTT detected result shows, effect has concentration dependent to anti-CD80-diabody to Raji cells in vitro inhibition of proliferation, when concentration is 25 μ g/ml, compare with the feminine gender group, can produce obvious retarding effect (P＜0.05) to the propagation of Raji cell, suitable therewith effect (P＜0.05 vs, 25 μ g/ml SQA-8) just can occur and SA-II concentration is 50 μ g/ml.To be that 50 μ g/ml effects reach maximum when SQA-8 concentration, and inhibiting rate is 31.27%, sees Fig. 9.

Embodiment nine: anti-CD80-diabody is to the blocking effect of APC surface C D80 molecule

Collect healthy human peripheral blood, separate peripheral blood mononuclear cell., at first it is hatched 40min(altogether with anti-CD80-diabody and combines sealing CD80 molecule as antigen presenting cell (APC) with the Raji cell by antigen-antibody), abandon supernatant.In cell, add mitomycin 50 μ g/1 * 10 ⁷Individual cell (inhibition cell proliferation), is hatched 40min by 37 ℃.Adjust concentration to 3.0 * 10 of Raji and PBMC after the washing respectively ⁶/ ml, both equal-volumes mix, and add 96 orifice plates, 200 μ l/ holes, 3 every group multiple holes.37 ℃, 5%CO ₂Cultivate 72h, mtt assay is measured.

The MTT experimental result shows, the Raji cell is handled the back through SQA-8 the short proliferation function of PBMC is obviously descended, handle the Raji cell with the anti-CD80-diabody of 3 μ g after, the obviously reduction of corresponding PBMC propagation is compared with the feminine gender group, difference have statistical significance ( P＜0.05), and compare with positive control 4E5 group, not statistically significant ( P＞0.05).The results are shown in Figure 10.

Nucleotide and/or aminoacid sequence table

＜110〉University Of Suzhou

＜120〉anti-people CD80 bivalent antibody and application thereof

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cgaagggggt tacgacgagg ttactctgct atggactact ggggtcaagg aacctcagtc 420

accgtctcct cagccggcgg cggaggtagc gacattgtgc tgacccaatc tccagcttct 480

ttggctgtgt ctctaggaca gagagccact atcttctgca gagccagcca gagtgtcgat 540

tataatggaa ttagttatat gcactggttc caacagaaac caggacagcc acccaaactc 600

ctcatctatg ctgcatccaa cctagaatct gggatccctg ccaggttcag tggcagtggg 660

tctgggacag acttcaccct caacatccat cctgtggagg aggaagatgc tgcaacctat 720

tactgtcagc aaagtattga ggatccgtac acgttcggag gggggaccaa gctggaaata 780

aaacggcacc accatcacca ccattaacag ctgtcc 816

<210> 4

<211> 33

<212> DNA

＜213〉synthetic

<400> 4

gtcgaattca tggacaggct tacttcctca ttg 33

<210> 5

<211> 42

<212> DNA

＜213〉synthetic

<400> 5

gtcgctacct ccgccgccgg ctgaggagac ggtgactgag gt 42

<210> 6

<211> 42

<212> DNA

＜213〉synthetic

<400> 6

gccggcggcg gaggtagcga cattgtgctg acccaatctc ca 42

<210> 7

<211> 51

<212> DNA

＜213〉synthetic

<400> 7

cctgtcgact taatggtggt gatggtggtg ccgttttgat ttccagcttg g 51

Claims (4)

1.. an anti-people CD80 bivalent antibody, described bivalent antibody is made up of variable region of heavy chain and variable region of light chain, it is characterized in that, between the two by connection peptides Gly ₄Ser links to each other, and described weight chain variable region nucleotide sequence is shown in SEQ ID NO.1; Described light chain variable region nucleotide sequence is shown in SEQ ID NO.2; The nucleotide sequence of bivalent antibody is shown in SEQ ID NO.3.

2. cell strain that produces anti-people CD80 bivalent antibody, the preservation information of described cell strain is: depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on 01 24th, 2011; Deposit number: CGMCCNo. 4571; Classification name: the cell strain of secreting anti-people CD80 molecule bivalent antibody.

3. the application of the described anti-people CD80 bivalent antibody of claim 1 in tumor imaging.

4. the application of the described anti-people CD80 bivalent antibody of claim 1 in the medicine of preparation inhibition tumor cell proliferation.

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