CN1603345A - A trivalent bispecific antibody, its preparation method and use - Google Patents
A trivalent bispecific antibody, its preparation method and use Download PDFInfo
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Abstract
本发明公开了一种三价双特异性抗体，其制备方法及用途。本发明的三价双特异性抗体包括人IgG1重链第一恒定区CH1、к链恒定区CL结构域、A抗体的重链可变区VH和轻链可变区VL、B抗体的单链可变区scFv。其中VH连接在CH1的N端，scFv连接在CH1的C端，VL连接在CL的N端，scFv连接在CL的C端。本发明的双特异性抗体制备方法包括表达载体的构建、双特异性抗体的表达及检测、双特异性抗体的纯化、双特异性抗体的结合活性分析等步骤。本发明的三价双特异性抗体具有副作用小、导向性好等优点，可以用于制备肿瘤的治疗药物。
The invention discloses a trivalent bispecific antibody, its preparation method and application. The trivalent bispecific antibody of the present invention comprises the first constant region CH1 of human IgG1 heavy chain, the CL domain of the constant region of the κ chain, the heavy chain variable region VH and the light chain variable region VL of the A antibody, and the single chain of the B antibody Variable region scFv. The VH is connected to the N-terminal of CH1, the scFv is connected to the C-terminal of CH1, the VL is connected to the N-terminal of CL, and the scFv is connected to the C-terminal of CL. The preparation method of the bispecific antibody of the present invention includes the steps of constructing an expression vector, expressing and detecting the bispecific antibody, purifying the bispecific antibody, and analyzing the binding activity of the bispecific antibody. The trivalent bispecific antibody of the invention has the advantages of less side effects, good orientation and the like, and can be used to prepare tumor therapeutic drugs.
Description
Technical field
The present invention relates to a kind of bi-specific antibody, specifically, relate to a kind of trivalent bi-specific antibody, also relate to Preparation Method And The Use.
Background technology
(bispecific antibody BsAb) is the artificial antibody of containing two species-specific antigen binding sites to bi-specific antibody, can erect bridge between target cell and functional molecular (cell), produces the effector function of guidance quality.BsAb particularly has broad application prospects in the immunotherapy of tumour in biomedicine.By BsAb mediated cell toxic action kill tumor cell is the focus of current immunotherapy applied research, its principal feature is that BsAb can be simultaneously in conjunction with the target molecule on tumor associated antigen and the effector cell, and directly the trigger effect cell is to the specific killing of tumour cell.Can prepare BsAb by methods such as chemical engineering, cell engineering and genetically engineereds at present.To be that antagonist carries out engineered but gene engineering research makes up the great advantage of BsAb, as making up humanization BsAb, small molecules BsAb, multivalence BsAb etc.Can adopt the genetic engineering technique design, make up two valency BsAb, its structure pattern comprises dual specific miniantibody (Bispecific miniantibody), double-stranded antibody (Diabody), single chain bispecific antibody (Sc-BsAb) etc.Two valency BsAb have that molecular weight is little, the tumor tissues good penetrability, be easy to make up and advantages such as expression, but because they only have a combined function territory to each specific antigens, not high to the avidity of target molecule, guide effect is relatively poor.Be stability and the treatment potential that improves BsAb, the structure pattern that had occurred tetravalence BsAb in recent years again, as dimerization dual specific miniantibody (Dimericbispecific miniantibody, Dibi Miniantibody), tetravalence bi-specific antibody Tandemdiabody (Tandab), IgG sample bi-specific antibody etc., they are except that the feature with dual specific, to each target antigen is again two valencys, thereby target cell there are higher avidity and stronger effector function, stability in human serum strengthens to some extent, and the residence time in circulation also obviously prolongs.But because tetravalence BsAb pairing effect cell (T lymphocyte, NK cell etc.) also is with two valency combinations, can cause during clinical application that effector cell's surface receptor is crosslinked in the blood circulation, cause the effector cell extensively to activate, discharge a large amount of cytokines (as TNF α etc.), cause unacceptable toxic reaction.Animal and the clinical test results of more than ten years in past show have the BsAb of bigger clinical value should have following characteristic, (1) must can highly selective and high-affinity ground in conjunction with tumor associated antigen; (2) in the recycle system, BsAb and effector cell or cytotoxicity trigger molecule should relatively low avidity, thereby can limit the side reaction that general effector cell activation causes, the necessary again cytotoxicity of inductive effect cell effectively when BsAb is incorporated into the tumour cell of expressing target antigen simultaneously; (3) BsAb should be humanized antibody, thereby reduces the anti-mouse immune response of people to greatest extent, makes repeat administration unrestricted; (4) the BsAb molecule should be of moderate size, and can be penetrated into tumor tissues inside, can guarantee the suitable circulation residence time again.The two special or trivalent three-specific antibodies of divalence that adopt human IgG1's heavy chain first constant region (CH1) and κ chain constant region (CL) structural domain to make up at present as the different dimerization structural domain, they only have a combined function territory to each specific antigens, avidity to target molecule is not high, and guide effect is relatively poor.So structure is reasonable more, the more powerful BsAb of functional effect still remains exploitation.
Summary of the invention
For solving the deficiency that present bi-specific antibody exists, the invention discloses a kind of trivalent BsAb.
The present invention adopts human IgG1's heavy chain first constant region CH1 and κ chain constant region CL structural domain as the different dimerization structural domain, designs a kind of novel trivalent BsAb.Trivalent BsAb of the present invention comprises the strand variable region scFv of the variable region of heavy chain VH of human IgG1's heavy chain first constant region CH1, κ chain constant region CL structural domain, A antibody and variable region of light chain VL, B antibody.At the variable region of heavy chain VH of the N of CH1 end connection A antibody, the strand variable region scFv (structure is VH-VL or VL-VH) at the C of CH1 end connection B antibody forms the VH-CH1-scFv structure; At the variable region of light chain VL of the N of CL end connection A antibody, the scFv at the C of CL end connection B antibody forms the VL-CL-scFv structure; Natural dimerization forms trivalent BsAb between CH1 and CL, and each structural domain of trivalent BsAb puts in order and structural representation is seen Fig. 1 and Fig. 2.In trivalent BsAb, A antibody recognition effector cell triggers molecule such as CD3, CD16, CD64 etc., B antibody recognition target cell surface marker such as Her2/neu, CD20, CD30, EGFR etc. in the surface.
The invention also discloses the preparation method of trivalent BsAb, this method may further comprise the steps:
(1) structure of expression vector is selected expression vector, makes up trivalent bi-specific antibody two-cistron expression vector.According to the design of the multiple clone site in VL, CL, scFv, RBS (ribosome bind site, sequence are aaggag), pelB (the bacterium signal peptide sequence is shown in sequence in the sequence table 15), VH and CH1 gene order and carrier primer.Adopt overlapping extension PCR method to be connected between wherein VL and CL, VH and CH1, RBS and pelB are blended in 5 of VH ' end by primer.Purifying reclaims the PCR product, and enzyme is cut the VL-CL fragment respectively, the scFv fragment, and the RBS-PelB-VH-CH1 fragment, each endonuclease bamhi inserts the multiple clone site in the carrier, construction of expression vector successively.
(2) expression of recombinant protein BsAb in intestinal bacteria, detect the recombinant vectors transformed into escherichia coli, picking list bacterium colony is cultivated, and adds IPTG and induces, and sample thief carries out the expression that SDS-PAGE and Western-blot detect BsAb.
(3) the medium centrifugal results thalline after the purifying of BsAb will be expressed, PBS is resuspended, freeze thawing, ultrasonic wave is broken bacterium, and is centrifugal, the results supernatant.Supernatant is crossed affinity column with the dilution of binding buffer liquid, the elution buffer wash-out, and SDS-PAGE detects protein purification.
(4) combination of facs analysis Preliminary detection BsAb is active
1. facs analysis BsAb is active with combining of SK-BR-3 cell.Digestion SK-BR-3 cell with cold PBA centrifuge washing, is hatched with the BsAb vibration, and centrifuge washing is hatched with goat anti-human igg's vibration of FITC mark, and centrifuge washing behind the re-suspended cell, is analyzed.
2. facs analysis BsAb is active with combining of CD16 surface antigen positive cell.Get healthy human peripheral blood, separate mononuclearcell, centrifuge washing is hatched with the BsAb vibration, and centrifuge washing is hatched with the mouse-anti people CD56 of phycoerythrobilin enzyme labelling, goat anti-human igg's vibration of FITC mark, and centrifuge washing behind the re-suspended cell, carries out the Two Colour Fluorescence analysis.
Trivalent bi-specific antibody of the present invention combines the advantage of bivalent, bispecific antibodies and tetravalence bi-specific antibody in the past, abandon its defective, have following advantage: (1) trivalent BsAb pairing effect cell is with the unit price combination, in the recycle system, the avidity that triggers molecule with the effector cell is lower, can avoid or alleviate the side reaction that general effector cell activation causes like this; (2) with two valency combinations, it has higher avidity to target cell to trivalent BsAb, has strengthened guide function to target cell, to be positioned tumor locus quickly when being used for interior therapeutic, both can the reinforcing effect function, can reduce bi-specific antibody concentration in the circulation again, alleviate side reaction; (3) the about 100kD of trivalent BsAb molecular weight, molecular size is moderate, can be penetrated into tumor tissues inside, can guarantee the suitable circulation residence time again.
The prepared antibody of the present invention can be used to prepare antineoplastic medicine, and particularly the removing for the residual kitchen range of tumour patient postoperative, metastasis has very important use value.
Description of drawings
Fig. 1 is each structural domain of trivalent BsAb synoptic diagram that puts in order.
Fig. 2 is each structural domain structural representation of trivalent BsAb
Fig. 3 is a bicistronic mRNA structural representation in the trivalent bi-specific antibody expression vector.Wherein P/O is T7 promotor/lac operator gene, RBS is a ribosome bind site, pelB is the bacterium signal peptide sequence, VL is anti-people CD16 light chain variable region sequence, CL is a people κ chain constant region sequence, scFv is anti-Her2/neu single-chain antibody variable region sequences, and VH is anti-people CD16 weight chain variabl area sequence, and CH1 is human IgG1's heavy chain first constant region.
Fig. 4 pcr amplification VL, VH, CL and CH1 fragment electrophorogram.Wherein
1 is VL; 2 is RBS-PelB-VH; 3 is DGL2000 DNA marker;
4 is CL; 5 is CH1
Fig. 5 is a pcr amplification scFv electrophorogram.Wherein
1 is scFv (EcoRI, HindIII); 2 is DGL2000 DNA marker;
3 is scFv (NotI, XhoI)
Fig. 6 is overlapping extension PCR method amplification VL-CL fragment electrophorogram.Wherein
1 is DGL2000 DNA marker; 2 is VL-CL;
Fig. 7 is overlapping extension PCR method amplification RBS-PelB-VH-CH1 fragment electrophorogram.Wherein
1 is RBS-PelB-VH-CH1; 2 is DGL2000 DNA.
Fig. 8 is recombinant vectors called after pET22b (+)/BsAb structural representation.
Fig. 9 detects the expression (precipitation) of BsAb in BL21 (DE3) for SDS-PAGE.Wherein
1 is LMW protein marker;
2 is control (empty carrier pET22b), does not induce;
3 is control, IPTG 0.5mmol/L;
4 is control, IPTG 0.2mmol/L;
5 is pET22b/VLCL+scFv, does not induce;
6 is VLCL+scFv, IPTG 0.5mmol/L;
7 is VLCL+scFv, IPTG 0.2mmol/L;
8 is pET22b/BsAb, does not induce;
9 is BsAb, IPTG 0.5mmol/L;
10 is BsAb, IPTG 0.2mmol/L.
Figure 10 detects the expression of BsAb in BL21 (DE3) for Western-blot, wherein
1 is LMW protein marker;
2 are induced precipitation not;
3 for not inducing supernatant;
4 is induced precipitation;
5 for inducing supernatant.
Figure 11 detects purifying BsAb for SDS-PAGE, wherein
1 is LMW protein marker, and 2 is purifying BsAb.
Figure 13 is that facs analysis BsAb is active with combining of CD16 surface antigen positive cell, wherein
1 negative contrast 2 is BsAb
Embodiment
The preparation of the trivalent BsAb of embodiment one anti-Her2/neu * anti-CD16
One, material
(VL VH) clones from hybridoma B88-9, commercial (experimental technique is with reference to " molecular cloning " (Science Press, second edition)) for mouse-anti people CD16 monoclonal antibody light chain, heavy chain variable region gene; Mouse-anti P185
ErbB2Monoclonal antibody VL and VH gene clone are synthesized scFv according to a conventional method from hybridoma Her2 (commercial), and the aminoacid sequence of connection peptides is (GGGGS) 3 (referring to " genetic engineering antibody ", Dong Zhiwei, Wang Yan chief editor) between VL and the VH; Human IgG1's heavy chain first constant region CH1 and κ chain constant region CL gene by doctor Tong Yigang of Military Medical Science Institute provide (GenBank AF027159, X95747); Coli strain JM109, BL21 (DE3), prokaryotic expression carrier pET22b (+) is type strain, and is commercial; High expression level P185
ErbB2Human breast cancer cell SK-BR-3 be type strain (ATCC Number:HTB-30), available from Shanghai cell biological institute of the Chinese Academy of Sciences.Affinity chromatography column packing rProtein G Sepharose Fast Flow is a Pharmacia Biotech company product; The T4 dna ligase is a Gibco BRL company product; VentDNA polysaccharase, restriction enzyme are Biolab company product; It is Beijing ancient cooking vessel state biotech company product that plasmid extraction kit, dna fragmentation reclaim test kit; IPTG is a SIGMA company product; Lymphocyte separation medium is river, a Tianjin page or leaf biochemical product company limited product; The goat anti-human igg is by this chamber self-control; The goat anti-human igg of horseradish peroxidase-labeled, sheep anti-mouse igg (HRP-GAH, HRP-GAM), the goat anti-human igg of marked by fluorescein isothiocyanate (FITC-GAH) is available from Beijing Zhong Shan biotech company; The mouse-anti people CD56 (PE-MAH CD56) of phycoerythrobilin enzyme labelling is available from the U.S. biotechnology of crystalline substance company limited.
Two, method and result
1, the structure of expression vector
Select pET22b (+) as expression vector, make up trivalent bi-specific antibody two-cistron expression vector, the bicistronic mRNA structure is seen Fig. 3.According to the design of the multiple clone site in VL, CL, scFv, ribosome bind site (RBS), bacterium signal peptide sequence (pelB), VH and CH1 gene order and pET22b (+) carrier primer, see Table 1.Adopt overlapping extension PCR method to be connected between wherein VL and CL, VH and CH1, RBS and pelB are blended in 5 of VH ' end by primer RBS/PelBl, PelB2, PelB3/VH5.
Table 1 PCR primer sequence
| Primer title (restriction endonuclease) | Sequence | Primer title (restriction endonuclease) | Sequence |
| VL5(NcoI) | | ?PelB2 | Sequence 8 in the sequence |
| VL3 | Sequence | ||
| 2 in the sequence table | ?PelB3/VH5 | Sequence 9 in the sequence | |
| CL5 | Sequence | ||
| 3 in the sequence table | ? | Sequence | 10 in the sequence table |
| CL3(EcoRI) | | ?CH15 | Sequence 11 in the sequence table |
| ScFv5(EcoRI) | | ?CH13(NotI) | Sequence 12 in the sequence table |
| ScFv3(HindIII) | Sequence 6 in the sequence table | ?ScFv5(NotI) | Sequence 13 in the sequence table |
| RBS/PelB1(HindIII) | Sequence 7 in the sequence table | ?ScFv3(XhoI) | Sequence 14 in the sequence table |
The condition of pcr amplification VL, VH, CL and CH1 is 95 ℃ of 2min, circulates 25 times by following parameter: 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 extend 45sec, and last circulation is extended 10min for 72 ℃.The 5` end primer of VH of wherein increasing is the mixture of RBS/PelB1, PelB2 and PelB3/VH5, with the 5` end that RBS and pelB are blended in VH, sees Fig. 4.
The condition of pcr amplification scFv is 95 ℃ of 2min, circulates 25 times by following parameter: 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 extend 75sec, and last circulation is extended 10min for 72 ℃ and is seen Fig. 5.
The condition that overlapping extension PCR method connects VL and CL be that the VL that reclaims with the equivalent purifying and CL (about 20ng) be template and primer each other, and all the other reagent circulate 7 times by following parameter with conventional PCR: 94 ℃ of sex change 1min, 53 ℃ of 1min that anneal, 72 ℃ of extension 40min; Add VL 5` end primer (VL5) and CL 3` end primer (CL3) then, circulate 25 times by following parameter: 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 75sec, and last circulation is extended 10min for 72 ℃, sees Fig. 6.
The condition that overlapping extension PCR method connects RBS-PelB-VH and CH1 be that the VH that reclaims with the equivalent purifying and CH1 (about 20ng) are also primer each other of template, all the other reagent are with conventional PCR, circulate 7 times by following parameter: 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 45min; Add VH 5` end primer (RBS/PelB1) and CH1 3` end primer (CH13) then, circulate 25 times by following parameter: 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 80sec, and last circulation is extended 10min for 72 ℃, sees Fig. 7.
Dna fragmentation reclaims the test kit purifying and reclaims the PCR product, NcoI, EcoRI double digestion VL-CL fragment, EcoRI, HindIII double digestion scFv fragment, HindIII, NotI double digestion RBS-PelB-VH-CH1 fragment, NotI, XhoI double digestion scFv fragment, each endonuclease bamhi inserts the multiple clone site in pET22b (+) carrier successively, and recombinant vectors called after pET22b (+)/BsAb sees Fig. 8.
2, expression, the detection of recombinant protein BsAb in e. coli bl21 (DE3)
With recombinant vectors pET22b (+)/BsAb transformed into escherichia coli BL21 (DE3), picking list colony inoculation is in the LB substratum that contains the 100mg/L acillin, and 37 ℃ of concussions are cultured to OD
600Be about 0.4, add IPTG and be respectively 0.2mmol/L and 0.5mmol/L to final concentration, room temperature, 130r/min continues to cultivate 3h.
SDS-PAGE detects expression.Each sample is got 1ml, the centrifugal 15min results of 10000g bacterium thalline, and resuspended thalline is in 200 μ l PBS, multigelation three times, ultrasonic wave is broken bacterium, 4 ℃ of centrifugal 15min of 10000g, collect respectively and go up cleer and peaceful precipitation, precipitate resuspended with 150 μ l, 1 * gel loading buffer.Get supernatant (10 μ l+10 μ l sds gel sample loading buffer) and precipitation respectively and carry out 10%SDS-PAGE.The result shows that the recombinant vectors transformed bacteria has newly-increased protein belt at the about 50 kD places of molecular weight, as shown in Figure 9.
The expression that Western-blot detects BsAb is transferred to the isolating protein electricity of SDS-PAGE on the nitrocellulose filter, spends the night in 4 ℃ of sealings with the PBS that contains 5% skim-milk.(pH7.5) washing is three times for NaCl 150mmol/L, Tris-HCl 50mmol/L, and each 10min adds the HRP-GAH that TBS dilutes, and hatches 1h for 37 ℃ for TBS.TBS washing three times, each 10min, the colour developing of DAB system.The result has the colour developing of specific protein band at the about 50 kD places of molecular weight, see Figure 10.
3, the purifying of BsAb
The activation of spending the night of 37 ℃ of frozen bacterial classifications, 120r/min is got 0.5ml activation bacterium and is seeded to (acillin 100mg/L) in the 120ml LB substratum, and 37 ℃, the 250rpm concussion is cultured to OD
600Be 0.5, adding IPTG is 0.25mmol/L to final concentration, 150r/min, and 30 ℃ are continued to cultivate 4h.4 ℃, the centrifugal 10min results of 4000g thalline is with the long-pending resuspended thalline of PBS of 20% initial incubation liquid.The suspension freeze thawing once, ultrasonic wave is broken bacterium.4 ℃, the centrifugal 20min of 12000g, results supernatant.Supernatant is crossed rProteinG Sepharose Fast Flow affinity column, 100mmol/L glycine, pH2.5 elution buffer wash-out with the binding buffer liquid dilution (50mmol/L Tris-HCl, pH7.4,150mmol/L NaCl) of 10 times of volumes.Elute protein is crossed the YM-30kD ultra-filtration membrane and is replaced by the PBS damping fluid.10%SDS-PAGE detects protein purification, the results are shown in Figure 11.
Embodiment two facs analysis detect anti-Her2/neu * anti-CD16's
The activity of trivalent BsAb detects
One, material
With embodiment one.
Two, method and result
1, facs analysis BsAb is active with combining of SK-BR-3 cell
0.02%EDTA digestion SK-BR-3 cell is with cold PBA (PBS, 2%BSA, 0.1%NaN
3) centrifuge washing 2 times; Cultivate 30min with 0 ℃ of vibration of BsAb (0.1mg/L); With cold PBA centrifuge washing 2 times, cultivate 30min with 0 ℃ of vibration of FITC-GAH; Cold PBA centrifuge washing 2 times, the PBS re-suspended cell is analyzed with Becton-Dickenson FACsort instrument.The result show BsAb can with complete S K-BR-3 cell-specific combine, relatively average fluorescent strength is 92.08, is 25.4 times of negative control, sees Figure 12.
2, facs analysis BsAb is active with combining of CD16 surface antigen positive cell
Get healthy human peripheral blood, isolate mononuclearcell, with cold PBA (PBS, 2%BSA, 0.1%NaN with lymphocyte separation medium
3) centrifuge washing 2 times; Cultivate 30min with 0 ℃ of vibration of BsAb (0.1mg/L); With cold PBA centrifuge washing 2 times, cultivate 30min with 0 ℃ of vibration of PE-MAH CD56, FITC-GAH; Cold PBA centrifuge washing 2 times, the PBS re-suspended cell carries out the Two Colour Fluorescence analysis with Becton-Dickenson FACsort instrument.The result shows that BsAb can combine specifically with the NK cell subsets, sees Figure 13.
Sequence table
＜110〉Institute of Basic Medical Sciences, Academy of Military Medical Sciences, PLA
＜120〉a kind of trivalent bi-specific antibody, Preparation Method And The Use
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Claims (6)
1, a kind of trivalent bi-specific antibody is characterized in that comprising the strand variable region scFv of the variable region of heavy chain VH of human IgG1's heavy chain first constant region CH1, κ chain constant region CL structural domain, A antibody and variable region of light chain VL, B antibody; The variable region of heavy chain VH of A antibody is connected the N end of CH1, and the strand variable region scFv of B antibody is connected the C end of CH1; The variable region of light chain VL of A antibody is connected the N end of CL, and the scFv of B antibody is connected the C end of CL.
2, trivalent bi-specific antibody according to claim 1 is characterized in that the antibody of A antibody for energy recognition effect cell surface triggering molecule, and B antibody is for discerning the antibody of target cell surface marker.
3, trivalent bi-specific antibody according to claim 2, wherein effector cell surface triggering molecule is CD3, CD16 or CD64, the target cell surface marker is Her2/neu, CD20, CD30 or EGFR.
4, bi-specific antibody according to claim 3, wherein effector cell surface triggering molecule is CD16, the target cell surface marker is Her2/neu.
5, the preparation method of the described bi-specific antibody of claim 1 comprises the steps:
(1) structure of expression vector: select expression vector, the design primer, the PCR method connects gene fragment, and after enzyme was cut respectively, each endonuclease bamhi inserted the multiple clone site in the carrier, construction of expression vector successively;
(2) expression of bi-specific antibody recombinant protein, detection: with the recombinant vectors transformed into escherichia coli, abduction delivering, sampling detects the bi-specific antibody expression;
(3) purifying of bi-specific antibody: collect the thalline after expressing, collect supernatant behind the broken bacterium, carry out affinitive layer purification;
(4) combination of analysis bi-specific antibody is active.
6, the application of the described bi-specific antibody of claim 1 in the preparation anti-tumor medicine.
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