CN102250245A - Bispecific antibody capable of resisting B cell lymphoma and application thereof - Google Patents
Bispecific antibody capable of resisting B cell lymphoma and application thereof Download PDFInfo
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Abstract
The invention relates to the technical fields of genetic engineering and protein engineering, in particular relates to DNA (deoxyribonucleic acid) for encoding recombinant fusion protein containing human CD19 antibody variable region and human CD3 antibody variable region fragments, fusion protein encoded by DNA, a production method of the fusion protein, pharmaceutical application of the fusion protein and a treatment method using the fusion protein. The invention provides bispecific antibody protein containing human CD19scFv and CD3scFv. The bispecific antibody protein can be combined with positive CD19 and CD3 positive cells, has good bioactivities in vivo and vitro, can activate human T lymphocyte, kill B lymphoma cells, and has good application prospects.
Description
Technical field
The present invention relates to genetically engineered and protein engineering field, specifically, relate to DNA, its coded fusion rotein, the production method of this fusion rotein, the pharmaceutical use of this fusion rotein and the methods of treatment of this fusion rotein that coding comprises people source CD19 antibody variable region and the segmental recombination fusion protein of people source CD3 antibody variable region.
Background of invention
B cell lymphoma is the higher malignant tumour of a kind of sickness rate, the serious harm human health, and its antibody drug therapy is affirmed at present, existing antibody drug such as Mabthera listings such as (Rituximab).Antibody drug target spot at B cell lymphoma mainly is the CD molecule that B lymphoma cell specifically expressing increases, as CD19, CD20 and CD22 etc.Can induce B lymphoma cell apoptosis at the antibody drug of these CD molecules, thereby reach the purpose of treatment disease.
Immunotherapy also is a kind of antitumour treatments relatively more commonly used, and wherein cellular immunization has a very important role at anti-tumor aspect.T lymphocyte in the immunocyte is the important cells of killing tumor cells, and there are surface moleculars such as CD3, interleukin-2 acceptor on its surface.Studies show that CD3 antibody and interleukin-2 part can activate the T lymphocyte effectively, induce its activation and propagation, thereby improve the activity of killing tumor cell, these have been applied to the immunotherapy of tumour.Yet owing to lack the function of target tumor cell, so activated T lymphocyte is difficult to very be enriched in tumor locus effectively, causes its killing tumor cells usefulness deficiency.Therefore, can molecule such as the CD19 antibody that CD3 antibody etc. can activate lymphocytic molecule of T and target B lymphoma cell be merged, so just can be effectively enrichment and activate the T lymphocyte around the B lymphoma cell, thereby improved the effect of killing tumor cell greatly.
Natural antibody is made up of heavy chain and light chain in the human body, and heavy chain wherein and light chain variable plot structure are brought into play keying action in antigenic identification.The appearance of human antibody has in recent years overcome HAMA (Human antimousantibody) generation that original mouse source antibody therapy causes, has improved clinical therapeutic efficacy greatly.Human antibody is compared with humanized antibody relatively, the aminoacid sequence that not only has the total man, and also consistent on the space structure with people's antibody structure, and its immunogenicity is very low, therefore the humanized antibody than the transformation of mouse source more has superiority, and has great potential applicability in clinical practice.
Given this, the invention provides a kind of bi-specific antibody, it not only can target B lymphoma cell, and can activate the T lymphocyte, has improved the effect of killing and wounding the B lymphoma cell greatly.And this bi-specific antibody is total man's heterologous protein sequence, has extremely low immunogenicity, has greatly improved the using value of bi-specific antibody of the present invention.
Summary of the invention
One of the technical problem to be solved in the present invention provides the people source bi-specific antibody albumen of a kind of CD19scFv of comprising and CD3scFv, it all has good biological activity and stability in vivo and in vitro, can be in conjunction with CD19 and CD3 positive cells, and can activate the human T lymphocyte, kill and wound the B lymphoma cell.
The technical scheme of technical solution problem of the present invention provides a kind of bi-specific antibody of anti-B cell lymphoma.The bi-specific antibody of this anti-B cell lymphoma, by constituting from the variable region of anti-CD 19 antibodies and the variable region of anti-cd 3 antibodies, between link to each other by a connection peptides (Linker).
Wherein, the CD19 antibody variable region in the bi-specific antibody of above-mentioned anti-B cell lymphoma is formed by variable region of light chain (VL) and variable region of heavy chain (VH) fusion.The CD19 antibody variable region can be expressed as CD19scFv, and the variable region of light chain of CD19 antibody variable region and the arrangement mode of variable region of heavy chain can be VL-VH, also can be VH-VL.Further, be connected to form antibody variable region by connection peptides between variable region of light chain (VL) and variable region of heavy chain (VH).The aminoacid sequence of connection peptides commonly used is GGGGSGGGGSGGGGS, can be expressed as (G4S) 3.
Preferably, the aminoacid sequence of the CD19 antibody variable region in the bi-specific antibody of above-mentioned anti-B cell lymphoma is shown in SEQID NO.2.
Wherein, the CD3 antibody variable region in the bi-specific antibody of above-mentioned anti-B cell lymphoma is formed by light chain and heavy chain fusion.Be expressed as CD3scFv.
The CD3 antibody variable region can be expressed as CD3scFv, and the variable region of light chain of CD3 antibody variable region and the arrangement mode of variable region of heavy chain can be VL-VH, also can be VH-VL.Further, be connected by connection peptides between variable region of light chain (VL) and variable region of heavy chain (VH).The aminoacid sequence of connection peptides commonly used is GGGGSGGGGSGGGGS, can be expressed as (G4S) 3.
Preferably, the aminoacid sequence of the CD3 antibody variable region in the bi-specific antibody of above-mentioned anti-B cell lymphoma is shown in SEQ IDNO.4.
Wherein, the purpose of connection peptides is to provide better snappiness in the bi-specific antibody of above-mentioned anti-B cell lymphoma, CD19scFv and CD3scFv or the variable region of light chain in scFv and heavy chain light chain variable are interval to be formed structural sterically hinderedly to avoid, so connection peptides aminoacid sequence and length all can have certain variation.The aminoacid sequence of connection peptides commonly used is GGGGSGGGGSGGGGS, can be expressed as (G4S) 3.
Further, the bi-specific antibody of above-mentioned anti-B cell lymphoma:
(1) its aminoacid sequence is shown in SEQ ID NO.8;
Or: in the proteic aminoacid sequence shown in the SEQ ID No.8 through replacing and/or disappearance and/or add one or several amino acid gained and the same or analogous antibody of the function bi-specific antibody shown in the SEQ ID No.8.
The present invention also provides the nucleotide sequence of the bi-specific antibody of the above-mentioned anti-B cell lymphoma of encoding.
Further, the encode nucleotide sequence of bi-specific antibody of anti-B cell lymphoma:
(1) nucleotides sequence is classified as shown in the SEQ ID NO.7; It perhaps is the degenerate sequence of SEQ ID NO.7;
Perhaps (2): in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of one or several Nucleotide, and with (1) in the same or analogous antibody of nucleotide sequence coded function.
The present invention also provides the genophore of the nucleotide sequence of the bi-specific antibody that contains the anti-B cell lymphoma of above-mentioned coding.This carrier can be selected from plasmid or virus.
The present invention also provides the host cell that contains the said gene carrier.This host cell can be selected from eukaryotic cell or prokaryotic cell prokaryocyte.
The present invention also provides the application of bi-specific antibody in the anti-B cell lymphoma medicine of preparation of above-mentioned anti-B cell lymphoma in addition.
The present invention also provides a kind of anti-B cell lymphoma pharmaceutical composition in addition, is that the bi-specific antibody by above-mentioned anti-B cell lymphoma is an activeconstituents.Certainly, it can add pharmaceutical excipient.The formulation of described anti-B cell lymphoma pharmaceutical composition can be common formulations such as injection, powder injection, lyophilized preparation.
Further, above-mentioned anti-B cell lymphoma pharmaceutical composition also comprises at least a other anti-B cell lymphoma medicine.
Above-mentioned anti-B cell lymphoma pharmaceutical composition can also be used for treating B cell lymphoma together with the other treatment method, and described other treatment method is selected from chemotherapy, radiotherapy, gene therapy.
The present invention designs and has made up the bi-specific antibody of a kind of CD19scFv of containing and CD3scFv, makes it have enough targets and biological activity.The more important thing is that this bi-specific antibody has total man's source sequence, avoided the HAMA in the clinical treatment application to take place effectively, thereby can more effectively reduce the generation of resistance phenomenon, improved the utilising efficiency and the result of treatment of medicine.
The intravital natural antibody of people is made up of heavy chain and light chain, wherein variable region of heavy chain and light chain variable plot structure for antigenic in conjunction with particularly important.Of the present invention studies show that, the fusion rotein of being made up of variable region of heavy chain and variable region of light chain (being single-chain antibody) still has good antigen-binding activity.The only about 25kD of this single-chain antibody molecular weight, volume only are 1/6 of natural antibody, therefore have the better tissues penetrating power.The heterodimer that the natural antibody variable region is made up of variable region of heavy chain and two peptide chains of variable region of light chain, and the fusion strand that scFv is made up of variable region of heavy chain and variable region of light chain may form sterically hinderedly, are difficult to form effective antigens in conjunction with conformation.Therefore, need between variable region of heavy chain and variable region of light chain, insert a flexible peptide linker, thereby the bonding chain antibody can form correct space conception, possesses effective antigens in conjunction with activity.In addition,, also need between two scFv, to insert a flexible peptide linker for two different scFv that guarantee bi-specific antibody have separately antigen-binding activity, with this avoid may forming between two scFv sterically hindered.
For effective purifying bi-specific antibody of the present invention, the present invention has been merged His6 and Flag label protein at the N of bi-specific antibody end, because Flag label aminoacid sequence (DYKDDDDK) includes the restriction enzyme site DDDDK of enteropeptidase (EK enzyme), therefore can be after utilizing His6 and Flag label purifying protein, further utilize the EK enzyme to cut away His6 and Flag label, to obtain not contain the bi-specific antibody of label protein.
ScFv and CD3scFv that the design of bi-specific antibody of the present invention also can be used for other anti-tumour antibodies merge, formed bi-specific antibody can be brought into play the effect of target tumor cell on the one hand, can activate the T lymphocyte on the other hand, thereby bring into play the effect of its killing tumor cells target cell.
The bi-specific antibody that the present invention describes can be constructed by conventional gene recombination technology, and concrete experimental procedure is put down in writing as " molecular cloning " third edition (Joseph Sambrook, Science Press) and similar laboratory manual.Used CD19 single-chain antibody, CD3 single-chain antibody and connection peptides can be respectively:
1.CD19 the variable region of human antibody is expressed as CD19scFv, aminoacid sequence is shown in SEQ ID NO.2 in the sequence table, and coding nucleotide sequence is shown in SEQ ID NO.1.
2.CD3 the variable region of human antibody is expressed as CD3scFv, aminoacid sequence is shown in SEQ ID NO.4 in the sequence table, and coding nucleotide sequence is shown in SEQ ID NO.3.
3.CD19scFv and the used connection peptides between the CD3scFv, be expressed as (G4S) 3, aminoacid sequence is as described in the SEQ IDNO.6 in the sequence table, coding nucleotide sequence is shown in SEQ ID NO.5.
The DNA of above-mentioned bi-specific antibody can obtain by conventional gene recombination technology.The dna sequence dna of required coding CD19scFv and CD3scFv is respectively from natural people source CD19 and CD3 antibody.Be cloned into respectively in the carrier after the dna sequence dna of the above-mentioned scFv of coding obtained with PCR, used carrier can be molecular biology plasmid, virus or a dna fragmentation commonly used.Dna sequence dna front end at the above-mentioned bi-specific antibody of coding adds the protein excretion signal peptide sequence, can secrete from cell to guarantee bi-specific antibody.Comprise the promotor, protein translation initial sum termination signal and polyadenylic acid (PolyA) sequence that are used for genetic expression in the carrier sequence.Contain antibiotics resistance gene in the carrier, be beneficial to carrier duplicating and expressing in host cell such as bacterium and eukaryotic cell.In addition, also comprise the eukaryotic cell selected gene in the carrier, be used for the selection of stable transfection host cell strain.
The section that bi-specific antibody of the present invention plays a role is two independently scFv, therefore guaranteeing under the complete situation of scFv, the aminoacid sequence at scFv two ends and length can have certain variation and not weaken its biological activity, and they all belong to category of the present invention.
The arrangement mode of variable region of light chain (VL) and variable region of heavy chain (VH) among the scFv in the bi-specific antibody of the present invention, can be VL-VH, also can be VH-VL, the arrangement mode of two scFv can be CD19scFv-CD3scFv, also can be CD3scFv-CD19scFv, they all belong to category of the present invention.
The purpose of the inner connection peptides of bi-specific antibody of the present invention is to provide better snappiness, structural sterically hindered to avoid CD19scFv and CD3scFv to form, therefore connection peptides aminoacid sequence and length all can have certain variation, and they all belong to category of the present invention.
After the plasmid construction of finishing the dna sequence dna that contains the above-mentioned bi-specific antibody of encoding, promptly available this recombinant vectors transfection or transformed host cell are expressed corresponding proteins matter.Can be used in the expression system of expressing these bi-specific antibodies has multiplely, can be eukaryotic cell, also can be prokaryotic cell prokaryocyte, and they comprise mammalian cell, insect cell, yeast, bacterium etc.Because the procaryotic cell expression bi-specific antibody forms inclusion body easily, so mammalian cell is to express this proteic vote.The mammalian cell that can be used for expressing on a large scale bi-specific antibody has multiple, for example Chinese hamster ovary celI, 293 cells, NS0 cell, COS cell etc., and they all are included in the row of the cell that the present invention can use.The recombinant plasmid that contains the above-mentioned bi-specific antibody gene of encoding can enter host cell through transfection, and the method for transfectional cell has multiple, comprising electroporation, liposome transfection method and calcium phosphate transfection method etc.
A kind of preferable protein expression is the host cell expression that utilizes stable transfection.For example, do not have the host cell of neomycin resistance with the recombinant vectors stable transfection that contains Xin Meisu (Neomycin) resistant gene after, the concentration that can increase Xin Meisu in cell culture fluid is to filter out the stable cell line of high expression level; After for example lacking the host cell of DHFR with the recombinant vectors stable transfection that contains Tetrahydrofolate dehydrogenase (DHFR) gene again, the concentration that can increase methotrexate (MTX) in cell culture fluid is to filter out the stable cell line of high expression level.
Other expression systems beyond the mammalian cell, for example insect cell, yeast, bacterium etc. also can be used to express bi-specific antibody of the present invention, the row of the host cell that their also involved the present invention can use.The protein output of these expression systems is more higher than the mammalian cell, but forms inclusion body easily, therefore needs further protein renaturation.
Because bi-specific antibody of the present invention contains His and Flag label, therefore after bi-specific antibody is expressed, available enzyme linked immunosorbent adsorption test (ELISA) or additive method are measured the bi-specific antibody protein concentration in the cell culture fluid, also can come the expressed bi-specific antibody of purifying with nickel post or immune-affinity chromatography.In addition, unite use, can be further purified bi-specific antibody of the present invention with other method for purifying proteins such as ion exchange chromatography etc.
After from the recombinant chou nutrient solution, obtaining corresponding bi-specific antibody, it is active to detect its combination to CD19 and CD3 with cell ELISA and flow cytometry, experimental result shows, bi-specific antibody of the present invention can be in conjunction with CD19 positive cells such as Raji cell, also can be in conjunction with CD3 positive cells such as Jurkat cell, so the constructed bi-specific antibody of the present invention can efficient targeting bone-marrow-derived lymphocyte and T lymphocyte.
After the application of purified method obtains highly purified bi-specific antibody, can vitro detection its to the lymphocytic activation of T, can utilize also that animal model detects its restraining effect to the B cell lymphoma growth in cell in vitro model and the body.In experiment in vitro, methods such as available MTT detect the proliferation function of bi-specific antibody of the present invention to CD3 expression male periphery blood T lymphocyte, or detect it expresses male lymphoma cell such as Raji cell to D19 growth-inhibiting effect with methods such as MTT.In the experiment, above-mentioned CD19 male tumour cell can be used for making up mouse tumor model in vivo, and methods such as tumor model can be subcutaneous by the abdomen dorsal part, abdominal cavity, tail vein make up, to be used to observe the antitumor or anti-metastasis experiment of bi-specific antibody.
Dual specific of the present invention is anti-can also to deliver and express by virus vector, and these virus vector include but not limited to adenovirus carrier (adenoviral vectors), gland relevant viral vector (adeno-associated viral vectors), retrovirus vector (retroviral vectors), herpes simplex virus vector (herpes simplex virus-based vectors), lentiviral vectors (lentiviral vectors).
The present invention also provides the pharmaceutical composition that contains bi-specific antibody of the present invention and pharmaceutical carrier.This pharmaceutical composition can be prepared into various forms of pharmaceutical preparations according to the pharmaceutics routine techniques, even more preferably injection, most preferably freeze drying injection.
Pharmaceutical composition of the present invention, wherein also comprise any or several other have a synergistic antitumor drug, described composition can be treated tumour together with other methods of treatment, and described other methods of treatment comprises chemotherapy, radiotherapy, biotherapy.
Beneficial effect of the present invention is: the CD19scFv-CD3scFv bi-specific antibody that a kind of people source is provided, can be in conjunction with CD19 and CD3 positive cells, and can activate the human T lymphocyte, thereby reach the effect of outstanding anti-B cell lymphoma, have good application prospects
Description of drawings
The structural pattern figure of Fig. 1 .CD19-CD3 bi-specific antibody.
Fig. 2 .CD19scFv-CD3scFv/pcDNA3.1 (+) recombinant vectors synoptic diagram.
Fig. 3. the recombinant vectors double digestion is identified.1, recombinant vectors; 2, carrier is through the HindIII/XhoI double digestion; M, molecule Marker.
Fig. 4. immunoblotting detects Chinese hamster ovary celI secreting, expressing bi-specific antibody.1, the Chinese hamster ovary celI culture supernatant of untransfected; 2: the Chinese hamster ovary celI culture supernatant of stable transfection.
Fig. 5. affinity chromatography obtains highly purified bi-specific antibody albumen.1, before the purifying; 2, behind the purifying.
Fig. 6. the Flow cytometry bi-specific antibody to Raji in conjunction with effect.A，PBS；B，CD19scFv-CD3scFv(10ug/ml)。
Fig. 7. the Flow cytometry bi-specific antibody to Jurkat in conjunction with effect.A，PBS；B，CD19scFv-CD3scFv(10ug/ml)。
Fig. 8. bi-specific antibody of the present invention is to the lymphocytic activation of T (48h).
Fig. 9. bi-specific antibody of the present invention is to the effect of Raji cells in vitro growth-inhibiting.(the T cell is 10: 1 with Raji cell number ratio; 96h).
Figure 10. bi-specific antibody of the present invention suppresses the experimental result of the growth of transplanted tumor in the mouse body, and ordinate zou is a gross tumor volume.1：PBMC+PBS；2：PBMC+CD19scFv-CD3scFv(0.1ug)；3：PBMC+CD19scFv-CD3scFv(1ug)；4：PBS；5：CD19scFv-CD3scFv(1ug)。
Thereby Figure 11. the growth of transplanted tumor prolongs the experimental result of the survival time of tumor-bearing mice in the bi-specific antibody inhibition mouse body, and X-coordinate is the time, and ordinate zou is the mouse number.1：PBMC+PBS；2：PBMC+CD19scFv-CD3scFv(0.1ug)；3：PBMC+CD19scFv-CD3scFv(1ug)；4：PBS；5：CD19scFv-CD3scFv(1ug)。
Figure 12. bi-specific antibody of the present invention is to the influence of the tissue of mouse important organ.The result shows that its tissue morphology to the mouse important organ does not have obvious influence.
Embodiment
Following example is to structure, the test of bi-specific antibody involved in the present invention and should be used as detailed description.But content of the present invention and purposes are not limited in the category of example.
The dna sequence dna of embodiment one clones coding bi-specific antibody and structure recombinant vectors
The gene fragment of coding bi-specific antibody can obtain by classical molecular biotechnology among the present invention, and this gene order can be at the optimization of lactation expression system, so that obtain better expression amount.Bi-specific antibody gene fragment and corresponding expression vector reconnect and can obtain recombinant vectors, with expression and the screening that adapts to mammalian cell.
1, obtains the gene fragment of coding CD19-CD3 bi-specific antibody
People source CD19 antibody variable gene among the present invention and people source CD3 antibody variable gene be respectively by screening total man source antibody library gained (total man source antibody library construction process can referring to
E, et al.Recombininggermline-derived CDR sequences for creating diverse single-framework antibodylibraries.Nat Biotechnol.2000 Aug; 18 (8): known technologies such as 852-6. are realized).The structure of the bi-specific antibody that this preferred embodiment is constructed is seen accompanying drawing 1.Bi-specific antibody of the present invention is merged by CD19scFv and CD3scFv and forms, and a connection peptides that comprises continuous 3 (GGGGS) tumor-necrosis factor glycoproteinss is arranged between CD19scFv and CD3scFv.Bi-specific antibody N end added interleukin-22 (IL-2) secreting signal peptide and and His6 and Flag label, can guarantee that not only it is secreted into outside the mammalian cell, but also can utilize affinitive layer purification, and can further utilize EK enzyme excision label and retentive activity bi-specific antibody albumen only.CD19-CD3 bi-specific antibody gene fragment is by splicing pcr amplification gained by above-mentioned several fragments among the present invention.
2, express the structure of bi-specific antibody recombinant vectors
The gene clone that fusion rotein is optimized in the code book invention is to be inserted into HindIII and the XhoI restriction enzyme site gained (see figure 2) of plasmid vector pcDNA3.1 (+) (Invitrogen company) by HindIII and XhoI restriction enzyme site by the above-mentioned bispecific antibody fragment of signal peptide that comprises.This recombinant plasmid utilizes the CMV promotor to come expressed fusion protein, and comprises the expression amount of polyadenylic acid (PolyA) sequence to guarantee that its expression is best of SV40.This recombinant plasmid comprises that also penbritin (Ampicillin) resistant gene is beneficial to duplicating in bacterium, and Xin Meisu (Neomycin) resistant gene is to be used for the screening of stable transfected cells.Recombinant plasmid transformed E.coli (DH5 α) the back adding LB culture medium culturing of the described bi-specific antibody of coding is spent the night, to obtain the copy of a large amount of recombinant plasmids, extract with plasmid extraction kit (Qiagen company) and to carry out the enzyme evaluation (see figure 3) of cutting and check order behind the plasmid, the dna sequence dna of the coding bi-specific antibody that is obtained is shown in SEQ ID NO.7.
Expression and the purifying of embodiment two bi-specific antibodies of the present invention in cell
Bi-specific antibody is expressed justacrine in nutrient solution among the present invention in Chinese hamster ovary celI, and utilizes the method purifying gained of nickel post affinity chromatography.
1, the transient expression of bi-specific antibody in Chinese hamster ovary celI
After obtaining the recombinant plasmid of high purity coding bi-specific antibody, utilize Lipofectamine 2000 plasmid transfection test kits (Invitrogen company) with recombinant plasmid transfection CHO cell (ATCC), in serum free medium, cultivate and collect the Chinese hamster ovary celI supernatant liquor after three days, can detect the expression (see figure 4) of bi-specific antibody with immunoblotting.This method can be used for obtaining apace a spot of bi-specific antibody albumen, and its concentration can detect with the ELISA standard measure, and used one anti-ly can be anti-His6 or Flag antibody.
2, the stably express of bi-specific antibody in Chinese hamster ovary celI
3, the purifying of bi-specific antibody
The cell culture fluid that comprises bi-specific antibody can adopt the method for nickel post affinity chromatography to carry out the purifying (see figure 5).With the nickel chromatography column with the damping fluid balance after, with the Chinese hamster ovary celI nutrient solution supernatant liquor sample introduction that ultra-fine filter concentrated, monitor with A280 (nm), be washed till unconjugated albumen all by wash-out with scavenging solution, use elutriant elution of bound albumen then.Bi-specific antibody behind the purifying can be used ELISA method detectable level.The elutriant that comprises bi-specific antibody can freeze-drying behind desalting and purifying, can place-20 ℃ of prolonged preservation after the freeze-drying.
Embodiment three bi-specific antibodies of the present invention combine experiment with cells in vitro
Bi-specific antibody of the present invention external can be in conjunction with corresponding target cell.The present invention as the CD19 positive cells, as the CD3 positive cells, and detects its cell in conjunction with activity with the bi-specific antibody among the present invention with the Jurkat cell with the Raji cell.
1, the Flow cytometry bi-specific antibody is active with combining of Raji cell
In bi-specific antibody and the Raji cytomixis bovine serum albumin (BSA) and the PBS (dyeing damping fluid) of 0.02% sodium azide in 1%, to hatching 1 hour on ice.Cell was extremely hatched 1 hour with mouse-anti His6 antibody then on ice with PBS damping fluid washing 2 times.After cell washs with the PBS damping fluid, in the dyeing damping fluid, hatched on ice 30 minutes with the rabbit anti-mouse antibody of fluorescein isothiocyanate (FITC) mark.Cell washs 2 times with the damping fluid that flows, and (ESP Elite Coulter) analyzes, and the percentage ratio that multiply by positive colony with average logarithm fluorescence calculates average fluorescent strength with flow cytometer.The result shows that bi-specific antibody of the present invention can be in conjunction with CD19 male Raji cell (see figure 6).
2, the Flow cytometry bi-specific antibody is active with combining of Jurkat cell
This example procedure and above-mentioned example are basic identical.After bi-specific antibody and Jurkat cell are extremely hatched 1 hour on ice,, continue then extremely to hatch 1 hour on ice with mouse-anti His6 antibody with PBS damping fluid washing 2 times.After cell washs with the PBS damping fluid, in the dyeing damping fluid, hatched on ice 30 minutes with the rabbit anti-mouse antibody of fluorescein isothiocyanate (FITC) mark.Cell washs 2 times with the damping fluid that flows, and (ESP Elite Coulter) analyzes, and the percentage ratio that multiply by positive colony with average logarithm fluorescence calculates average fluorescent strength with flow cytometer.The result shows that bi-specific antibody of the present invention can be in conjunction with CD3 male Jurkat cell (see figure 7).
Embodiment four: the tumor cell in vitro growth inhibitory activity of bi-specific antibody of the present invention detects
The present invention utilizes periphery blood T lymphocyte and Raji cell to detect bi-specific antibody to the effect of Raji cells in vitro growth-inhibiting as cell model.
1, bi-specific antibody effectively activates the T lymphocyte
Extract the peripheral blood of healthy premenopausal volunteers, separate periphery blood T lymphocyte with the T lymphocyte separation medium.Inoculation T lymphocyte cell (1 * 10 in 96 porocyte culture plates
4/ hole 200ul), joins the cell cultures hole with the bi-specific antibody of different concns.Continue culturing cell after 48 hours, every hole adds the MTT tetrazolium bromide solution that 20ul concentration is 5mg/ml.After continuing to hatch 4 hours, inhale the supernatant liquor of abandoning in the cell cultures hole, every hole adds 150ul dimethyl sulfoxide (DMSO) (DMSO), and jolting 10 minutes is fully melted crystallisate.Measure the absorption value of each hole 490nm wavelength with enzyme-linked immunosorbent assay instrument, draw cell growth curve.The result shows that bi-specific antibody of the present invention can effectively activate T lymphocyte (see figure 8).
2, bi-specific antibody effectively suppresses the growth of Raji cells in vitro
Inoculation Raji cell (1 * 10 in 96 porocyte culture plates
4/ hole, 200ul), treat cell attachment after, according to T lymphocyte and 10: 1 ratio of Raji cell, periphery blood T lymphocyte is joined in the culture plate, the bi-specific antibody with different concns joins the cell cultures hole simultaneously.Continue culturing cell after 96 hours, every hole adds the MTT tetrazolium bromide solution that 20ul concentration is 5mg/ml.After continuing to hatch 4 hours, inhale the supernatant liquor of abandoning in the cell cultures hole, every hole adds 150ul dimethyl sulfoxide (DMSO) (DMSO), and jolting 10 minutes is fully melted crystallisate.Measure the absorption value of each hole 490nm wavelength with enzyme-linked immunosorbent assay instrument, draw cell growth curve.The result shows that bi-specific antibody of the present invention can effectively bring out the growth-inhibiting (see figure 9) of T lymphocyte to the Raji cell.
Embodiment five: the detection that bi-specific antibody of the present invention suppresses growth of xenografted in the mouse body
The present invention utilizes Raji cell and human peripheral blood single nucleus cell PBMCs to inoculate the SCID mouse detecting bi-specific antibody to growth-inhibiting effect in the body of Raji cell, and observes the tissue morphology of important organ.
1, bi-specific antibody can suppress the growth of transplanted tumor in the mouse body
Female non-pregnant SCID mouse in 5-6 age in week (body weight is 18-20g/) is divided into 2 groups.One group of abdomen dorsal part inoculation Raji cell (1 * 10
5/ only), another group abdomen dorsal part combined inoculation Raji cell (1 * 10
5/ only) and nonactivated human peripheral blood single nucleus cell PBMCs (1 * 10
6/ only).The Raji inoculation group is accepted vehicle (PBS) or the processing of 1 μ g bi-specific antibody (CD19/CD3BsAb) separately; Raji and PBMCs combined inoculation winding are handled by vehicle (PBS), 0.1 μ g or 1 μ g bi-specific antibody; Processing scheme is continuous tail vein injection 7 times (1 times/day, 100ul/ only).Gross tumor volume calculates and adopts following formula: (wide footpath
2* major diameter)/2.The result shows that bi-specific antibody of the present invention can effectively suppress the growth (see figure 10) of Raji transplanted tumor, and can prolong the existence (seeing Figure 11) of tumor-bearing mice.
2, bi-specific antibody does not have obvious influence to the tissue morphology of mouse important organ
Get the mouse after bi-specific antibody is treated, put to death important organs such as the back branch is centrifugal, liver, spleen, lung, kidney, 10% neutral formalin is fixed 24 hours, carries out paraffin embedding after the gradient alcohol dehydration.After adopting tissue slice and Hematorylin Yihong (HE) dyeing, the neutral gum mounting is in microscopically tissues observed form.The result shows that bi-specific antibody of the present invention does not have obvious influence (seeing Figure 12) to the tissue morphology of mouse important organ.
Above-mentioned example shows that people of the present invention source CD19-CD3 bi-specific antibody can be expressed, and can further pass through affinitive layer purification in Chinese hamster ovary celI.Resulting bi-specific antibody can be in conjunction with CD19 male Raji cell and CD3 male Jurkat cell, and can activate human peripheral T lymphocyte, thereby reaches the effect of anti-B cell lymphoma.Has good effect.
Claims (14)
1. the bi-specific antibody of anti-B cell lymphoma, by constituting from the variable region of anti-CD 19 antibodies and variable region from anti-cd 3 antibodies, between link to each other by a connection peptides.
2. the bi-specific antibody of anti-B cell lymphoma as claimed in claim 1 is characterized in that: described CD19 antibody variable region is merged by variable region of light chain and variable region of heavy chain and forms.
3. the bi-specific antibody of anti-B cell lymphoma as claimed in claim 2, it is characterized in that: the aminoacid sequence of described CD19 antibody variable region is shown in SEQ ID NO.2.
4. the bi-specific antibody of anti-B cell lymphoma as claimed in claim 1 is characterized in that: described CD3 antibody variable region is merged by variable region of light chain and variable region of heavy chain and forms.
5. the bi-specific antibody of anti-B cell lymphoma as claimed in claim 4, it is characterized in that: the aminoacid sequence of its described CD3 antibody variable region is shown in SEQ ID NO.4.
6. as the bi-specific antibody of each described anti-B cell lymphoma of claim 1～5, it is characterized in that: the aminoacid sequence of described connection peptides is GGGGSGGGGSGGGGS.
7. as the bi-specific antibody of each described anti-B cell lymphoma of claim 1～6, it is characterized in that:
(1) its aminoacid sequence is shown in SEQ ID NO.8;
Perhaps (2): in the proteic aminoacid sequence shown in the SEQ ID No.8 through replacing and/or disappearance and/or add one or several amino acid gained and the same or analogous antibody of the function bi-specific antibody shown in the SEQ ID No.8.
8. the nucleotide sequence of the bi-specific antibody of coding claim 1～6 each described anti-B cell lymphoma.
9. nucleotide sequence as claimed in claim 8 is characterized in that:
(1) nucleotides sequence is classified as shown in the SEQ ID NO.7; It perhaps is the degenerate sequence of SEQ ID NO.7;
Perhaps (2): in the nucleotide sequence that (1) limits through replacing, lack or add the derive nucleotide sequence of gained of one or several Nucleotide, and the same or analogous antibody of nucleotide sequence coded function that limits with (1).
10. the genophore that contains claim 8 or 9 each described nucleotide sequences.
11. contain the host cell of the described genophore of claim 10.
12. the application of the bi-specific antibody of each described anti-B cell lymphoma of claim 1-7 in the anti-B cell lymphoma medicine of preparation.
13. anti-B cell lymphoma pharmaceutical composition is characterized in that: the bi-specific antibody by each described anti-B cell lymphoma of claim 1-7 is an activeconstituents.
14. pharmaceutical composition as claimed in claim 13 is characterized in that, also comprises at least a other anti-B cell lymphoma medicine.
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| CN201110139081.XA CN102250245B (en) | 2010-05-27 | 2011-05-26 | Bispecific antibody capable of resisting B cell lymphoma and application thereof |
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| CN201110139081.XA CN102250245B (en) | 2010-05-27 | 2011-05-26 | Bispecific antibody capable of resisting B cell lymphoma and application thereof |
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| CN102250245B CN102250245B (en) | 2014-05-14 |
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| CN104342453A (en) * | 2013-08-06 | 2015-02-11 | 深圳先进技术研究院 | Minicircle DNA recombinant parent plasmid containing genetically engineered antibody gene expression cassette, minicircle DNA containing the expression cassette and application thereof |
| CN104558181A (en) * | 2014-12-31 | 2015-04-29 | 四川大学 | Humanized single-chain variable fragments of targeted B lymphoma cells |
| CN104592393A (en) * | 2015-01-21 | 2015-05-06 | 武汉友芝友生物制药有限公司 | Construction method and application of bispecific antibody CD19*CD3 |
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