CN101628940A - Monoclonal antibody and application thereof - Google Patents
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Abstract
The invention discloses a monoclonal antibody and application thereof. The monoclonal antibody consists of light chains and heavy chains, wherein amino acid residue sequences of the variable area of the heavy chains are showed in a sequence 1 in the sequence table, and amino acid residue sequences of the variable area of the light chains are showed in a sequence 2 in the sequence table. The invention combines and constructs anti-CTLA-4 antibodies and anti-4-1BB antibodies to form antihuman 4-1BB and CTLA-4 monoclonal antibodies. Experimental result shows that the monoclonal antibody has an antibody structure and can be respectively combined with human 4-1BB and CTLA-4.
Description
Technical field
The present invention relates to a kind of monoclonal antibody and application thereof.
Background technology
Theoretically, human body all has a large amount of cells to undergo mutation every day, but has only the cell of only a few sudden change can continue merisis, and escapes immune supervision and attack and form tumour.Tumour derives from the cell of body self sudden change, and the composition of most of mutant cell is identical with the composition of organism normal cell, has only the protein of only a few unconventionality expression and lopsided polysaccharide to have immunogenicity.Because have immunogenic difference between the tumour cell, the tumour that those immunogenicities are stronger can be induced the effective antitumour immunne response, is easily eliminated by body; And the relative more weak tumour of those immunogenicities then can be escaped immune supervision and propagation optionally, and this process is called immunoselection; A little less than the continuous selection of process, the immunogenicity of tumour more and more.
Body has multiple antineoplastic immune mechanism, comprises cellular immunization and humoral immunization, can be divided into specific immunity and non-specific immunity again.Antineoplastic immune is based on cellular immunization, wherein have the immunological memory function and specific mainly be the T cell, be subject to people's attention, but not specificity antineoplastic immunity cell-natural killer cell (NK) also is subject to people's attention day by day always.The T cell mainly contains two classes: CD4+T helper and CD8+ cytotoxic T cell (CTL).The CD4+T cell is behind the MHC antigenic compound and costimulatory molecules dual signal accepted on the full-time antigen presenting cell (APC), the increment of clone's property takes place, and discharge the various kinds of cell factor, wherein be mainly: interleukin II (IL-2), g Interferon, rabbit (INF-g), tumour necrosis factor (TNF) and lymphotoxin (LT).These cytokines play an important role in the anti-tumour effect of regulating and activate CTL, scavenger cell and B cell.CD8+CTL is activated and clones increment under the dual signal effect, CTL must directly contact with target cell could produce lethal effect.Present research is thought, CTL lethal effect mode has three kinds: 1. CTL contacts with target cell and produces degranulation, discharging pore-forming protein (perferin) is inserted on the target cell membrane, and make it form passage, and effect molecules such as granzyme (granzymes), TNF, secretion ATP enter target cell, cause its death.Wherein, pore-forming protein causes the target cell membrane damage, and granzyme makes dna break, causes apoptosis (PCD).2. CTL activates the back and expresses Fas part (FasL), and it can be released to, and born of the same parents are outer to be combined with the Fas molecule on target cell surface, and the conduction dead signal enters in the born of the same parents, and the dna degradation enzyme in the activation target cell causes the target cell apoptosis.Activate interleukin-11 b conversion enzyme (ICE) or the proteolytic enzyme relevant, cause apoptosis with ICE.3. above-mentioned dual mode coexistence.The CD8+CTL killing activity has 2/3 to come from the pore-forming protein approach approximately, and Fas/FasL inductive PCD accounts for 1/3.
The activation of T cell also must have costimulatory molecules (costimulator) to participate in except by TXi Baoshouti (TCR) and CD3 mixture antigen being discerned.Lack costimulatory signal at the t cell activation induction period, not only can not activating T cell, also can cause T cell clone specificity anergy, cause immunological tolerance.Costimulatory molecules B7 is second stimulus signal of generally acknowledging at present, the contactin member is found on the activatory B cell at first, and it also is expressed on dendritic cell and the activatory scavenger cell, except that the tumour in B cell source, other tumours are seldom expressed B7.The shortage of costimulatory molecules also is the weak immunogenic important factor of tumour.B7 has two kinds of acceptors to be present in the T cell surface, CD28 low-affinity receptor and CTLA-4 high-affinity receptor.CD28 is the immunoglobulin superfamily member, is that molecular weight is the homodimer of 44kD, and all there is the constructive expression on the two male thymocyte surfaces of the CD4+T lymphocyte 95%, 50%CD8+T lymphocyte and CD4+CD8+.CTLA-4 is the homolgous molecule of CD28, and their coexpression is in the activated T cell surface.Though the expression amount of CTLA-4 is lower than CD28, the avidity of it and B7 is 20 times of CD28.The B7:CD28 path provides key signal for the generation of IL-2.B7 combines with CD28 on the T cell and produces positive signal, and enhancing immunity is replied; B7 combines with CTLA-4 and then produces negative signal, has sealed the t cell activation that CD28 relies on, the downward modulation immune response.
4-1BB/4-1BBL stimulation approach altogether found in recent years that 4-1BB (CDw137) was the tumor necrosis factor receptor super family member, mainly is expressed on the T cell.Its high-affinity part 4-1BBL is expressed on scavenger cell and the activatory B cell.But the activation and the propagation of 4-1BBL or anti-4-1BB antibody and inducing T cell after the 4-1BB of T cell combines.4-1BB can work in coordination with the common hormesis of CD28 to the T cell, also can not rely on CD28 and brings into play common stimulatory effect.
Because the keying action of costimulatory molecules in the T cell activation utilize antibody to stimulate these molecules, thereby it is not reactive to the immunity of tumour cell to break immunocyte, becomes an important directions of developing anti-tumor medicaments.CTLA-4 antibody tremelimumab and ipilimumab receive the concern of industry in the medicine that grinds.Tremelimumab is developed by Pfizer, and ipilimumab is by Bristol Myers Squibb and the exploitation of Medarex company.Tremelimumab and ipilimumab have been in the final stage of clinical trial at present, and hundreds of melanoma patients have added this test.
Stimulate path altogether according to 4-1BB/4-1BBL, but use anti-4-1BB antibody direct activation T cell in the mouse body, eradicate the big tumour that some has been planted.Mouse experiment shows: use anti-4-1BB monoclonal antibody and can eradicate the Ag104A sarcoma of immunogenicity difference becomes knurl with height P815 mastocytoma.
Major side effects by immune cell activated treatment tumour is the immune response of self, and in the clinical experiment of anti-CTLA-4 antibody, stronger autoimmune disease appears in tumour patient.But anti-4-1BB antibody not only has the effect that activates the CD8+T cell, suppresses the B cell function in addition, thereby shows the effect that suppresses autoimmune disorder.
Summary of the invention
The purpose of this invention is to provide a kind of monoclonal antibody.
Monoclonal antibody name provided by the present invention is called anti-4-1BB-CTLA4-IgG2a, contains heavy chain and light chain; The amino acid residue sequence of the variable region of described heavy chain is shown in sequence in the sequence table 1, and the amino acid residue sequence of the variable region of described light chain is shown in sequence in the sequence table 2.
Concrete, in the said monoclonal antibody, the constant region of described heavy chain is the constant region of isotype (isotype) mouse IgG2a, the constant region of described light chain is the constant region of the Kappa of mouse.
The amino acid residue sequence of the heavy chain of described monoclonal antibody anti-4-1BB-CTLA4-IgG2a is shown in sequence in the sequence table 3, and the amino acid residue sequence of the light chain of described monoclonal antibody anti-4-1BB-CTLA4-IgG2a is shown in sequence in the sequence table 4.
The encoding gene of said monoclonal antibody also belongs to protection scope of the present invention.
The Fab fragment that obtains with the papain enzymolysis said monoclonal antibody also belongs to protection scope of the present invention.
The segmental encoding gene of above-mentioned Fab also belongs to protection scope of the present invention.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said monoclonal antibody or the segmental encoding gene of Fab also belong to protection scope of the present invention.
Another object of the present invention provides a kind of by said monoclonal antibody deutero-humanized antibody.
Described humanized antibody is made up of light chain and heavy chain, and the amino acid residue sequence of described variable region of heavy chain is shown in sequence in the sequence table 1, and the amino acid residue sequence of described variable region of light chain is shown in sequence in the sequence table 2.
The present invention will resist CTLA-4 antibody and the coupling of anti-4-1BB antibody to be built into the monoclonal antibody of anti-people 4-1BB and CTLA-4, and experimental result shows that said monoclonal antibody has antibody structure, can combine with people 4-1BB and CTLA-4 specificity respectively.
Description of drawings
Fig. 1 detects the binding curve of monoclonal antibody anti-4-1BB-CTLA4-IgG2a and people CTLA-4 for ELISA
Fig. 2 detects monoclonal antibody anti-4-1BB-CTLA4-IgG2a and the binding curve that is illustrated in the people 4-1BB extracellular fragment of yeast surface for flow cytometer
Embodiment
Following experimental technique if no special instructions, is ordinary method.
The preparation of embodiment 1, monoclonal antibody anti-4-1BB-CTLA4-IgG2a
1, the acquisition of monoclonal antibody anti-4-1BB-CTLA4-IgG2a heavy chain and light chain
The encoding gene of the variable region of heavy chain of difference anti-CTLA-4 antibody of synthetic and anti-4-1BB antibody.Adopt the method for overlapping extension PCR to be connected the anti-CTLA-4 antibody that obtains and the encoding gene of the variable region of heavy chain of anti-4-1BB antibody, obtain the dna sequence dna of monoclonal antibody variable region of heavy chain, the amino acid residue sequence of the dna sequence encoding of this monoclonal antibody variable region of heavy chain is shown in sequence 1.
Encoding gene with the monoclonal antibody variable region of heavy chain of above-mentioned acquisition is connected with the method for overlapping extension PCR respectively with isotype mouse IgG2a constant region encoding gene again, obtain the dna sequence dna of monoclonal antibody heavy chain, the amino acid residue sequence of the dna sequence encoding of this monoclonal antibody heavy chain is shown in sequence 3.
The encoding gene of the variable region of light chain of difference anti-CTLA-4 antibody of synthetic and anti-4-1BB antibody.The method of the overlapping extension PCR of encoding gene of the anti-CTLA-4 antibody that obtains and the variable region of light chain of anti-4-1BB antibody is connected, obtain the dna sequence dna of monoclonal antibody variable region of light chain, the amino acid residue sequence of the dna sequence encoding of this monoclonal antibody variable region of light chain is shown in sequence 2.
Encoding gene with the monoclonal antibody variable region of light chain of above-mentioned acquisition is connected with the method for overlapping extension PCR respectively with isotype mouse Kappa constant region encoding gene again, obtain the dna sequence dna of monoclonal antibody light chain, the amino acid residue sequence of the dna sequence encoding of this monoclonal antibody light chain is shown in sequence 4.
2, the structure of monoclonal antibody anti-4-1BB-CTLA4-IgG2a expression vector
The encoding gene of the monoclonal antibody heavy chain that above-mentioned steps 1 is obtained is inserted between the NotI and SalI restriction enzyme site of expression vector pCI after NotI and SalI enzyme are cut, and obtains the recombinant expression vector pCI-DHFR-4-1BB-CTLA4-H of monoclonal antibody anti-4-1BB-CTLA4-IgG2a heavy chain.
Between the SalI and XbaI enzyme cutting site of encoding gene through being inserted into expression vector pCI behind SalI and the XbaI enzyme cutting of the monoclonal antibody light chain that above-mentioned steps 1 is obtained, obtain the recombinant expression vector pPCI-4-1BB-CTLA4-L of monoclonal antibody anti-4-1BB-CTLA4-IgG2a light chain.
3, the preparation of monoclonal antibody anti-4-1BB-CTLA4-IgG2a
The monoclonal antibody anti-4-1BB-CTLA4-IgG2a heavy chain of above-mentioned steps 2 acquisitions and the recombinant expression vector pCI-4-1BB-CTLA4-H and the pCI-4-1BB-CTLA4-L cotransfection of light chain are entered in the Mammals COS-7 cell, and expression, purifying obtain monoclonal antibody anti-4-1BB-CTLA4-IgG2a.
The binding ability of embodiment 2, monoclonal antibody anti-4-1BB-CTLA4-IgG2a is identified
1, the binding ability of monoclonal antibody anti-4-1BB-CTLA4-IgG2a and CTLA-4 is identified
With the CTLA-4-Fc monoclonal antibody (available from R﹠amp; D company, article No.: 325-CT) wrapper sheet, every hole bag is by 1ug/ml.The monoclonal antibody anti-4-1BB-CTLA4-IgG2a gradient dilution that embodiment 1 makes up is hatched, the two anti-sheep anti-mouse iggs of horseradish peroxidase-labeled of using are (available from JacksonImmunoResearch, article No.: 115-035-062), with OPD is substrate, as terminator, ELISA detects the binding ability of monoclonal antibody anti-4-1BB-CTLA4-IgG2a and CTLA-4 with 2mmol/L sulfuric acid.The result as shown in Figure 1, among Fig. 1, ordinate zou is the absorbance value after the ELISA colour developing.The result shows that monoclonal antibody anti-4-1BB-CTLA4-IgG2a can combine with CTLA-4.
2, the binding ability of monoclonal antibody anti-4-1BB-CTLA4-IgG2a and 4-1BB is identified
Express 4-1BB molecule extracellular fragment in yeast cell surface, (mRNA with the human peripheral blood cell is a template with 4-1BB molecule extracellular fragment, through oligo (dT) reverse transcription, with 5 '-TTTGAGAGGACAAGATCATTG-3 ' is 5 ' primer, with 5 '-GGAGATGATCTGCGGAGAGTG-3 is 3 ' primer, carry out pcr amplification) cut the back and pass through pYD1 plasmid (available from Invitrogen company) that same enzyme cuts and be connected and obtain recombinant plasmid with EcoR1 and NotI enzyme, with this recombinant plasmid transformed yeast saccharomyces cerevisiae EBY100.Choose mono-clonal from the SD flat board and be inoculated into (final concentration of glucose is 2% in the substratum) the 2ml SD substratum, when 30 ℃ of shaking tables are cultivated OD value to 2.0, get 1ml bacterium liquid, 5000 rev/mins centrifugal 5 minutes, abandon supernatant.With 1ml SG substratum (final concentration of semi-lactosi is 2% in the substratum) suspension thalline, 5000 rev/mins centrifugal 5 minutes, abandon supernatant, use 2ml SG substratum suspension thalline then, change test tube over to, 20 ℃ of shaking tables were induced 36～48 hours, confirm the expression of yeast surface 4-1BB molecule extracellular fragment with anti-V5 antibody, FACS detects the binding ability of monoclonal antibody anti-4-1BB-CTLA4-IgG2a and 4-1BB molecule, the result as shown in Figure 2, among Fig. 2, ordinate zou is the per-cent that the antibody labeling positive cells accounts for total cell.The result shows that monoclonal antibody anti-4-1BB-CTLA4-IgG2a can combine with 4-1BB, and the binding ability of binding ability and anti-4-1BB antibody and 4-1BB is suitable.
The segmental preparation of the Fab of embodiment 3, monoclonal antibody anti-4-1BB-CTLA4-IgG2a
Utilize
Fab prepares the monoclonal antibody anti-4-1BB-CTLA4-IgG2a of the Mammals COS-7 emiocytosis of the immobilized papain digestion transient transfection in the test kit (Pierce), and full length monoclonal antibodies is degraded to Fab and Fc fragment.Product behind the enzymolysis is obtained the Fab fragment with the immobilization albumin A column purification that provides in the test kit.Carrying out ELISA according to the method for embodiment 2 identifies.The result shows that the Fab fragment is suitable with the full length antibody binding ability.
Sequence table
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1 5 10 15
Val?Gln?Cys?Lys?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Gly?Leu?Val?Lys
20 25 30
Pro?Gly?Ala?Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe
35 40 45
Thr?Asp?Tyr?Ile?Ile?Gln?Trp?Ile?Lys?Gln?Arg?Ser?Gly?Gln?Gly?Leu
50 55 60
Glu?Trp?Ile?Gly?Trp?Phe?Tyr?Pro?Gly?Ser?Gly?Gly?Ile?Asn?Tyr?Asn
65 70 75 80
Glu?Lys?Phe?Lys?Asn?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Lys?Ser?Ser?Ser
85 90 95
Thr?Val?Tyr?Leu?Asp?Leu?Ser?Lys?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100 105 110
Tyr?Phe?Cys?Val?Arg?His?Glu?Gly?Ser?Tyr?Gly?Tyr?Phe?Asp?Tyr?Trp
115 120 125
Gly?Gln?Gly?Thr?Thr?Leu?Thr?Val?Ser?Ser?Ala?Lys?Thr?Thr?Pro?Pro
130 135 140
Gln?Val?Gln?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Leu?Val?Ala?Pro?Ser?Gln
145 150 155 160
Ser?Leu?Ser?Ile?Thr?Cys?Thr?Val?Ser?Gly?Phe?Ser?Leu?Thr?Ser?Tyr
165 170 175
Gly?Leu?Ser?Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Leu
180 185 190
Gly?Val?Ile?Trp?Tyr?Asp?Gly?Asn?Thr?Asn?Phe?His?Ser?Ala?Leu?Ile
195 200 205
Ser?Arg?Leu?Thr?Ile?Ser?Lys?Asp?Asn?Ser?Lys?Ser?Gln?Val?Phe?Leu
210 215 220
Glu?Leu?Asn?Ser?Leu?Gln?Thr?Asp?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys?Ala
225 230 235 240
Lys?Thr?Glu?Gly?His?Tyr?Tyr?Gly?Ser?Asn?Tyr?Gly?Tyr?Tyr?Ala?Leu
245 250 255
Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Ser?Val?Thr?Val?Ser?Ser
260 265
<210>2
<211>249
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Met?Asp?Met?Arg?Val?Pro?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Leu?Leu?Trp
1 5 10 15
Phe?Pro?Gly?Ser?Arg?Cys?Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ser
20 25 30
Leu?Ala?Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Ser?Cys?Lys?Ala?Ser
35 40 45
Gln?Ser?Val?Asp?Tyr?Asp?Gly?Tyr?Ser?Tyr?Met?Asn?Trp?Tyr?Gln?Gln
50 55 60
Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?SerIle?Leu
65 70 75 80
Asp?Ser?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp
85 90 95
Phe?Thr?Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Glu?Asp?Val?Ala?Thr?Tyr
100 105 110
Tyr?Cys?Gln?Gln?Ser?Asn?Glu?Asp?Pro?Phe?Thr?Phe?Gly?Ser?Gly?Thr
115 120 125
Lys?Leu?Glu?Ile?Lys?Arg?Ala?Asp?Ala?Ala?Pro?Thr?Val?Asp?Ile?Gln
130 135 140
Met?Thr?Gln?Ser?Pro?Ala?Ser?Leu?Ser?Val?Ser?Val?Gly?Glu?Thr?Val
145 150 155 160
Thr?Ile?Thr?Cys?Arg?Ala?Ser?Glu?Asn?Ile?Tyr?Ser?Asn?Leu?Ala?Trp
165 170 175
Tyr?Gln?Gln?Lys?Gln?Gly?Lys?Ser?Pro?Gln?Leu?Leu?Val?Tyr?Ala?Ala
180 185 190
Thr?Asn?Leu?Ala?Asp?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser
195 200 205
Gly?Thr?Gln?Tyr?Ser?Leu?Lys?Ile?Asn?Ser?Leu?Gln?Ser?Glu?Asp?Phe
210 215 220
Gly?Thr?Tyr?Phe?Cys?Gln?His?Leu?Trp?Gly?Thr?Pro?Tyr?Thr?Phe?Gly
225 230 235 240
Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
245
<210>3
<211>599
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Met?Glu?Phe?Gly?Leu?Ser?Trp?Leu?Phe?Leu?Val?Ala?Ile?Leu?Lys?Gly
1 5 10 15
Val?Gln?Cys?Lys?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Gly?Leu?Val?Lys
20 25 30
Pro?Gly?Ala?Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe
35 40 45
Thr?Asp?Tyr?Ile?Ile?Gln?Trp?Ile?Lys?Gln?Arg?Ser?Gly?Gln?Gly?Leu
50 55 60
Glu?Trp?Ile?Gly?Trp?Phe?Tyr?Pro?Gly?Ser?Gly?Gly?Ile?Asn?Tyr?Asn
65 70 75 80
Glu?Lys?Phe?Lys?Asn?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Lys?Ser?Ser?Ser
85 90 95
Thr?Val?Tyr?Leu?Asp?Leu?Ser?Lys?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100 105 110
Tyr?Phe?Cys?Val?Arg?His?Glu?Gly?Ser?Tyr?Gly?Tyr?Phe?Asp?Tyr?Trp
115 120 125
Gly?Gln?Gly?Thr?Thr?Leu?Thr?Val?Ser?Ser?Ala?Lys?Thr?Thr?Pro?Pro
130 135 140
Gln?Val?Gln?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Leu?Val?Ala?Pro?Ser?Gln
145 150 155 160
Ser?Leu?Ser?Ile?Thr?Cys?Thr?Val?Ser?Gly?Phe?Ser?Leu?Thr?Ser?Tyr
165 170 175
Gly?Leu?Ser?Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Leu
180 185 190
Gly?Val?Ile?Trp?Tyr?Asp?Gly?Asn?Thr?Asn?Phe?His?Ser?Ala?Leu?Ile
195 200 205
Ser?Arg?Leu?Thr?Ile?Ser?Lys?Asp?Asn?Ser?Lys?Ser?Gln?Val?Phe?Leu
210 215 220
Glu?Leu?Asn?Ser?Leu?Gln?Thr?Asp?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys?Ala
225 230 235 240
Lys?Thr?Glu?Gly?His?Tyr?Tyr?Gly?Ser?Asn?Tyr?Gly?Tyr?Tyr?Ala?Leu
245 250 255
Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Ser?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr
260 265 270
Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser
275 280 285
Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu
290 295 300
Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His
305 310 315 320
Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser
325 330 335
Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys
340 345 350
Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu
355 360 365
Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro
370 375 380
Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys
385 390 395 400
Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val
405 410 415
Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp
420 425 430
Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr
435 440 445
Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp
450 455 460
Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu
465 470 475 480
Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg
485 490 495
Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys
500 505 510
Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp
515 520 525
Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys
530 535 540
Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser
545 550 555 560
Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser
565 570 575
Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser
580 585 590
Leu?Ser?Leu?Ser?Pro?Gly?Lys
595
<210>4
<211>355
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>4
Met?Asp?Met?Arg?Val?Pro?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Leu?Leu?Trp
1 5 10 15
Phe?Pro?Gly?Ser?Arg?Cys?Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ser
20 25 30
Leu?Ala?Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Ser?Cys?Lys?Ala?Ser
35 40 45
Gln?Ser?Val?Asp?Tyr?Asp?Gly?Tyr?Ser?Tyr?Met?Asn?Trp?Tyr?Gln?Gln
50 55 60
Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Ile?Leu
65 70 75 80
Asp?Ser?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp
85 90 95
Phe?Thr?Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Glu?Asp?Val?Ala?Thr?Tyr
100 105 110
Tyr?Cys?Gln?Gln?Ser?Asn?Glu?Asp?Pro?Phe?Thr?Phe?Gly?Ser?Gly?Thr
115 120 125
Lys?Leu?Glu?Ile?Lys?Arg?Ala?Asp?Ala?Ala?Pro?Thr?Val?Asp?Ile?Gln
130 135 140
Met?Thr?Gln?Ser?Pro?Ala?Ser?Leu?Ser?Val?Ser?Val?Gly?Glu?Thr?Val
145 150 155 160
Thr?Ile?Thr?Cys?Arg?Ala?Ser?Glu?Asn?Ile?Tyr?Ser?Asn?Leu?Ala?Trp
165 170 175
Tyr?Gln?Gln?Lys?Gln?Gly?Lys?Ser?Pro?Gln?Leu?Leu?Val?Tyr?Ala?Ala
180 185 190
Thr?Asn?Leu?Ala?Asp?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser
195 200 205
Gly?Thr?Gln?Tyr?Ser?Leu?Lys?Ile?Asn?Ser?Leu?Gln?Ser?Glu?Asp?Phe
210 215 220
Gly?Thr?Tyr?Phe?Cys?Gln?His?Leu?Trp?Gly?Thr?Pro?Tyr?Thr?Phe?Gly
225 230 235 240
Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val
245 250 255
Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser
260 265 270
Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln
275 280 285
Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val
290 295 300
Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu
305 310 315 320
Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu
325 330 335
Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg
340 345 350
Gly?Glu?Cys
355
Claims (7)
1, a kind of monoclonal antibody is made up of light chain and heavy chain, it is characterized in that: the amino acid residue sequence of the variable region of described heavy chain is shown in sequence in the sequence table 1, and the amino acid residue sequence of the variable region of described light chain is shown in sequence in the sequence table 2.
2, monoclonal antibody according to claim 1 is characterized in that: the amino acid residue sequence of described monoclonal antibody heavy chain is shown in sequence in the sequence table 3, and the amino acid residue sequence of described monoclonal antibody light chain is shown in sequence in the sequence table 4.
3, claim 1 or 2 described monoclonal antibody encoding genes.
4, the Fab fragment that obtains with papain enzymolysis claim 1 or 2 described monoclonal antibodies.
5, the segmental encoding gene of the described Fab of claim 4.
6, the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 3 or 5 described encoding genes.
7, by claim 1 or 2 described monoclonal antibody deutero-humanized antibodies, form by light chain and heavy chain, it is characterized in that: the amino acid residue sequence of described variable region of heavy chain is shown in sequence in the sequence table 1, and the amino acid residue sequence of described variable region of light chain is shown in sequence in the sequence table 2.
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| CN2009101575927A CN101628940B (en) | 2008-07-15 | 2009-07-15 | Monoclonal antibody and application thereof |
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| CN200810116696 | 2008-07-15 | ||
| CN200810116696.9 | 2008-07-15 | ||
| CN2009101575927A CN101628940B (en) | 2008-07-15 | 2009-07-15 | Monoclonal antibody and application thereof |
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| CN101628940B CN101628940B (en) | 2011-11-23 |
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| CN101628940B (en) | 2011-11-23 |
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