CN103880958B - Antibody L4H6 for resisting CD20 antigen and application thereof - Google Patents
Antibody L4H6 for resisting CD20 antigen and application thereof Download PDFInfo
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Abstract
The invention discloses an antibody L4H6 for resisting CD20 antigen and application thereof. The anti-CD 20 antigen provided by the invention is named as L4H6 antibody, and is IgG, wherein the light chain of the IgG consists of a light chain variable region and a light chain constant region, and the heavy chain of the IgG consists of a heavy chain variable region, a hinge region and a heavy chain constant region; the CDR1, CDR2 and CDR3 in the light chain variable region are the amino acid residues at the 61 st to 79 th positions, the amino acid residues at the 89 th to 102 th positions and the amino acid residues at the 112 th and 123 th positions from the N tail end of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the heavy chain variable region are the 57 th-67 th amino acid residue, the 81 th-96 th amino acid residue and the 106 th-119 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence. The invention also protects the application of the L4H6 antibody in preparing a medicament for treating B cell lymphoma. The invention has great application value for treating B cell lymphoma.
Description
Technical field
The present invention relates to a kind of antibody L4H6 and application thereof of anti-CD20 antigen.
Background technology
World Health Organization's hematopoiesis in 2008 and lymphoproliferative dissorders classification determine the B cell lymphoma histological subtypes more than 25 kinds with extensive biology and Clinical symptoms.ACS estimates, the U.S. in 2012 has 70130 routine B cell lymphoma new cases and diagnosed, and 18940 routine patients will die from this disease.In U.S. adults, B cell lymphoma accounts for 4% of all cancers and 3% of cancer related mortality.In Europe and North America, diffuse large B cell lymphoma is the most common type (accounting for the case of about 30%) of non-Hodgkin lymphoma (NHL), is regarded as a kind of invasive cancer needing to treat immediately.Follicular lymphoma is the second the most common histological type (accounting for the case of about 25%-30%) of non-Hodgkin lymphoma.
Traditionally, lymph atomization is all pick out the cell surface marker of expressing in lymphocyte generation specified phase by histologic analysis to identify.CD20 antigen is a kind of B cell specific differentiation antigen, expresses at mature B cell with more than 95%B cell non-Hodgkin's, but not B cell progenitor cell or mature plasme cell expression in late period in early days.CD20 antigen is made up of 297 amino-acid residues, and molecular weight is 33kD, and film compares exposure, easily close, without remarkable internalization and come off after being combined with monoclonal antibody, also can not there is antigenic modulation because of the combination with antibody, therefore become the promising target for the treatment of B cell lymphoma.
Rituximab (Rituximab) is a kind of chimeric monoclonal antibodies (incorporating human immunoglobulin G 1 heavy chain and mouse immune globulin variable zone) of identifiable design mankind CD20 antigen.The approval of in November, 1997 Rituximab acquisition food and drug administration is used for the treatment of lymphadenomatous antibody.1998, European Union also ratified Rituximab listing, and commodity are called MabThera.Rituximab has remarkable result for the treatment of in recurrent, inertia non-Hodgkin lymphoma case, has started the epoch of mab treatment cancer.
Summary of the invention
The object of this invention is to provide a kind of antibody L4H6 and application thereof of anti-CD20 antigen.
Anti-CD20 antigen provided by the invention, called after L4H6 antibody, be a kind of IgG, its light chain is made up of variable region of light chain and constant region of light chain, and its heavy chain is made up of variable region of heavy chain, hinge area and CH; CDR1, CDR2 and CDR3 in described variable region of light chain are followed successively by the sequence 1 of sequence table from N-terminal 61-79 amino acids residue, 89-102 amino acids residue and 112-123 amino acids residue; CDR1, CDR2 and CDR3 in described variable region of heavy chain are followed successively by the sequence 3 of sequence table from N-terminal 57-67 amino acids residue, 81-96 amino acids residue and 106-119 amino acids residue.
Described light chain specifically can be following (a) or (b): the protein that the sequence 1 of (a) sequence table forms from N-terminal 21-234 amino acids residue; The protein shown in sequence 1 of (b) sequence table;
Described heavy chain specifically can be following (c) or (d): the protein that the sequence 3 of (c) sequence table forms from N-terminal 20-470 amino acids residue; The protein shown in sequence 3 of (d) sequence table.
The present invention also protects the gene of described L4H6 antibody of encoding, and it is characterized in that:
The gene of described light chain of encoding is following (1) or (2) or (3):
(1) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end 80-721 position Nucleotide;
(2) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end 20-724 position Nucleotide;
(3) DNA molecular shown in sequence 2 of sequence table;
The gene of described heavy chain of encoding is following (4) or (5) or (6):
(4) sequence 5 of sequence table is from the DNA molecular shown in 5 ' end 58-1410 position Nucleotide;
(5) DNA molecular shown in sequence 5 of sequence table;
(6) DNA molecular shown in sequence 4 of sequence table.
The present invention also protects described L4H6 antibody for the preparation of the application killed and wounded in the medicine of B cell lymphoma cell.Described B cell lymphoma cell is the B cell lymphoma cell of expressing CD20 antigen.Described B cell lymphoma cell specifically can be Daudi cell.
The present invention also protects a kind of medicine for killing and wounding B cell lymphoma cell, and its activeconstituents is described L4H6 antibody.Described B cell lymphoma cell is the B cell lymphoma cell of expressing CD20 antigen.Described B cell lymphoma cell specifically can be Daudi cell.
The present invention also protects described L4H6 antibody for the preparation of the application in B cell lymphoma medicine.
The present invention has major application value for the treatment of B cell lymphoma.
Accompanying drawing explanation
Fig. 1 is the elution curve in embodiment 2.
Fig. 2 is the 10%SDS-PAGE collection of illustrative plates in embodiment 2.
Fig. 3 is the result of the ADCC in embodiment 4.
Fig. 4 is the result of the CDC in embodiment 4.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
PcDNA-3.3 carrier (linear plasmid): Invitrogen company, catalog number (Cat.No.) K8300-01.POptiVEC carrier (linear plasmid): Invitrogen company, catalog number (Cat.No.) 12744-017).CHO-DG44 cell: Invitrogen company, catalog number (Cat.No.) A13737-01.OptiPRO
tMsFM nutrient solution: Invitrogen company, catalog number (Cat.No.) 12309-050.Liposome (FreeStyle
tMmAXReagent): Invitrogen company, catalog number (Cat.No.) 16447-100.CDDG44Medium:Invitrogen company, catalog number (Cat.No.) 12610-010.CDOptiCHO
tMmedium:Invitrogen company, catalog number (Cat.No.) 12681-011.Rituximab (solution form; 100mg/10ml, colourless transparent liquid; IgG, its light chain is as shown in the sequence 6 of sequence table, and its heavy chain is as shown in the sequence 7 of sequence table): German RocheDiagnosticsGmbH, lot identification mark H0119, search sequence number: 10000661731142).
Rituximab has the mouse source protein of about 30%, and life-time service can cause humanized murine antibodies react and cannot use.Therefore, the antibody seeking more efficiently anti-CD20 antigen seems particularly urgent for the lymphadenomatous treatment of B.Contriver, based on groping in a large number, analyzing, verify, carries out microcomputer modelling and humanization modified, devises a kind of antibody (called after L4H6 antibody) of anti-CD20 antigen.L4H6 antibody is IgG, and its light chain (called after light chain L4) is as shown in the sequence 1 of sequence table, and its heavy chain (called after heavy chain H6) is as shown in the sequence 3 of sequence table.
In the sequence 1 of sequence table, from N-terminal 1-20 amino acids residue composition leading peptide, 21-131 amino acids residue composition variable region of light chain VL(wherein, 61-79 amino acids residue composition CDR1,89-102 amino acids residue composition CDR1,112-123 amino acids residue composition CDR3), 132-234 amino acids residue composition constant region of light chain CL.
In the sequence 3 of sequence table, from N-terminal 1-19 amino acids residue composition leading peptide, 20-140 amino acids residue composition variable region of heavy chain VH(wherein, 57-67 amino acids residue composition CDR1,81-96 amino acids residue composition CDR2,106-119 amino acids residue composition CDR3), 141-238 amino acids residue composition CH CH1,239-253 amino acids residue composition hinge area Hinge, 254-364 amino acids residue composition CH CH2,365-470 amino acids residue composition CH CH3.
Embodiment 1, construction recombination plasmid
1, the DNA molecular shown in the sequence 2 of sequence table is inserted pcDNA-3.3 carrier, obtain recombinant plasmid pcDNA-3.3-L4.
The DNA molecular shown in sequence 2 of sequence table is from 5 ' end 20-79 position nucleotide coding leading peptide, and 80-412 position nucleotide coding VL, 413-721 position nucleotide coding CL, 722-724 position Nucleotide is terminator codon.
2, the DNA molecular shown in the sequence 4 of sequence table is inserted pOptiVEC carrier, obtain recombinant plasmid pOptiVEC-H6.
The DNA molecular shown in sequence 4 of sequence table is from 5 ' end 14-70 position nucleotide coding leading peptide, 71-433 position nucleotide coding VH, 434-727 position nucleotide coding CH1 regional code CH1,728-1115 position Nucleotide is intron, 1116-1160 position nucleotide coding Hinge, 1161-1278 position Nucleotide is intron, 1279-1611 position nucleotide coding CH2,1612-1708 position Nucleotide is intron, 1709-2026 position nucleotide coding CH3,2027-2029 position Nucleotide is terminator codon.Namely, after removing intron, the coding region in the sequence 4 of sequence table is as shown in the sequence 5 of sequence table.
Embodiment 2, preparation L4H6 antibody
1, get 10 μ g recombinant plasmid pcDNA-3.3-L4 and 10 μ g recombinant plasmid pOptiVEC-H6, use OptiPRO
tMsFM nutrient solution is settled to 500 μ l, and room temperature places 5min.
2, get the liposome of 20 μ l, use OptiPRO
tMsFM nutrient solution is settled to 500 μ l, and room temperature places 5min.
3, mixed by the solution of the solution of step 1 and step 2, room temperature places 20 minutes.
4, inoculation CHO-DG44 cell (about 1 × 10 in the 125ml Tissue Culture Flask (20ml substratum is housed in bottle) of band vent filter
6individual cell/bottle), when to be cultured to cell density be 80-90%, the centrifugal 4min of 800rpm, inhales and abandons culture supernatant, and every bottle to add the fresh CDDG44Medium of 4ml resuspended.
5, the dropwise that step 3 obtains is added in the Tissue Culture Flask of completing steps 4, mixing, containing 8%CO
2shaking table in 37 DEG C, 150rpm shaking culture 5 days (first cultivates 6-8 hour, the then centrifugal 4min of 800rpm, inhales and abandon supernatant, rejoin the CDOptiCHO that 30ml is fresh
tMcontinue after Medium to cultivate), the centrifugal 15min of 12000rpm, collects supernatant liquor, adjusts pH to 6.0-7.0, then uses 0.45 μm of membrane filtration, collects filtrate.
6, get the filtrate that step 5 obtains, adopt rProteinASepharose4B affinity column to carry out purifying.
RProteinASepharose4B affinity column (Pharmacia Corp, XK16 pillar): column volume is 40 milliliters, and internal diameter is 16 millimeters.
Binding buffer liquid (pH7.0): containing the 20mM phosphate buffered saline buffer of 0.15MNaCl.
Elution buffer (pH3.0): 0.1M citrate buffer solution.
Flow velocity: 1-3ml/min.
Process: (1) balances pillar with 400ml binding buffer liquid; (2) loading; (3) with 400ml binding buffer liquid washing pillar; (4) use 100ml elution buffer wash-out target protein, elution curve is shown in Fig. 1, and collecting retention volume is solution after the post excessively of 30-64ml.
That 7, gets that step 6 obtains crosses solution after post, pH to 7.0 is adjusted with the Tris aqueous solution (pH9.0), then use the super filter tube (Millipore company) that molecular weight cut-off is 30kd to concentrate, the solution obtained is the solution containing L4H6 antibody, called after L4H6 solution.Fig. 2 is shown in by the 10%SDS-PAGE collection of illustrative plates of L4H6 solution.
8, replace recombinant plasmid pcDNA-3.3-L4 with pcDNA-3.3 carrier, replace recombinant plasmid pOptiVEC-H6 with pOptiVEC carrier, carry out step 1 successively to 6, in elution process, do not show any elution peak.
Embodiment 3, avidity detect
Entrust Beijing company limited of Ke Nuo Xingcheng Technology to complete, concise and to the point flow process is as follows: utilize Fortebio instrument, and by biotinylated CD20 epitope, (aminoacid sequence of CD20 epitope is: CEP
aNPSeKNSPSTQ
yCYSIq) immobilization is on Steptavidin chip, respectively flow through test antibodies solution (the L4H6 solution prepared by the PBS damping fluid dilution embodiment 1 of pH7.2,0.01M or the Rituximab of 5 different concns, obtain the test antibodies solution of different concns, protein concn in each test antibodies solution be respectively 0,25,50,75 or 100nmol/L), detect and combine (Kon) and (Koff) numerical value that dissociates, thus calculate avidity (Kd).
The avidity (Kd) of Rituximab is 6.48 × 10
-9the avidity (Kd) of M, L4H6 antibody is 3.19 × 10
-9m.
Embodiment 4, cell experiment
ADCC(antibody-dependentcell-mediatedcytotoxicity, the cell-mediated cytotoxic effect of antibody-dependant) and CDC(complementdependentcytotoxicity, the cytotoxic effect of Complement Dependent) be the vital role mode that antibody drug plays that targeting eliminates target cell.These two kinds of mechanism of action can be applicable to study in vitro in antibody drug R&D process, evaluate, screen high performance antibody drug, have very important significance.ADCC refers to that effector cell's (NK cell, scavenger cell and neutrophil leucocyte etc.) of expressing IgGFc acceptor is by being combined with the Fc section of the IgG antibody being combined in tumor cell surface, and kills and wounds the effect of these target cells.CDC is combined with surface of cell membrane corresponding antigens by specific antibody, forms mixture and activating complement classical pathway, and the membrane attack complex formed plays cracking effect to target cell.Daudi cell (human lymphoma cell system): ATCC catalog number (Cat.No.) CCL-213, through Flow cytometry (be purchased from BD Products catalog number (Cat.No.) be the FITCMouseAnti-HumanCD20 of 556632 for detecting the traget antibody of CD20 antigen), CD20 antigen presentation rate is 92%.DIO cytolemma green fluorescence probe: Sheng Ruitai scientific & technical corporation in Beijing, catalog number (Cat.No.) 60011.PI dye liquor: Sigma company, catalog number (Cat.No.) P4170-250MG.
One, the optimum amount of Rituximab is determined
Rituximab and 37 DEG C, Daudi cell are acted on 30 minutes, and (reaction system is DMEM in high glucose substratum; The concentration of Rituximab in reaction system is respectively 10 μ g/ml, 20 μ g/ml, 30 μ g/ml, 40 μ g/ml or 50 μ g/ml, in protein concentration; The concentration of Daudi cell in reaction system is 1 × 10
6/ ml), then adopt the CD20 antigen presentation on Flow cytometry Daudi cell, Rituximab concentration when expressing close to zero with it is most suitable activity.
The most suitable interaction in vitro concentration of Rituximab is 30 μ g/ml.
Two, ADCC
1, Ficoll-Hapaque is adopted to be separated peripheral blood lymphocyte (PBL).
2, with DIO cytolemma green fluorescence probe mark Daudi cell (operating by the specification sheets of DIO cytolemma green fluorescence probe), the cell of DIO mark is obtained.
3, get the cell of the DIO mark that step 2 obtains, in 37 DEG C of incubators, hatching 30min(reaction system with test antibodies is DMEM in high glucose substratum; Test antibodies is L4H6 solution or the Rituximab of embodiment 1 preparation, and the concentration of test antibodies in reaction system is 30 μ g/ml, in protein concentration; The concentration of cell in reaction system is 1 × 10
6/ ml), then collecting cell, with the PBS buffer solution 2 times of pH7.2,0.01M.
4, the cell (target cell) that step 3 obtains is got, the PBL(effector cell obtained with step 1) in 37 DEG C of incubators, hatching 4 hours, (reaction system is DMEM in high glucose substratum; The concentration of target cell in reaction system is 1 × 10
4/ ml; The concentration of effector cell in reaction system is respectively 1 × 10
6/ ml, 5 × 10
5/ ml or 2.5 × 10
5/ ml, namely target effect is than being respectively 1:100,1:50 or 1:25), then add PI dye liquor and pass through Flow cytometry.
Each process arranges three repeated sample, results averaged.
The intact cell display of DIO mark is green.Under the effect of effector cell, target cell is broken, and PI enters nucleus and dyes, and display is red.Target cell lysis rate=red cell quantity ÷ (red cell quantity+green cell quantity) × 100%.
The results are shown in Figure 3.Result shows, in target effect than equal, add the target cell lysis rate of L4H6 antibody higher than the target cell lysis rate adding Rituximab, namely the action effect of L4H6 antibody is better than Rituximab, can promote target cell lysis more significantly.
Three, CDC
1, with DIO cytolemma green fluorescence probe mark Daudi cell (operating by the specification sheets of DIO cytolemma green fluorescence probe), the cell of DIO mark is obtained.
2, get Healthy Human Serum, deactivation, obtain inactivated serum.
3, the inactivated serum get the cell of the DIO mark that step 1 obtains, obtaining with test antibodies (L4H6 solution prepared by embodiment 1 or Rituximab) and step 2 hatches 4 hours in 37 DEG C of incubators, and (reaction system is DMEM in high glucose substratum; The concentration of target cell in reaction system is 1 × 10
6/ ml; The concentration of test antibodies in reaction system is 30 μ g/ml, in protein concentration; The concentration expressed in percentage by volume of inactivated serum in reaction system is respectively 20%, 10%; Arrange and do not add the control group of inactivated serum, represent with 0% serum-concentration), then collecting cell, with the PBS buffer solution 2 times of pH7.2,0.01M.
4, the cell of the DIO mark that step 1 obtains is got, with test antibodies (L4H6 solution prepared by embodiment 1 or Rituximab) with Healthy Human Serum hatches 4 hours in 37 DEG C of incubators, and (reaction system is DMEM in high glucose substratum; The concentration of target cell in reaction system is 1 × 10
6/ ml; The concentration of test antibodies in reaction system is 30 μ g/ml, in protein concentration; The concentration expressed in percentage by volume of Healthy Human Serum in reaction system is respectively 20%, 10%; Arrange and do not add the control group of Healthy Human Serum, represent with 0% serum-concentration), then collecting cell, with the PBS buffer solution 2 times of pH7.2,0.01M.
5, get the cell that step 3 or step 4 obtain, resuspended with the PBS damping fluid of pH7.2,0.01M, obtaining cell concn is 1 × 10
5the cell suspension of/ml, then adds PI dye liquor and passes through Flow cytometry.
Each process arranges three repeated sample, results averaged.
The intact cell display of DIO mark is green.Under the effect of effector cell, target cell is broken, and PI enters nucleus and dyes, and display is red.Target cell lysis rate=red cell quantity ÷ (red cell quantity+green cell quantity) × 100%.
The results are shown in Figure 4.Result shows, when adding Healthy Human Serum, add the target cell lysis rate of L4H6 antibody higher than the target cell lysis rate adding Rituximab, namely the action effect of L4H6 antibody is better than Rituximab, can promote target cell lysis more significantly.
In sum, L4H6 antibody has good anti-Daudi cell (expressing the B cell lymphoma cell of CD20 antigen) growth, is expected to become a kind of antibody drug for clinical treatment.
Claims (7)
1. an IgG; The light chain of described IgG is following (a) or (b): the protein that the sequence 1 of (a) sequence table forms from N-terminal 21-234 amino acids residue; The protein shown in sequence 1 of (b) sequence table; The heavy chain of described IgG is following (c) or (d): the protein that the sequence 3 of (c) sequence table forms from N-terminal 20-470 amino acids residue; The protein shown in sequence 3 of (d) sequence table.
2. the gene of IgG described in coding claim 1, is characterized in that:
The gene of described light chain of encoding is following (1) or (2) or (3):
(1) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end 80-721 position Nucleotide;
(2) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end 20-724 position Nucleotide;
(3) DNA molecular shown in sequence 2 of sequence table;
The gene of described heavy chain of encoding is following (4) or (5) or (6):
(4) sequence 5 of sequence table is from the DNA molecular shown in 5 ' end 58-1410 position Nucleotide;
(5) DNA molecular shown in sequence 5 of sequence table;
(6) DNA molecular shown in sequence 4 of sequence table.
3. IgG described in claim 1 is for the preparation of the application killed and wounded in the medicine of B cell lymphoma cell; Described B cell lymphoma cell is the B cell lymphoma cell of expressing CD20 antigen.
4. apply as claimed in claim 3, it is characterized in that: described B cell lymphoma cell is Daudi cell.
5., for killing and wounding a medicine for B cell lymphoma cell, its activeconstituents is IgG described in claim 1; Described B cell lymphoma cell is the B cell lymphoma cell of expressing CD20 antigen.
6. medicine as claimed in claim 5, is characterized in that: described B cell lymphoma cell is Daudi cell.
7. IgG described in claim 1 is for the preparation of the application in B cell lymphoma medicine; Described B cell lymphoma cell is the B cell lymphoma cell of expressing CD20 antigen.
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| CN103897059B (en) * | 2014-03-27 | 2016-03-23 | 中国人民解放军军事医学科学院生物工程研究所 | The antibody L5H7 of anti-CD20 antigen and application thereof |
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| CN1662557A (en) * | 2002-02-14 | 2005-08-31 | 免疫医疗公司 | Anti-cd20 antibodies and fusion proteins thereof and methods of use |
| CN1729203A (en) * | 2002-10-17 | 2006-02-01 | 根马布股份公司 | Human monoclonal antibodies against CD20 |
| CN1747969A (en) * | 2002-12-16 | 2006-03-15 | 健泰科生物技术公司 | Immunoglobulin variants and uses thereof |
| CN1896229A (en) * | 2005-06-29 | 2007-01-17 | 北京天广实生物技术有限公司 | Production of anti-CD20 intrinsic antibody and its use |
| CN101282993A (en) * | 2005-06-02 | 2008-10-08 | 阿斯利康公司 | Antibodies directed to cd20 and uses thereof |
| CN101583626A (en) * | 2006-10-10 | 2009-11-18 | 瓦西尼斯公司 | Anti-CD20 antibodies and methods of use |
| CN102933231A (en) * | 2010-02-10 | 2013-02-13 | 伊缪诺金公司 | Cd20 antibodies and uses thereof |
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| CN1662557A (en) * | 2002-02-14 | 2005-08-31 | 免疫医疗公司 | Anti-cd20 antibodies and fusion proteins thereof and methods of use |
| CN1729203A (en) * | 2002-10-17 | 2006-02-01 | 根马布股份公司 | Human monoclonal antibodies against CD20 |
| CN1747969A (en) * | 2002-12-16 | 2006-03-15 | 健泰科生物技术公司 | Immunoglobulin variants and uses thereof |
| CN101282993A (en) * | 2005-06-02 | 2008-10-08 | 阿斯利康公司 | Antibodies directed to cd20 and uses thereof |
| CN1896229A (en) * | 2005-06-29 | 2007-01-17 | 北京天广实生物技术有限公司 | Production of anti-CD20 intrinsic antibody and its use |
| CN101583626A (en) * | 2006-10-10 | 2009-11-18 | 瓦西尼斯公司 | Anti-CD20 antibodies and methods of use |
| CN102933231A (en) * | 2010-02-10 | 2013-02-13 | 伊缪诺金公司 | Cd20 antibodies and uses thereof |
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