CN1292418A - Heavy chain and light chain variable region gene of anti-C D 20 monoclonal antibody and its application - Google Patents
Heavy chain and light chain variable region gene of anti-C D 20 monoclonal antibody and its application Download PDFInfo
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- CN1292418A CN1292418A CN991217284A CN99121728A CN1292418A CN 1292418 A CN1292418 A CN 1292418A CN 991217284 A CN991217284 A CN 991217284A CN 99121728 A CN99121728 A CN 99121728A CN 1292418 A CN1292418 A CN 1292418A
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Abstract
The present invention discloses anti-CD20 monoclonal antibody HI47 heavy chain and light chain variable region gene, polypeptide coded by the described gene, carrier containing the described gene and application of the described gene and polypeptide in the preparation of medicine for curing and diagnosing B lymphocytoma.
Description
The present invention relates to anti-CD
20Monoclonal antibody heavy chain and chain variable region gene by the polypeptide of described genes encoding, contain the carrier of described gene and described gene and polypeptide are used for the medicine of the treatment of bone-marrow-derived lymphocyte knurl and diagnosis in preparation application.Particularly, heavy chain of the present invention and chain variable region gene are from anti-CD
20Monoclonal antibody HI
47
The bone-marrow-derived lymphocyte knurl is a kind of common disease in the blood system, and its sickness rate is about 2/10000, and by this estimation, China has 20,000 multiple-cases every year approximately.Methods of treatment to the bone-marrow-derived lymphocyte knurl mainly contains chemotherapy at present, three kinds of therapies of radiotherapy and biological assisting therapy, biotherapy as adjuvant therapy be mainly used in that chemotherapy substitutes, the killing and wounding and the treatment of early stage potential metastasis of combined utilization treatment, minimal residue tumour cell, improve patient's more back quality.CD
20Be the bone-marrow-derived lymphocyte surface membrane protein, CD
20With Ca
2+From closely related in stride film transportation at bone-marrow-derived lymphocyte, CD
20To the differentiation of bone-marrow-derived lymphocyte with bred regulating effect (BubienJK, Zhou LJ, Bell PD, et a1 J.Cell Biol.1993,121 (5): 1121-1132; Golay JT, Clark EA, Beverley PC, et al J Immuno1.1985; 135:3795).CD
20As treatment bone-marrow-derived lymphocyte knurl target spot following advantage is arranged: at first, CD
20Only at pre-B lymphocyte, immature B lymphocyte, ripe bone-marrow-derived lymphocyte, the activation bone-marrow-derived lymphocyte is expressed, and at its hetero-organization and multipotency B lymphoid stem cell, plasmocyte is not expressed (Stashenko P Nadler LM, Hardv R, et al.J.Immun, 1980; 125:1678-1685; Nadler LM, Karsmeyer SJ, Anderson KC, et al Clin.Invest.1984; 74:332-340; Stashenko P Nadler LM, Hardy R, et al.Proc.Natl.Acad.Sci.USA 1981; 78:3848-3852; Rosenthal P, Rimm IJ, Umiel T, et al.J.Immunol.1983; 131:232-237).Secondly, CD
20With anti-CD
20Endocytosis does not take place after the antibodies, thereby cell surface CD
20Quantity does not reduce (Liu AY, Robinson RJr, Murvay ED, et al.J.Immunol.1987 because of the combination of antibody; 139:3521).The 3rd, CD
20Relatively expose, do not covered by other surface molecular, thereby easily near (Einleld DA, Brown JP, Vakebtube MA, et al.EMBOJ.1988; 7:711).The 4th, no free CD in human serum
20There are (EinleldDA, Brown JP, Valentine MA, et al.EMBOJ.1988; 7:711).
Anti-CD
20Studies on Monoclonal Antibody has the history of more than ten years, as far back as the anti-CD eighties
20Antibody I F5, B1 have been used for clinical experiment, but produce little effect, the progress of making a breakthrough property just until 1997.Be the anti-CD of year drugs approved by FDA
20Chimeric antibody Rituxan is applied to the clinical treatment of bone-marrow-derived lymphocyte knurl, evident in efficacy, little (the Magic bulletsat last:Finally-approval of a monoclonal antibody for the treatment ofcancer.Cancer Biotherapy ﹠amp of toxic side effect; Radio pharmaceuticals 1997; 14 (4): 223-225).Different anti-CD
20Antibody and CD
20In conjunction with after the effect difference that causes, the made bone-marrow-derived lymphocyte that has is from G
0Phase enters G
1Phase, cause the activation of bone-marrow-derived lymphocyte; The activation of the inhibition bone-marrow-derived lymphocyte that has.Anti-CD
20Antibody mainly is to kill and wound the bone-marrow-derived lymphocyte knurl by three kinds of mechanism such as combination of antibody-dependent cytotoxicity effect (ADDC), complement-dependent cytotoxicity (CDDC) and antibody and bone-marrow-derived lymphocyte.
HI
47(IgG
3) be that Inst. of Hematology, Chinese Academy of Medical Sciences is in the 1990 anti-CD that succeed in developing
20Mouse monoclonal antibody, the 4th the human leukocyte differentiation antigen meeting in the world is with HI
47Called after CD
20+ X, HI
47Tissue reactive slightly different (Yang Xifeng, Shen Decheng, golden space light etc., HI with IF5, B1
47: a kind of anti-mature B cell Monoclonal Antibody, evaluation and biological characteristic research thereof; The Shanghai Journal of Immunology, 1990:10 (2): 65-68).HI
47Can suppress the synthetic of SAC inductive bone-marrow-derived lymphocyte DNA, RNA, and Anti-IgM and PMA are caused that the propagation of B cell activation and clone do not have obvious effect, HI
47SAC activated B cell HI antigen presentation is obviously reduced, but PMA activating cells HI antigen presentation is not obviously influenced (Yang Xifeng, Shen Decheng, old barrier etc., HI
47(CD
20) monoclonal antibody is to the influence of B cell activation propagation; China's Journal of Immunology, 1991:7 (1): 21-24).This and opposite (the Tedder TF et al.Eur J Immunol.1986 of report such as Tedder; 16:881), these results indicate HI
47Action site different with the action site of B1.HI
47The inhibition activity be the specific effect of antibody.
Human body is the major obstacle that the mouse endogenous antibody is applied to clinical treatment to the immune response (HAMA reaction) of mouse endogenous antibody, and ADDC, CDDC that the mouse endogenous antibody can not Mediated Human reaction.The humanization of antibody and the structure of chimeric antibody can reduce the immunogenicity of mouse endogenous antibody greatly.
Therefore, one object of the present invention is to provide a kind of anti-CD
20Monoclonal antibody chain variable region gene and heavy chain variable region gene and expression product thereof are expressed the inosculating antibody CD that produces after both recombinate
20Antibody and Fab fragment thereof can reduce the anti-CD of mouse source property
20The immunogenicity of antibody.
Another object of the present invention is anti-CD
20Chimeric antibody and Fab fragment thereof are used for the application of the medicine of the treatment of bone-marrow-derived lymphocyte knurl and diagnosis in preparation.
According to an aspect of the present invention, the present invention relates to a kind of anti-CD
20Monoclonal antibody chain variable region gene and heavy chain variable region gene, wherein, described heavy chain variable region gene total length is 366bp, and its nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence coded is shown in SEQ ID NO:3.Described chain variable region gene total length is 318bp, and its nucleotide sequence is shown in SEQ ID NO:2, and its amino acid sequence coded is shown in SEQ IDNO:4.
We adopt the phage antibody display technique, successfully are cloned into anti-CD
20Monoclonal antibody HI
47The weight chain variable region gene, and the anti-CD that will obtain
20Monoclonal antibody weight chain variable region gene is cloned in the Fab expression vector, makes up inosculating antibody CD
20The Fab fragment of antibody has reduced the anti-CD of mouse source property
20The immunogenicity of antibody.And can in intestinal bacteria, carry out efficient secretion expression, secretion expression's antibody capable improves anti-CD
20The output of genetic engineering antibody reduces production costs, and is beneficial to suitability for industrialized production.The Fab fragment is compared with whole antibody and had the following advantages: at first, Fab is little than the whole antibody molecular weight, and penetration power is more intense, and secondly, Fab can be beneficial to suitability for industrialized production at expression in escherichia coli, and production cost is low.
Following with reference to the present invention of drawings and Examples detailed description.Wherein
Fig. 1 illustrates anti-CD
20The segmental structure of chimeric antibody Fab.
Fig. 2 illustrates anti-CD
20The SDS-PAGE analytical results of chimeric antibody Fab fragment separation and purification product, wherein swimming lane 1 is the preceding product of purifying under the non-reduced condition, swimming lane 2 is the product behind the purifying under the non-reduced condition, swimming lane 3 is the product before the purifying under the reductive condition, the product of swimming lane 4 after for the purifying under the reductive condition.
Fig. 3 illustrates anti-CD
20Chimeric antibody Fab fragment and HI
47Monoclonal antibody competitive immunization fluorescence suppresses experiment FACS measurement result.
Use the RT-PCR method from anti-CD
20Antibody hybridoma cell strain HI
47The anti-CD of clone in (Yang Xifeng etc., Shanghai Journal of Immunology, the 10th the 2nd phase of volume, nineteen ninety)
20Antibody weight chain variable region gene.Concrete operations are as follows: anti-CD
20Antibody weight chain variable region gene clone: the RT-PCR anti-CD that increases
20Antibody weight chain variable region gene:
1) total RNA extracts: take the guanidinium isothiocyanate single stage method (Chomczynski P, SacchiN, Anal.Biochem.1987,162:156).In brief, with 10
7Individual anti-CD
20Monoclonal antibody hybridoma HI
47Cell, add successively solution D (storing solution (50ml): guanidinium isothiocyanate 23.8g, 0.75mol/L Trisodium Citrate (PH 7.0) 2.2ml, 10% dodecyl propylhomoserin sodium 3.3ml, adding distilled water to liquor capacity is 50ml; Solution D: add 72 μ l 2 mercapto ethanols in the 10ml storing solution), 3M NaAc, phenol, the chloroform isoamyl alcohol mixing, ice bath is centrifugal after 10 minutes, and supernatant adds the equal-volume Virahol, placed 1 hour for-20 ℃, centrifugal, get precipitation and be dissolved in the solution D, add isopyknic Virahol again, placed 1 hour for-20 ℃, centrifugal, abandon supernatant, wash precipitation with 70% ethanol, the ddH that handled with DEPC
2The O dissolution precipitation.
2) reverse transcription: get the total RNA of 2 μ g and place 0.5ml Eppendorf pipe, add 600ng random primer, each 10nmol of 4 * dNTP, RNasin 20 μ l successively, in 65 ℃ of water-bath 5-10 minutes, the M-MLV reversed transcriptive enzyme that adds 200U then, 37 ℃ of water-baths were placed 1 hour, were transferred to 70 ℃ of water-baths and placed 10 minutes.
3) the anti-CD of pcr amplification
20Monoclonal antibody weight chain variable region gene:
Chain variable region gene pcr amplification reaction system (100 μ l):
Reverse transcription product 10 μ l, 10mM dNTP 2 μ l, 10 * PCR damping fluid, 10 μ l, Taq archaeal dna polymerase 2U purchases in light chain amplimer 1,2 each 2 μ l of the recombinant phages antibody system of Pharmacia company, adds water to final volume 100 μ l.Reaction conditions is 95 ℃ of pre-sex change after 5 minutes, carries out 30 round-robin pcr amplifications (72 ℃ were extended 2 minutes for 94 ℃ of sex change 1 minute, 55 ℃ of annealing 2 minutes) again, extends 10 minutes in 72 ℃ at last.
Heavy chain variable region gene pcr amplification reaction system (100 μ l):
Reverse transcription product 10 μ l, 10mM dNTP 2 μ l, 10 * PCR damping fluid, 10 μ l, Taq archaeal dna polymerase 2U purchases in heavy chain amplimer 1,2 each 2 μ l of the recombinant phages antibody system of Pharmacia company, adds water to final volume 100 μ l.Reaction conditions is 95 ℃ of pre-sex change after 5 minutes, carries out 30 round-robin pcr amplifications (72 ℃ were extended 2 minutes for 94 ℃ of sex change 1 minute, 55 ℃ of annealing 2 minutes) again, extends 10 minutes in 72 ℃ at last.
Oligonucleotide sequence with coding joint (GGGGS) 3 connects the weight chain variable region gene that is obtained, and the weight chain gene of connection is assembled into after enzyme is cut in the corresponding restriction enzyme site of single-chain antibody expression vector pCANTAB 5E.Adopt electroporation with single-chain antibody (ScFv) the expression vector transformed into escherichia coli bacterial strain TG1 (preservation of laboratory, inventor place) that assembles, set up a recombinant phages antibody library.With the screening of ELISA method antagonist storehouse, obtain several strains and secrete anti-CD
20The clone strain of single-chain antibody.Through nucleic acid sequencing, the feature amino acid homology is analyzed in immunoglobulin (Ig) (IgG) primary structure, proves that our cloned genes is a goal gene.Embodiment 2 anti-CD
20The segmental structure of chimeric antibody Fab
With the best anti-CD of biological activity
20The single-chain antibody clone carries out the segmental assembling of Fab, the following (see figure 1) of its assembling process brief description for template:
With the PCR method from anti-CD
20Single-chain antibody (ScFv) expression vector amplification variable region of heavy chain (V
H), variable region of light chain (V
L) gene, with agarose electrophoresis separation and purification V
H, V
LGene.Respectively with recombinate respectively after MluI+ApaI, the Mlul+StyI digestion pYZH, pYZL carrier (pYZH carrier: contain human normal immunoglobulin γ
1Chain CH
1, for laboratory, inventor place makes up, pYZL carrier: contain human normal immunoglobulin K chain C
LBe that laboratory, inventor place makes up) in the corresponding restriction enzyme site, recon pYZHcd20, pYZLcd20 transformed into escherichia coli 16C9 bacterial strain (preservation of laboratory, inventor place) with gained, extract pYZHcd20, pYZLcd20 after the amplification cultivation, with MluI+NheI, SpeI+SphI digestion pYZLcd20, pYZHcd20, the agarose electrophoresis separation and purification contains V respectively
L, V
HThe corresponding DNA fragments of gene connects into anti-CD with these two fragments with through the carrier segments of MluI+SphI digestion Fab expression vector pYZF (laboratory, inventor place structure) gained
20Chimeric antibody Fab fragment expression carrier pYZFcd20.Embodiment 3 anti-CD
20Chimeric antibody Fab fragment solubility expression and analysis in intestinal bacteria
With the carrier pYZFcd20 transformed into escherichia coli 16C9 bacterial strain that embodiment 2 obtains, picking list colony inoculation 5ml contains 2 * YT substratum (peptone 16g/L, the yeast extract paste 10g/L of penbritin 50 μ g/ml, sodium-chlor 5g/L, pH7.4), 37 ℃, 200rpm, shaking culture 6 hours.6000rpm collected thalline in centrifugal 10 minutes, and thalline is suspended in the AP that 20ml contains penbritin 50 μ g/ml again
5(every liter of substratum contains substratum: casein acid hydrolysis thing 11g, yeast extract paste 0.6g, MgSO
40.19g, glucose 1.5g, NH
4Cl 1.07g, KCl3.73g, NaCl 1.2g, 1M trolamine pH 7.4 120ml), 30 ℃, 250rpm, shaking culture 20 hours.6000rpm, 4 ℃, centrifugal 10 minutes collection thalline, with thalline in-20 ℃ frozen 1 hour, add 1ml bacterium pericentral siphon chamber protein extract (Tutofusin tris 25mM, ethylenediamine tetraacetic acid (EDTA) 1mM after thawing, phenylmethylsulfonyl fluoride 0.1mM, sucrose 20% (W/V), sodium-chlor 200mM, pH7.5), mixing was in 4 ℃ of jogs 1 hour, thereafter 4 ℃, 12000rpm, centrifugal 20 minutes, get supernatant, with ProteinG post (GIBCO BRL) separation and purification Fab fragment.SDS-PAGE analyzes:
Press the sds page of the method preparation 12% on 881 pages of the molecular cloning experimental implementation guides (T.Maniatis etc. write for J.Sambrook, E.F.Fritsch, and Jin Dongyan etc. translate).Mix 100 ℃ of water-baths 3 minutes with sample buffer through the Fab of ProteinG column separating purification fragment sample with above-mentioned.Go up sample in order, arrange the standard substance of known molecular amount.Voltage was 8v/cm when electrophoresis began, and after dyestuff enters separation gel, voltage is increased to 15v/cm continuation electrophoresis arrives at separation gel bottom, deenergization up to dyestuff.Take off gel, with the staining fluid of at least 5 times of volumes (the 25g Xylene Brilliant Cyanine G is dissolved in 90ml methyl alcohol: the immersion gel solution of making in water (1: 1) and the 10ml glacial acetic acid solution), put on the decolorization swinging table room temperature and slowly rotated 3-4 hour.Change staining fluid, soak gel, slowly shook 4-8 hour, change destainer therebetween 3-4 time, till satisfaction, the gel after the decolouring is taken a picture with destainer (7% acetate, 5% methanol solution).Fab separation and purification product S DS-PAGE analyzes and sees Fig. 2, from Fig. 2 as seen under non-reduced condition the molecular weight of (2 road) Fab be about 48kDa, identical with the molecular weight of expection, and under reductive condition (4 road), disulfide bonds between the weight chain, the weight chain separates, and therefore the electrophoresis band at the 48kDa place disappears.Show that being in the 48kDa place is the Fab product.Embodiment 4 competitive immunization fluorescence suppress experiment
With 1 * 10
6Daudi cell (CD
20 +, CD
3 -, preserve in laboratory, inventor place) and to be suspended from 30 μ l concentration be the HI of 60 μ g/ml
47The monoclonal anti liquid solution was hatched under 4 ℃ 1 hour.2000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, PBS washes cell 3 times, and cell is resuspended in the solution of Fab that 30 μ l concentration are the embodiment 3 described Protein G purifying of 40 μ g/ml, places 1 hour for 4 ℃, 2000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, PBS washes cell 3 times, cell is resuspended in 30 μ l dilutes the solution of good sheep anti-mouse igg-FITC by the school valency, places 1 hour for 4 ℃, 2000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, PBS washes cell 3 times, and FACS measures.Simultaneously with anti-CD
3The negative contrast of mouse endogenous antibody is with anti-CD
20HI
47The positive contrast of mouse monoclonal antibody is carried out FACS equally and is measured.
Anti-CD
20Chimeric antibody Fab fragment and HI
47Monoclonal antibody competitive inhibition experiment FACS measurement result is seen Fig. 3, and when adding Fab, positive peak is offset left, shows anti-CD
20Chimeric antibody Fab fragment can competitive inhibition HI
47With CD
20Combination, i.e. Fab and CD
20Specific combination.
SEQ ID NO：1：366bp：：cDNA：SEQ ID NO：11 CAGGTGAAGC TGCAGCAGTC AGGGGCTGAG CTGGTGAAGC CTGGGGCCTC AGTGAAGATG61 TCCTGCAAGG CTTCTGGCTA CACATTTATC AGTTACAATA TGCACTGGGT AAAGCAGACA121 CCTGGACAGG GCCTGGAATG GATTGGAGGT ATTTATCCAG GAAATGGTGA TACTTCCTAC181 AATCAGAAAT TCAAAGGCAA GGCCACATTG ACTGCAGACA AATCCTCCAG CGCAGCCTAC241 ATGCAGCTCA GCAGCCTGAC ATCTGAGGAC TCTGCGGTCT ATTACTGTGC AAGATGGAAC301 TATGGTAACT TCGGGGGGGG TACTATGGAC TACTGGGGCC AAGGGATCAC GGTCACCGTC361 TCCTCASEQ ID NO：2：318bp：：cDNA：SEQ ID NO：21 GACATCGAGC TCACTCAGTC TCCAGCAATC CTGTCTGCAT CTCCAGGGGA GAAGGTCACA61 ATGACTTGCA GGGCCAGCTC AAGTGTAAGT TACATGCTCT GGTACCAGCA GAAGCCAGGA121 TCCTCCCCCA AACCCTGGAT TTATGCCACA TCCCACCTGG CTTCTGGAGT CCCTACTCGC181 TTCAGTGGCA GTGGGTCTGG GACCTCTTAC TCTCTCACAA TCAGCAGAGT GGAGGCTGAA241 GATGCTGCCA CTTATTACTG CCAGCAGTGG ACTAGTAACC CACCCACGTT CGGTGCTGGG301 ACCAAGCTGG AGCTGAAASEQ ID NO：3：122：：：SEQ ID NO：3 1 Q V K L Q Q S G A E L V K P G A S V K M S C K A S G Y T F I 31 S Y N M H W V K Q T P G Q G L E W I G G I Y P G N G D T S Y 61 N Q K F K G K A T L T A D K S S S A A Y M Q L S S L T S E D 91 S A V Y Y C A R W N Y G N F G G G T M D Y W G Q G I T V T V121 S SSEQ ID NO：4：108：：：SEQ ID NO：41 D I E L T Q S P A I L S A S P G E K V T M T C R A S S S V S31 Y M L W Y Q Q K P G S S P K P W I Y A T S H L A S G V P T R61 F S G S G S G T S Y S L T I S R V E A E D A A T Y Y C Q Q W91 T S N P P T F G A G T K L E L K R A
Claims (8)
1, a kind of anti-CD
20The heavy chain variable region gene of antibody, it has the sequence of SEQ ID NO:1.
2, by the polypeptide of the genes encoding of claim 1, it has the sequence of SEQ ID NO:3.
3, a kind of anti-CD
20The chain variable region gene of antibody, it has the sequence of SEQ ID NO:2.
4, by the polypeptide of the genes encoding of claim 3, it has the sequence of SEQ ID NO:4.
5, a kind of expression vector, it contains the anti-CD of claim 1
20The anti-CD of the heavy chain variable region gene of antibody and claim 3
20The chain variable region gene of antibody.
6, expression vector as claimed in claim 5, it is pYZFcd20.
7, the polypeptide expressed as the expression vector of claim 5 or 6.
8, the described polypeptide of claim 7 is used for the application of the medicine of the treatment of bone-marrow-derived lymphocyte knurl and diagnosis in preparation.
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|---|---|---|---|
| CNB991217284A CN1169952C (en) | 1999-10-10 | 1999-10-10 | Heavy chain and light chain variable region gene of anti-C D 20 monoclonal antibody and its application |
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|---|---|---|---|
| CNB991217284A CN1169952C (en) | 1999-10-10 | 1999-10-10 | Heavy chain and light chain variable region gene of anti-C D 20 monoclonal antibody and its application |
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| CN1169952C CN1169952C (en) | 2004-10-06 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011011973A1 (en) * | 2009-07-28 | 2011-02-03 | 中国医学科学院医药生物技术研究所 | Fusion protein of anti-cd20 antibody fab fragment and lidamycin, preparation method and use thereof |
| CN106397608A (en) * | 2016-10-19 | 2017-02-15 | 中国医学科学院血液病医院（血液学研究所） | CD20 specificity chimeric antigen receptor and application thereof |
| CN113549155A (en) * | 2020-04-23 | 2021-10-26 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Chimeric antigen receptor simultaneously targeting CD19 and CD20 and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA036531B1 (en) | 2003-11-05 | 2020-11-19 | Роше Гликарт Аг | Type ii anti-cd20 humanized antibody (variants), pharmaceutical composition comprising these antibody variants, and use thereof |
-
1999
- 1999-10-10 CN CNB991217284A patent/CN1169952C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011011973A1 (en) * | 2009-07-28 | 2011-02-03 | 中国医学科学院医药生物技术研究所 | Fusion protein of anti-cd20 antibody fab fragment and lidamycin, preparation method and use thereof |
| CN106397608A (en) * | 2016-10-19 | 2017-02-15 | 中国医学科学院血液病医院（血液学研究所） | CD20 specificity chimeric antigen receptor and application thereof |
| CN113549155A (en) * | 2020-04-23 | 2021-10-26 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Chimeric antigen receptor simultaneously targeting CD19 and CD20 and application thereof |
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