CN1421459A - Humanized CD3-resisting monoclonal antibody - Google Patents
Humanized CD3-resisting monoclonal antibody Download PDFInfo
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Abstract
The present invention provides one new kind of humanized CD3-resisting monoclonal antibody, its DNA encoding sequence and the host cell containing the encoding sequence. The present invention obtains effective immunodepressant with powerful rejection capacity, and has imunogenicity and toxic side effect, raises the expression of the antibody in mammal cell, and may be used widely in the diagnosis and treatment of CD3 relative diseases.
Description
Invention field
The present invention relates to monoclonal antibody technique, antibody humanization's technology, and gene recombination technology.Especially the Humanized monoclonal antibodies of anti-CD3.
Background of invention
The CD3 molecule is the membrane antigen that is distributed widely in people's mature T lymphocytic cell surface, and it and T cell surface membrane receptor TCR form complex body, play an important role in cell internal information transmittance process.The existing monoclonal antibody of anti-human T-cell CD3 that studies show that has the two-way function of activation and suppressor T cell.Its curative effect is confirmed fully in the preventative desensitization before acute immunologic rejection shock therapy, treatment graft-vs-host reaction and organ transplantation, and demonstrated wide application prospect in the treatment of antitumor, anti-multiple organ transplant rejection and aplastic anemia.
MOKT3 is the monoclonal antibody by the anti-people CD3 in mouse source of U.S. Ortho pharmaceutical companies production, and it has good effect to prevention and treatment organ transplantation immunity rejection.But mOKT3 is as mouse source property monoclonal antibody, and its immunogenicity causes the patient more than 50% to produce anti-mOKT3 antibody, and these antibody mainly contain two kinds: anti-idiotype and anti-isotype.During the former passes through and antigen binding site block combining of mOKT3 and CD3, the latter also may cause serious side reaction except that the removing of acceleration monoclonal antibody.
Thereby, overcome the key that mouse source property and first-dose response become its wide clinical application.Using human antibody or humanized antibody is two kinds of possible methods that overcome the anti-mouse of people source antibody response (HAMA reaction).Because high specificity, the human antibody that avidity is high are difficult to obtain, and mainly take the humanized method of mouse resource monoclonal antibody at present.
Summary of the invention
The object of the present invention is to provide Humanized anti-human CD3 monoclonal antibody, its associated dna sequence and the host cell that contains this sequence with potential medical science and pharmacy value.
To achieve these goals, the invention provides following technology incidence of criminal offenses.
The invention provides a kind of humanized anti-people CD3 monoclonal antibody, wherein the variable region of heavy chain of protoplast's antibody and variable region of light chain are replaced by inhuman source variable region of heavy chain and variable region of light chain respectively, described inhuman source variable region of heavy chain is selected from SEQ IN NO:1 or SEQ IN NO:5, and described inhuman endogenous light chain variable region is selected from SEQ INNO:2 or SEQ IN NO:6.
The present invention also provides a kind of dna molecular, wherein comprises the encoding sequence of above-mentioned inhuman source variable region of heavy chain and/or the encoding sequence of above-mentioned inhuman endogenous light chain variable region.
In one of embodiments of the present invention, the encoding sequence of above-mentioned inhuman source variable region of heavy chain is selected from SEQ INNO:3 or SEQ IN NO:7, and the encoding sequence of described inhuman endogenous light chain variable region is selected from SEQ IN NO:4 or SEQ IN NO:8.
In one of embodiments of the present invention, the form of described dna molecular a kind of carrier, especially expression vector.
The present invention also provides a kind of host cell, and it contains dna molecular of the present invention, especially is contained in the described dna molecular in host's consistency expression system.Described host cell can be for example to the various suitable cells of mammalian cells such as CHO from prokaryotic cell prokaryocytes such as intestinal bacteria.Its selection is the ordinary skill in the art.
The present invention also provides antibody of the present invention and the purposes of dna molecular of the present invention in the preparation of preparation diagnosis and/or treatment CD3 relative disease.
The present invention is undertaken humanization modified by antagonism CD3 monoclonal antibody, obtained that the immunologic rejection ability is strong, immunogenicity and the little effective immunosuppressor of toxic side effect, and has improved and merge the expression of antibody in mammalian cell.
Description of drawings
Fig. 1 is the synoptic diagram that the present invention is used for the overlapping PCR of synthetic humanization reorganization VH and VL encoding sequence.
Fig. 2 is the collection of illustrative plates of the expression vector pMG18-3K that uses among the present invention.Wherein, Ck represents the constant region encoding sequence of people's antibody kappa light chain; IgG1 Heng Qu represents the CH encoding sequence of people's IgG antibody 1; PA represents that SV40 adds poly (A) site.
Fig. 3 reflects that antibody engages affinity fluorescent dye result.Wherein ordinate zou is the cell flow of fluorescent dye, and unit is cell count/second, and X-coordinate is a fluorescence intensity, and unit is 10-4W/cm
2The main antibody that panel A is used is the anti-CD-20 monoclonal antibody Rituxan (available from Genentech) as negative control, and second antibody is the anti-people-FITC of rabbit; The main antibody that component B uses is the mOKT3 as the anti-CD3 of positive control, and second antibody is the anti-mouse FITC of rabbit; The main antibody that component C uses is cGD3 of the present invention, and second antibody is the anti-people-FITC of rabbit; The main antibody that component D uses is huGD of the present invention, and second antibody is the anti-people-FITC of rabbit.
Preferred implementation one. the preparation of anti-CD3 monoclonal antibody and evaluation
Employing is isozygotied CD3 albumen as immunogen, adds complete Freund's adjuvant, fully is ground to conventional subcutaneous multi-point injection 10 age in days Balb/c mouse after the complete emulsification, injection 50 microlitres, totally 6 points at every.Then with 1 weekly interval totally 5 duplicate injections react with booster immunization, and the serum antibody level is monitored.Last intravenous injection 5 * 10
6New fresh cell/PBS mixed solution killed mouse after 3 days, took out spleen.
The ordinary method separating spleen is unicellular, and definite cytoactive＞90%.Spleen cell is mixed centrifugal co-precipitation with the SP2/0 murine myeloma cell at 10: 1, add PEG (1400MW) and carry out cytogamy in the 37C water-bath, time of fusion is 1 minute, 2 minutes, 5 minutes and 10 minutes.Added at interval 1 milliliter of fresh serum free medium, 2 milliliters, 5 milliliters and 10 milliliters in 5 minutes.After the centrifugation cell is joined in the 20%FCSRPMI-1640 nutrient solution, change 96 orifice plates over to and cultivate.Add 1 * HAT after 24 hours and select nutrient solution.Changed fresh culture 1 time in per 3 days, to growing the clone.
Select mono-clonal to form the hole, took out supernatant liquor, measure the antibody expression amount, select positive hole subclone, under normal condition, go down to posterity with the ELISA method at the 14th day, stable to the 5th generation.Picking out the highest Position Number of expression amount in view of the above is the clone of GD3, as the material that continues research.Two. the amplification and the order-checking of anti-CD3 monoclonal antibody variable region
The method of employing 5 ' RACE (Rapid amplification of cDNA ends) is obtained the variable region encoding sequence of anti-CD3 mouse source monoclonal antibody mGD3.
Design and synthesize following because
1. design of primers:
GSP1-H：5’-AGC?TGG?GAA?GGT?GTG?CAC?ACC?A?CT-3’(SEQ?ID?NO：9)
GSP2-H：5’-CAG?AGT?TCC?AGG?TCA?AGG?TCA-3’(SEQ?ID?NO：10)
GSP3-H：5’-CTT?GAC?CAG?GCA?TCC?TAG?AGT-3’(SEQ?ID?NO：11)
GSP1-L：5’-TTG?CTG?TCC?TGA?TCA?GTC?CAA?CT-3’(SEQ?ID?NO：12)
GSP2-L：5’-TGT?CGT?TCA?CTG?CCA?TCA?ATC?TT-3’(SEQ?ID?NO：13)
GSP3-L：5’-TTG?TTC?AAG?AAG?CAC?ACG?ACT?GA-3’(SEQ?ID?NO：14)
AAP：5’-GGC?CAC?GCG?TCG?ACT?AGT?ACG?GGI?IGG?GII?GGG?IIG-3’(SEQ?IDNO：15)
AUAP：5’-GGC?CAC?GCG?TCG?ACT?AGT?AC-3’(SEQ?ID?NO：16)
Wherein, GSP represents gene-specific primer, and AAP represents 5 '-RACE brachymemma anchor primer, and AUAP represents brachymemma universal amplification primer.GSP1 distance variable district gene is used for reverse transcription reaction farthest.GSP2 is used for nested PCR with GSP3, and wherein GSP3 is in GSP2.
Trizol Reagent reagent with Gibco company extracts 2 * 10 respectively
6Total RNA of hybridoma mGD3.According to 5 '-RACE test kit (Pharmacia company product) specification sheets is that primer becomes cDNA with total RNA reverse transcription with GSP1.Adding poly (C) tail for then 3 ' the end of the first chain cDNA, is that primer carries out pcr amplification with GSP2 and AAP behind the tailing, and it is that primer carries out nested pcr amplification with AUAP and GSP3 again that amplified production is diluted 100 times.Warm start, reaction conditions are all adopted in twice PCR reaction: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 1 minute 10 seconds, 30 circulations; 72 ℃ 7 minutes.Nested PCR product reclaims purifying purpose fragment (the about 320bp of light chain length, the about 360bp of heavy chain length) after 1% agarose gel electrophoresis separates.Be cloned in pGEM-T (Promega) carrier, at the dull and stereotyped enterprising row filter of IPTG/X-gal, get 8 white bacterial plaques and be inoculated in the LB liquid nutrient medium that contains penbritin and increase behind the transformed into escherichia coli TG1 cell.Screening positive clone with the plasmid extraction test kit extracting plasmid of QIAGEN and check order, has been determined the heavy chain of mGD3 and the dna sequence dna of variable region of light chain, and has been drawn the deduction aminoacid sequence in view of the above
The aminoacid sequence of mGD3 variable region of heavy chain (VH) (119 amino acid): QVQKSDSGGGVKQPGRSLRLSGKASGYTFTSYTAHWVRQAPGKGWNPLAYINPSSG YTKSGDKFKDRFTISRKKDKNTLFLQMDSLRPEDTGDYFCARWQDYDVYFDYWGQA CLVTVSS (SEQ ID NO:1)
The aminoacid sequence of mGD3 variable region of light chain (VL) (107 amino acid): DIKLNQSPSSMNASVGDRVTISKLDSSSVSYMDDYQQTPIKAPKLLIYATSNLASG VPSTFSGSGSGTDYTFTISSQDPEDIATYYCQQWSSDNPTFGQGTKSDKTR (SEQ ID NO:2)
The dna sequence dna of mGD3 VH (357 nucleotides): CAGGTTCAGAAATCTGACTCTGGTGGTGGTGTTAAACAGCCGGGTCGTTCTCTGCG TCTGTCTGGTAAAGCTTCTGGTTACACCTTCACCTCTTACACCGCTCACTGGGTTC GTCAGGCTCCGGGTAAAGGTTGGAACCCGCTGGCTTACATCAACCCGTCTTCTGGT TACACCAAATCTGGTGACAAATTCAAAGACCGTTTCACCATCTCTCGTAAAAAAGA CAAAAACACCCTGTTCCTGCAGATGGACTCTCTGCGTCCGGAAGACACCGGTGACT ACTTCTGCGCTCGTTGGCAGGACTACGACGTTTACTTCGACTACTGGGGTCAGGCT TGCCTGGTTACCGTTTCTTCT (SEQ ID NO:3)
The dna sequence dna of mGD3 VL (321 nucleotides): GACATCAAACTGAACCAGTCTCCGTCTTCTATGAACGCTTCTGTTGGTGACCGTGT TACCATCTCTAAACTGGACTCTTCTTCTGTTTCTTACATGGACGACTACCAGCAGA CCCCGATCAAAGCTCCGAAACTGCTGATCTACGCTACCTCTAACCTGGCTTCTGGT GTTCCGTCTACCTTCTCTGGTTCTGGTTCTGGTACCGACTACACCTTCACCATCTC TTCTCAGGACCCGGAAGACATCGCTACCTACTACTGCCAGCAGTGGTCTTCTGACA ACCCGACCTTCGGTCAGGGTACCAAATCTGACAAAACCCGT (SEQ ID NO:4) three. the structure of humanization chimeric mAb expression vector
Adopt the PCR reaction to add suitable restriction enzyme site for respectively above antibody variable region.Wherein 5 of variable region of heavy chain ' end designs Xba I restriction enzyme site, and 3 ' end adds Nhe I site, and in variable region of light chain encoding sequence 5 ' end design HindIII site, 3 ' end adds the BsiWI site.
Used PCR primer is:
Heavy chain forward primer: 5 '-ctctctagaCAGGTTCAGAAATCTG-3 ' (SEQ ID NO:17)
Heavy chain reverse primer: 5 '-gacgctagcAGAAGAAACGGTAAC-3 ' (SEQ ID NO:18)
Light chain forward primer: 5 '-ctgaagcaaGACATCAAACTGAACCAG-3 ' (SEQ ID NO:19)
Light chain reverse primer: 5 '-gaccgtacgacgggttttgtc-3 ' (SEQ ID NO:20)
With the aforementioned plasmid pGEM-T that contains variable region of heavy chain and variable region of light chain is that template is carried out regular-PCR.Gained PCR product cloning is carried out the sequencing conclusive evidence in the pGEM-T (Promega product) be required correct aim sequence.With Xba I and Nhe I the antibody heavy chain variable region encoding sequence is downcut from carrier pGEM-T then and be inserted into the corresponding site of expression vector pMG18-3K, with Hind III and Bsi WI antibody chain variable region is downcut from carrier pGEM-T again and be inserted into the corresponding site of expression vector pMG18-3K (referring to DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ONINCP-9 PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, C.M.Thomas Schoolof Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK andFaculty of Biology, Dept of Microbiology, Belarus State University Scorina Av.4, Minsk220080Belarus).Constituted the expression vector pcGD3 of chimeric antibody at last.Four. the design of humanization recombinant monoclonal antibodies VL and VH
The present invention has carried out the humanization design to VL and the VH of CD 3-resisting monoclonal antibody mGD3, take into account simultaneously mammalian cell access to your password the son preferences, the design incidence of criminal offenses as follows:
The weight chain variable region amino acid sequence of humanized antibody huGD3 (119 amino acid) QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSG YTKYNQKFKDRFTISADNSKSTAFLQMDSLRPEDTAVYYCARWQDYDVYFDYWGQG TPVTVSS (SEQ ID NO:5)
The light chain variable region amino acid sequence of humanized antibody huGD3 (107 amino acid) DIVLTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASG VPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR (SEQ ID NO:6)
The variable region of heavy chain coded sequence of humanized antibody huGD3 (5 ' → 3 ', 357bp) 001 CAGGTGCAGC TGGTGCAGTC TGGCGGTGGA GTGGTCCAGC CCGGCCGCAG051 CCTGAGGCTG TCCTGCAAGG CCAGCGGCTA CACCTTCACC AGCTACACGA101 TGCACTGGGT GCGCCAAGCC CCCGGAAAGG GCCTCGAATG GATTGGCTAC151 ATTAATCCTA GCAGTGGTTA TACTAAGTAC AATCAGAAGT TCAAGGACAG201 ATTTACAATA TCAGCCGACA ACAGCAAGTC CACCGCCTTC CTACAAATGG251 ACAGCTTGCG TCCAGAGGAC ACCGCCGTAT ACTACTGTGC GCGCTGGCAG301 GATTACGACG TCTACTTTGA CTACTGGGGC CAAGGCACTC CAGTCACCGT351 CTCCTCT (SEQ ID NO:7)
The variable region of light chain coded sequence of humanized antibody huGD3 (5 ' → 3 ', 321bp) 001 GACATCGTTC TCACTCAGAG CCCATCCAGC TTGAGCGCAT CAGTAGGCGA051 CCGCGTAACG ATCACTTGCA GGGCCAGCTC AAGTGTAAGT TACATGCACT101 GGTACCAGCA GACTCCCGGC AAAGCCCCAA AGCCCTGGAT TTATGCCACA151 TCCAACCTGG CTTCTGGCGT GCCATCACGC TTTAGCGGCA GCGGGTCCGG201 TACAGATTAC ACGTTCACCA TTAGCAGTCT GCAGCCTGAG GACATAGCCA251 CCTACTACTG TCAGCAGTGG AGTAGTAACC CACCGACGTT TGGCCAGGGA301 ACTAAACTGC AGATTACTCG A (SEQ ID NO:8) five. synthesizing of humanized antibody variable region coded sequence
We are in 5 of humanized antibody heavy chain encoding sequence ' end design XbaI enzyme cutting site, and 3 ' end adds the NheI site, and in light chain encoding sequence 5 ' end design HindIII site, 3 ' end adds the BsiWI site.Dna sequence dna at above humanization heavy chain and light chain, synthetic respectively 8 segment lengths are about 75bp and 20bp eclipsed Oligonucleolide primers are arranged each other, through three overlapping PCR (see figure 1)s: 1. with primer a and b, c and d, e and f, g and h are mixed into the performing PCR reaction respectively, obtain product ab, cd, ef, gh.2. primer a, d are increased after mixing mutually with last round of PCR product ab, cd, primer e, h are mixed mutually with last round of PCR product ef, gh increase equally, obtain product ad and eh respectively.3. primer a, h and last round of PCR product ad, eh are mixed into mutually the performing PCR amplification, obtain product ah.Above-mentioned PCR adopts the high-fidelity amplification system of Roche company, synthetic respectively above variable region encoding sequence.The PCR reaction conditions is: 95C5 minute; 94C 45 seconds, 55C 45 seconds, 72C 55 seconds, 30 circulations; 72C 7 minutes.Agarose gel electrophoresis separates amplified production, reclaim the purpose band respectively (about heavy chain length 360bp, light chain length about 320) and be cloned in the pGEM-T carrier, the screening positive clone order-checking confirms to have obtained respectively the VH of above-mentioned humanization monoclonal antibody huGD3 and the encoding sequence of VL.Six. the structure of humanization recombinant monoclonal antibodies expression vector
With XbaI and NheI the variable region of heavy chain encoding sequence is downcut the corresponding site that is inserted into expression vector pMG18-3K from carrier pGEM-T.With HindIII and BsiWI variable region of light chain is downcut from carrier pGEM-T again and be inserted into corresponding site (referring to Fig. 2) the expression vector pMG18-3K, obtain humanization recombinant monoclonal antibodies expression vector phuGD3.Seven. the expression of humanized antibody
With the pcGD3 of preamble step 3 structure and the phuGD3 transfection Escherichia coli DH5a of above structure.In the inoculation 100ml LB substratum, increase according to ordinary method.The results culture is with the UltraPure plasmid DNA of the Qiagen company test kit extracting and purifying plasmid DNA of isozygotying.The plasmid DNA of above-mentioned purifying is adopted the liposome method test kit transfection CHO cell of Invitrogen company, and operation is carried out with reference to the specification sheets of producer.
The Chinese hamster ovary celI that transforms carries out the extreme dilution at last and cultivates in the selection of selecting to carry out on the substratum (HAT that contains 0.05～10mM aminomethyl pterin selects substratum) continuous 9 weeks on 96 orifice plates, carry out continuously 3 times, carry out mono-clonalization.
The monoclonal cell of choosing ties up on the RPM1641 substratum and cultivates.Supernatant is carried out the Western Blot experiment, judge expression intensity according to staining reaction.Select high-expression clone to carry out follow-up study.The result selects respectively with the clone huGD3 of phuGD3 transfection with the clone cGD3 of pcGD3 transfection.
With albumin A affinity column direct separation and purification Humanized monoclonal antibodies of the present invention from the culture supernatant of above-mentioned 6 clones.SDS-PAGE electrophoretic examinations proves that products therefrom purity is greater than 90%.
The product of above affinity chromatography has obtained the sample of purity＞98% once more through sieve chromatography research.Gained antibody can be used for following further researching and analysing.Eight. the research of humanized antibody expression amount
With the ELISA method expression amount of the Chinese hamster ovary celI strain of above-mentioned little murine hybridoma (mGD3), chimeric monoclonal antibody (cGD3) and humanization monoclonal antibody (huGD3) is studied.Get the 5ml cell with cultivating in the described cell strain 37 degree 5%CO2 incubators after 72 hours, in 5000RPM centrifugal 10 minutes, get supernatant and carry out DOT ELISA.Its second antibody is the anti-people's antibody of the rabbit of HRP mark.DAB system (available from Ameresco company) is adopted in colour developing, dyes with reference to the explanation of producer.
The result of study of polyclonal antibody expression amount
Expression amount (mg/ml)
Positive control 1 (mOKT3) 25.6
Positive control 2 (Rituxan, Genetech product) 38.8
mGD3???????????????????????????????????20.1
cGD3???????????????????????????????????66.9
huGD3??????????????????????????????????224.3
The The above results explanation, still difference is little not as two contrasts for the expression amount of our mouse hybridoma cell, but the expression amount comparison of the chimeric cell that we make up system exceeds more than 70% according to the chimeric antibody expression amount, our humanized antibody clone than two positives to exceeding 8.8 times and 5.8 times respectively.
As described in preamble the 6th part,, obtain mouse source antibody mGD3 and humanized antibody cGD3 and huGD3 respectively with Protein A affinity chromatography column separating purification hybridoma excretory polyclonal antibody.The biological activity of nine .CD3 Humanized monoclonal antibodies is identified
The avidity of humanized antibody is measured
With FITC mark mouse GD3 (mGD3), after the unlabelled huGD3 mixing of FITC-mGD3 and a series of different extent of dilution of saturation concentration, with target cell (5 * 10
5Human peripheral blood mononuclear cell (PBMC)) at 4 ℃ of incubation 60min.Cell is fixing with PBS flushing back, detect with flow cytometer, and calculate the fluorescencepositive cell percentage.The affinity constant Ka of mGD3 does the figure method with Scatchard and draws.The affinity constant of humanized antibody calculates with formula [X]-[mGD3]=(1/Kx)-(1/Ka), wherein Ka is the affinity constant of mGD3, [X] is the concentration that traget antibody " engage/free " value is competed antibody during for R0/2, and R0 represents the maximum fluorescence cell count.The result is as follows:
The affinity constant of mGD3 is: 1.7 * 10
9M
-1
The affinity constant of huGD3 is: 1.6 * 10
9M
-1
From the result, the affinity constant of huGD3 of the present invention has reached 1.6 * 10
9M
-1, reached the avidity of mouse source antibody fully.Proof thus, like this, the humanized antibody of our gained has kept the avidity of former mouse source antibody.
Fluorescent dye
With T lymphocyte oncocyte HPBLL or Jarket cell is the CD3 positive target cell, 5 * 10
6Individual cell is divided into 5 pipes, average every pipe 1 * 10
6Cell.The result as shown in Figure 3, the chimeric and humanization CD3 monoclonal antibody of reorganization of our development can significantly reduce consumption clinically and reduce possible side reaction than high 2 orders of magnitude of avidity of positive control mOKT3, has great economic worth.
In order to determine the dose,equivalent of cGD3 and two kinds of monoclonal antibodies of huGD3 and Rituxan, carry out the fluorescent dye test in addition.The result is as follows
The relation of different monoclonal antibody avidity and its concentration
| Antibody | Antibody concentration μ g/ml | ||
| ?0.1 | ?1 | ?10 | |
| ?Rituxan | ?57MFI | ?88MFI | ?101MFI |
| ?cGD3 | ?171MFI | ?224MFI | ?320MFI |
| ?huGD3 | ?55MFI | ?76MFI | ?97MFI |
The above results explanation, the chimeric monoclonal antibody of our development when concentration is 0.1ug/ml, the twice of effect in the time of can reaching positive control Rituxan 1 μ g/ml, i.e. 20 times of being equivalent to contrast of our affinity of antibody.And the avidity and the positive control of our humanization monoclonal antibody are about the same.
The test of T cell film CD3 antigenic modulation
To the antigenic promotor action of human lymphocyte CD3 is the main immunologic function of CD3, whether also has the ability of modulation CD3 molecule in order to detect humanized antibody, mix back 37 ℃ of incubation 24h with the dilution antibody of difference mutually with human PBMC (1ml), harvested cell (selects mOKT3-FITC to be because its bonded epi-position is different from mOKT3 and other anti-CD3 monoclonal antibody with mOKT3 (available from Genetech company)-FITC, but visual representation is not by the CD3 molecule of modulation) dyeing, cell is redyed (to distinguish out the T cell) with the anti-CD5 monoclonal antibody of PE mark, analyze with flow cytometer then, each result is the mean value of 3 experiments.
The calculating different antibodies to the formula of the modulation ability of CD3 is:
Humanized antibody is to the promotor action test-results of human lymphocyte
| CD3 modulation (%) | |||||||
| [antibody] ng/ml | ?10 -1 | ?10 | ?10 1 | ?10 2 | ?10 3 | ?10 4 | ?10 5 |
| mGD3 | ?10.56 | ?15.45 | ?16.78 | ?67.67 | ?78.89 | ?85.86 | ?85.34 |
| huGD3 | ?9.52 | ?13.25 | ?13.57 | ?56.87 | ?70.00 | ?85.36 | ?85.64 |
From above data as can be seen, huGD3 has reached the intensity of mouse source antibody to the promotor action of human lymphocyte, is a humanized antibody that keeps the main immunologic function of mGD3.
Sequence table＜110〉Lansheng Guojian Pharmaceutic Ind. Co., Ltd., Shanghai＜120〉Humanized CD 3-resisting monoclonal antibody＜130〉016014＜160〉20＜170〉PatentIn version 3.0＜210〉1＜211〉119＜212〉PRT＜213〉mouse (Mus musculus)＜400〉1Gln Val Gln Lys Ser Asp Ser Gly Gly Gly Val Lys Gln Pro Gly Arg1,5 10 15Ser Leu Arg Leu Ser Gly Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20??????????????????25??????????????????30Thr?Ala?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Trp?Asn?Pro?Leu
35??????????????????40??????????????????45Ala?Tyr?Ile?Asn?Pro?Ser?Ser?Gly?Tyr?Thr?Lys?Ser?Gly?Asp?Lys?Phe
50??????????????????55??????????????????60Lys?Asp?Arg?Phe?Thr?Ile?Ser?Arg?Lys?Lys?Asp?Lys?Asn?Thr?Leu?Phe65??????????????????70??????????????????75??????????????????80Leu?Gln?Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Gly?Asp?Tyr?Phe?Cys
85??????????????????90??????????????????95Ala?Arg?Trp?Gln?Asp?Tyr?Asp?Val?Tyr?Phe?Asp?Tyr?Trp?Gly?Gln?Ala
100?????????????????105?????????????????110Cys?Leu?Val?Thr?Val?Ser?Ser
115＜210〉2＜211〉107＜212〉PRT＜213〉mouse (Mus musculus)＜400〉2Asp Ile Lys Leu Asn Gln Ser Pro Ser Ser Met Asn Ala Ser Val Gly1,5 10 15Asp Arg Val Thr Ile Ser Lys Leu Asp Ser Ser Ser Val Ser Tyr Met
20??????????????????25??????????????????30Asp?Asp?Tyr?Gln?Gln?Thr?Pro?Ile?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr
35??????????????????40??????????????????45Ala?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ser?Thr?Phe?Ser?Gly?Ser
50??????????????????55??????????????????60Gly?Ser?Gly?Thr?Asp?Tyr?Thr?Phe?Thr?Ile?Ser?Ser?Gln?Asp?Pro?Glu65??????????????????70??????????????????75??????????????????80Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asp?Asn?Pro?Thr
85??????????????????90??????????????????95Phe?Gly?Gln?Gly?Thr?Lys?Ser?Asp?Lys?Thr?Arg
100 105＜210〉3＜21l〉357＜212〉DNA＜213〉 ( Mus nlusculus )＜400〉3caggttcaga aatctgactc tggtggtggt gttaaacagc cgggtcgttc tctgcgtctg 60tctggtaaag cttctggtta caccttcacc tcttacaccg ctcactgggt tcgtcaggct 120ccgggtaaag gttggaaccc gctggcttac atcaacccgt cttctggtta caccaaatct 180ggtgacaaat tcaaagaccg tttcaccatc tctcgtaaaa aagacaaaaa caccctgttc 240ctgcagatgg actctctgcg tccggaagac accggtgact acttctgcgc tcgttggcag 300gactacgacg tttacttcga ctactggggt caggcttgcc tggttaccgt ttcttct 357＜210〉4＜211〉321＜212〉DNA＜213〉 ( Mus nusculus )＜400〉4gacatcaaac tgaaccagtc tccgtcttct atgaacgctt ctgttggtga ccgtgttacc 60atctctaaac tggactcttc ttctgtttct tacatggacg actaccagca gaccccgatc 120aaagctccga aactgctgat ctacgctacc tctaacctgg cttctggtgt tccgtctacc 180ttctctggtt ctggttctgg taccgactac accttcacca tctcttctca ggacccggaa 240gacatcgcta cctactactg ccagcagtgg tcttctgaca acccgacctt cggtcagggt 300accaaatctg acaaaacccg t 321＜210〉5＜211〉119＜212〉PRT＜213〉＜220〉＜221〉misc_feature＜223〉CD3＜400〉5Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20??????????????????25??????????????????30Thr?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile
35??????????????????40??????????????????45Gly?Tyr?Ile?Asn?Pro?Ser?Ser?Gly?Tyr?Thr?Lys?Tyr?Asn?Gln?Lys?Phe
50??????????????????55??????????????????60Lys?Asp?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Asn?Ser?Lys?Ser?Thr?Ala?Phe65??????????????????70??????????????????75??????????????????80Leu?Gln?Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85??????????????????90??????????????????95Ala?Arg?Trp?Gln?Asp?Tyr?Asp?Val?Tyr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly
100?????????????????105?????????????????110Thr?Pro?Val?Thr?Val?Ser?Ser
115＜210〉6＜211〉107＜212〉PRT＜213〉artificial sequence＜220〉＜221〉misc feature＜223〉humanized CD 3-resisting monoclonal antibody variable region of light chain＜400〉6Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1,5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20??????????????????25??????????????????30His?Trp?Tyr?Gln?Gln?Thr?Pro?Gly?Lys?Ala?Pro?Lys?Pro?Trp?Ile?Tyr
35??????????????????40??????????????????45Ala?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser
50??????????????????55??????????????????60Gly?Ser?Gly?Thr?Asp?Tyr?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu65??????????????????70??????????????????75??????????????????80Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asn?Pro?Pro?Thr
85??????????????????90??????????????????95Phe?Gly?Gln?Gly?Thr?Lys?Leu?Gln?Ile?Thr?Arg
100 105＜210〉7＜211〉357＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜223〉CD3＜400〉7caggtgcagc tggtgcagtc tggcggtgga gtggtccagc ccggccgcag cctgaggctg 60tcctgcaagg ccagcggcta caccttcacc agctacacga tgcactgggt gcgccaagcc 120cccggaaagg gcctcgaatg gattggctac attaatccta gcagtggtta tactaagtac 180aatcagaagt tcaaggacag atttacaata tcagccgaca acagcaagtc caccgccttc 240ctacaaatgg acagcttgcg tccagaggac accgccgtat actactgtgc gcgctggcag 300gattacgacg tctactttga ctactggggc caaggcactc cagtcaccgt ctcctct 357＜210〉8＜211〉321＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜223〉CD3＜400〉8gacatcgttc tcactcagag cccatccagc ttgagcgcat cagtaggcga ccgcgtaacg 60atcacttgca gggccagctc aagtgtaagt tacatgcact ggtaccagca gactcccggc 120aaagccccaa agccctggat ttatgccaca tccaacctgg cttctggcgt gccatcacgc 180tttagcggca gcgggtccgg tacagattac acgttcacca ttagcagtct gcagcctgag 240gacatagcca cctactactg tcagcagtgg agtagtaacc caccgacgtt tggccaggga 300actaaactgc agattactcg a 321＜210〉9＜211〉24＜212〉DNA＜213〉＜400〉9agctgggaag gtgtgcacac cact 24＜210〉10＜211〉21＜212〉DNA＜213〉＜400〉10cagagttcca ggtcaaggtc a 21＜210〉11＜211〉21＜212〉DNA＜213〉＜400〉11cttgaccagg catcctagag t 21＜210〉12＜211〉23＜212〉DNA＜213〉＜400〉12ttgctgtcct gatcagtcca act 23＜210〉13＜211〉23＜212〉DNA＜213〉＜400〉13tgtcgttcac tgccatcaat ctt 23＜210〉14＜211〉23＜212〉DNA＜213〉＜400〉14ttgttcaaga agcacacgac tga 23＜210〉15＜211〉36＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜223〉n＝＜400〉15ggccacgcgt cgactagtac gggnngggnn gggnng 36＜210〉16＜211〉20＜212〉DNA＜213〉＜400〉16ggccacgcgt cgactagtac 20＜210〉17＜211〉25＜212〉DNA＜213〉＜400〉17ctctctagac aggttcagaa atctg 25＜210〉18＜211〉24＜212〉DNA＜213〉＜400〉18gacgctagca gaagaaacgg taac 24＜210〉19＜211〉27＜212〉DNA＜213〉＜400〉19ctgaagcaag acatcaaact gaaccag 27＜210〉20＜211〉21＜212〉DNA＜213〉＜400〉20gaccgtacga cgggttttgt c 21
Claims (9)
1. humanized anti-people CD3 monoclonal antibody, wherein former I variable region of heavy chain and the variable region of light chain of antibody are replaced by inhuman source variable region of heavy chain and variable region of light chain respectively, described inhuman source variable region of heavy chain is selected from SEQ IN NO:1 or SEQ IN NO:5, and described inhuman endogenous light chain variable region is selected from SEQ IN NO:2 or SEQ IN NO:6.
2. a dna molecular wherein comprises the encoding sequence of the described inhuman source of claim 1 variable region of heavy chain and/or the encoding sequence of the described inhuman endogenous light chain of claim 1 variable region.
3. dna molecular according to claim 2, the encoding sequence of described inhuman source variable region of heavy chain is selected from SEQ IN NO:3 or SEQ IN NO:7, and the encoding sequence of described inhuman endogenous light chain variable region is selected from SEQ INNO:4 or SEQ IN NO:8.
4. dna molecular according to claim 2, it is a kind of carrier.
5. host cell, it contains each described dna molecular in the claim 2 to 4.
6. limit is according to the described cell of claim 5, and contained described dna molecular is included in host's consistency expression system.
7. cell according to claim 5, it is a mammalian cell.
8. the purposes of the described antibody of claim 1 in the preparation of preparation diagnosis and/or treatment CD3 relative disease.
9. the purposes of the described dna molecular of claim 2 in the preparation of preparation diagnosis and/or treatment CD3 relative disease.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132281 CN1189483C (en) | 2001-11-23 | 2001-11-23 | Humanized CD3-resisting monoclonal antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132281 CN1189483C (en) | 2001-11-23 | 2001-11-23 | Humanized CD3-resisting monoclonal antibody |
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| CN1421459A true CN1421459A (en) | 2003-06-04 |
| CN1189483C CN1189483C (en) | 2005-02-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01132281 Expired - Lifetime CN1189483C (en) | 2001-11-23 | 2001-11-23 | Humanized CD3-resisting monoclonal antibody |
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| CN (1) | CN1189483C (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7777017B2 (en) | 1998-06-15 | 2010-08-17 | Biosynexus Incorporated | Nucleic acids encoding opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of gram positive bacteria |
| CN1984931B (en) * | 2004-06-03 | 2012-11-28 | 诺维莫尼公司 | Anti-CD3 antibodies and methods of use thereof |
| CN104098698A (en) * | 2013-04-08 | 2014-10-15 | 中国人民解放军第二军医大学 | Antibody against CD3, and preparation method and application thereof |
| US9782478B1 (en) | 2011-04-22 | 2017-10-10 | Aptevo Research And Development Llc | Prostate-specific membrane antigen binding proteins and related compositions and methods |
| CN109913493A (en) * | 2017-12-12 | 2019-06-21 | 百奥赛图江苏基因生物技术有限公司 | The preparation method and application of humanization CD3 genetic modification animal model |
| US11352426B2 (en) | 2015-09-21 | 2022-06-07 | Aptevo Research And Development Llc | CD3 binding polypeptides |
| WO2024152963A1 (en) * | 2023-01-18 | 2024-07-25 | 贝达药业股份有限公司 | Humanized anti-cd3 antibody and use thereof |
| US12122850B2 (en) | 2023-03-14 | 2024-10-22 | LamKap Bio gamma AG | Bispecific GPC3xCD28 and GPC3xCD3 antibodies and their combination for targeted killing of GPC3 positive malignant cells |
-
2001
- 2001-11-23 CN CN 01132281 patent/CN1189483C/en not_active Expired - Lifetime
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7777017B2 (en) | 1998-06-15 | 2010-08-17 | Biosynexus Incorporated | Nucleic acids encoding opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of gram positive bacteria |
| CN1984931B (en) * | 2004-06-03 | 2012-11-28 | 诺维莫尼公司 | Anti-CD3 antibodies and methods of use thereof |
| US9782478B1 (en) | 2011-04-22 | 2017-10-10 | Aptevo Research And Development Llc | Prostate-specific membrane antigen binding proteins and related compositions and methods |
| CN104098698A (en) * | 2013-04-08 | 2014-10-15 | 中国人民解放军第二军医大学 | Antibody against CD3, and preparation method and application thereof |
| CN104098698B (en) * | 2013-04-08 | 2019-04-05 | 中国人民解放军第二军医大学 | A kind of anti-cd 3 antibodies and its preparation method and application |
| US11352426B2 (en) | 2015-09-21 | 2022-06-07 | Aptevo Research And Development Llc | CD3 binding polypeptides |
| CN109913493A (en) * | 2017-12-12 | 2019-06-21 | 百奥赛图江苏基因生物技术有限公司 | The preparation method and application of humanization CD3 genetic modification animal model |
| US10945420B2 (en) | 2017-12-12 | 2021-03-16 | BiocytogenPharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric CD3e |
| US12016315B2 (en) | 2017-12-12 | 2024-06-25 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric CD3e |
| WO2024152963A1 (en) * | 2023-01-18 | 2024-07-25 | 贝达药业股份有限公司 | Humanized anti-cd3 antibody and use thereof |
| US12122850B2 (en) | 2023-03-14 | 2024-10-22 | LamKap Bio gamma AG | Bispecific GPC3xCD28 and GPC3xCD3 antibodies and their combination for targeted killing of GPC3 positive malignant cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1189483C (en) | 2005-02-16 |
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