CN1775808A - Anti CD19 engineered antibody for target conjugated lymphocyte, leuco cyte and its use - Google Patents
Anti CD19 engineered antibody for target conjugated lymphocyte, leuco cyte and its use Download PDFInfo
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Abstract
The invention discloses an anti-CD19 engineering antibody and its application which is used in target direction combining lymphocytic leukemia cell. It lays a foundation for the next trituration genetic engineering medicine of the target direction therapy leukemia. It relates to anti-CD19 monoclonal antibody HI19a heavy and light chain variable region gene, and application of the gene code polypeptide, the gene carrier, and using the gene and polypeptide to make leukemia therapy medicine. The heavy and light chain variable region gene is come from the anti-CD19 monoclonal antibody HI19a. The invention successfully adopts gene engineering technique to make anti-CD19 gene engineering antibody, and lays a foundation for the target direction therapy of the leukemia.
Description
Technical field
The present invention relates to a kind of engineered antibody, in particular for engineered antibody of target conjugated lymphocyte leukemia cell's anti-CD19 and uses thereof.
Background technology
Acute lymphoblastic leukemia (ALL) is leukemic a kind of hypotype, based on B cell ALL (B-ALL), accounts for the 70-80% of ALL, and annual neopathy rate is 0.98/10 ten thousand people.The treatment of adult ALL is based on chemotherapy at present, and curative effect is relatively poor, though can obtain temporary transient alleviation, lifetime is short.China's adult ALL curative effect is poorer, so that can't calculate its long-term survival rate.Therefore, reduce leukemic case fatality rate, carrying out and improve leukemic methods of treatment is the emphasis that world medicine is being captured.
Along with modern molecular biology and immunologic development, the birth of genetic engineering antibody is that the diagnosis and the treatment of tumour brought hope, especially the application of single-chain antibody becomes the focus of tumor immune gene therapy, it is little with molecular mass, penetration power by force, better keeps antigen affinity and specificity, immunogenicity is low, in the body transformation period short, the antibody molecule that constitutes multiple new function etc. of easily linking to each other with effector molecule is characteristics, and single-chain antibody becomes the important means of immunotherapy of tumors.
The nineties in 20th century, the appearance of humanized's monoclonal antibody (monoclonal antibody) has been started monoclonal antibody and has been treated hemopathic new era.The multiple antigen of ALL cell surface expression, as CD19, CD20, CD22 and CD52 etc., these surface antigens all can become the action target spot of monoclonal antibody.The IDEC-C2B8 that has gone on the market at present can make 93% preceding B-ALL and ripe B-ALL patient obtain alleviation (CR) fully, and survival rate reached 86% in 1 year.Application IDEC-C2B8s such as Thomas add CVAD treatment Burkitt lymphoma, and 89% patient obtains CR, and disease free survival (DFS) rate reached 86% in 1 year, did not have the treatment associated death.In addition, anti-CD19 monoclonal antibody and anti-CD52 monoclonal antibody also have been used for human body.Seibel etc. have contrasted simple chemotherapy and chemotherapy adds anti-CD19 monoclonal antibody to just sending out the curative effect of ALL.The result shows that it only is 43% that simple chemotherapy group 2 all CR lead, and the combined chemotherapy group reaches 93%.Anti-CD22 monoclonal antibody has obvious lethal effect external to MDR-1 (multidrug resistance gene) positive B urkitt lymphoma cell strain and BCP-ALL cell strain EU-1.
In sum, antibody drug brings profound influence to the tumour especially treatment of blood system malignant tumour.Antibody had proved effectively already as independent medicine, and had synergy with other chemotherapy drugs in combination application.Utilize the cell-specific of antibody, antibody as the delivery of cells cytotoxic drug or in conjunction with other proteic carrier, can effectively be killed targeted cells, and normal cell is not had lethal effect.Therefore, if utilize the surface markers of CD19 surface antigen as ALL leukemia cell, make up the CD19 single-chain antibody, as carrier, in conjunction with other protein molecular of biological action is arranged, constitute fusion rotein, fusion rotein is attached to ALL leukemia cell by the CD19 single-chain antibody in the fusion rotein, stimulate the CTL cell-stimulating, can play specific killing leukemia cell's effect.
Summary of the invention
Technical problem to be solved by this invention is, a kind of engineered antibody (ScFv) that is used for target conjugated lymphocyte leukemia cell's anti-CD19 is provided, obtain a kind of new, can with B-ALL leukemia cell's bonded activity expression product.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of engineered antibody that is used for target conjugated lymphocyte leukemia cell's anti-CD19, contain the described anti-CD19 monoclonal antibody HI19a variable region of light chain VL gene nucleotide series of described anti-CD19 monoclonal antibody HI19a variable region of heavy chain VH gene nucleotide series of SEQ ID NO.1 and SEQ ID NO.2.
The expressed aminoacid sequence of described anti-CD19 monoclonal antibody HI19a variable region of heavy chain VH gene comprises and contains the partly or entirely fragment of this sequence C DRs shown in SEQ ID NO.3.
The expressed aminoacid sequence of described anti-CD19 monoclonal antibody HI19a variable region of light chain VL gene comprises and contains the partly or entirely fragment of this sequence C DRs shown in SEQ ID NO.4.
The pMD-18T carrier that comprises above-mentioned cDNA.
Constructed pET28a (+) expression vector that comprises above-mentioned cDNA.
PET28a (+) expression vector that described cDNA is constructed, expressed cDNA aminoacid sequence comprises and contains the partly or entirely fragment of this sequence C DRs.
PET28a (+) expression vector that described cDNA is constructed, described expression vector at the aminoacid sequence of expression in escherichia coli as described in SEQ ID NO.3 and the SEQ ID NO.4.
The described engineered antibody that is used for target conjugated lymphocyte leukemia cell's anti-CD19, the application in the leukemic medicine of preparation treatment.
The present invention is from light, the heavy chain variable region gene of the hybridoma cell strain amplification monoclonal antibody of mouse-anti people CD19 monoclonal antibody, and connect into single-chain antibody (ScFv) gene, in intestinal bacteria, express, with obtain a kind of new, can with B-ALL leukemia cell's bonded activity expression product.
Description of drawings
Fig. 1 is pcr amplification VH, and VL gene fragment electrophorogram (M:Marker, H:VH, L:VL).
Fig. 2 is pcr amplification single-chain antibody (ScFv) gene fragment electrophorogram (M:Marker).
Fig. 3 is anti-CD19 ScFv expression vector pET28a (+) ScFv structural representation.
Fig. 4 is SDS-PAGE (10%) electrophorogram (M:Marker of ScFv expression product; O: do not induce tropina; 1-4: induce the back tropina).
Fig. 5 is the proteic SDS-PAGE electrophorogram of anti-CD19-ScFv behind the purifying.
Fig. 6 is that the Western blot of anti-CD19 ScFv analyzes.
Fig. 7 a suppresses experiment negative control group (AB serum+mouse IgG) for competitive immunization fluorescence.
Fig. 7 b suppresses experiment positive controls (HI19a+PBS) for competitive immunization fluorescence.
Fig. 7 c suppresses experimental group (anti-CD19-ScFv+HI19a) for competitive immunization fluorescence
Positive control group of Fig. 7 d and experimental group FACS overlay chart.
Fig. 8 is that anti-CD19ScFv binding curve and Scatchard analyze
Embodiment
Below in conjunction with the drawings and specific embodiments the engineered antibody that is used for target conjugated lymphocyte leukemia cell's anti-CD19 of the present invention is described in further detail:
HI19a is the hybridoma cell strain that Inst. of Hematology, Chinese Academy of Medical Sciences developed, had the mouse-anti people CD19 monoclonal antibody of independent intellectual property right voluntarily, and the anti-CD19 monoclonal antibody of this cell strain excretory is discerned a 95KDI type and worn membrane glycoprotein.B-ALL leukemia cell almost all expresses the CD19 surface antigen.
Use the RT-PCR method and from secrete anti-CD19 monoclonal antibody hybridoma cell HI19a, cloned heavy chain, variable region of light chain (VH, the VL) gene of antibody, long respectively 366bp of institute's clone gene (SEQID NO.1) and 324bp (SEQ ID NO.2), there is not the codon of termination in the gene, be opening code-reading frame, encode respectively 122 (SEQ ID NO.3) and 108 (SEQ ID NO.4) amino acid.Compare with the GeneBank database, find that our institute's cloned genes fragment and mouse endogenous antibody have higher homology.
With (Gly
4Ser)
3The connection peptides gene is with VH, and VL is spliced into single-chain antibody (ScFv) gene, is cloned into the pET28a expression plasmid, and with PET28a (+)-CD19ScFv plasmid transformation escherichia coli BL21 that makes up, the IPTG inducible protein is expressed, and expressed proteins is positioned at inclusion body.Inclusion body protein carries out the SDS-PAGE electrophoresis through sex change, purifying, renaturation, and coomassie brilliant blue R250 dyeing confirms to obtain the single protein band of molecular weight for about 30Kd behind the purifying.Further carry out Western blot and detect, confirm that the purifying protein band is a CD19ScFv albumen with anti-His-tag antibody.
The combination that detects CD19ScFv with immune competitive assay is active, through single-chain antibody sealing target cell---after CD19 expression male people pre B cell leukemia cell is the Nalm6 cell, add parental antibody (the anti-CD19 monoclonal antibody of HI19a hybridoma excretory) again, cells were tested by flow cytometry demonstration fluorescent mark positive rate reduces to 55.17% from 92.64%, show that but anti-CD19ScFv competitive inhibition HI19a combines with the Nalm6 cell, promptly anti-CD19ScFv can combine with Nalm6 cell surface CD19 antigen-specific.In addition, also use dissociation constant (Kd) method of calculation to measure the combination activity of CD19ScFv, the Kd value is 1.7 * 10
-8Mol/L (R
2=0.99).
The present invention adopts the RT-PCR method to clone single-chain antibody gene from the hybridoma cell strain HI19a of secretion anti human CD 19 monoclonal antibody, and in protein expression vector pET28a, express, obtained to combine active ScFv albumen, be its basic work that should be used as early stage in treatment in the future with the bone-marrow-derived lymphocyte leukemia cell.
Describe specific implementation process of the present invention below in detail:
1. light, the heavy chain variable region gene of anti-CD 19 antibodies are cloned
Use the RT-PCR method from secreting light, the heavy chain variable region gene of anti-CD19 monoclonal antibody hybridoma cell HI19a amplification anti-CD 19 antibodies:
(1) RNA extraction: adopt the Trizol single stage method, 1) gets hybridoma about 10
6, add 1mlTrizol, the piping and druming mixing, room temperature left standstill 5 minutes.2) add the 0.2ml chloroform, thermal agitation 15 seconds, room temperature left standstill 2-3 minute.3) 12000rpm, 4 ℃, centrifugal 15 minutes.4) get supernatant, add 0.5ml Virahol room temperature and left standstill 15 minutes.5) 12000rpm, 4 ℃, centrifugal 15 minutes.6) abandon supernatant, the ethanol that adds 1ml 75% is washed 7500rpm, 4 ℃, centrifugal 5 minutes.7) abandon supernatant, precipitation is dried, and adds the water dissolution RNA that 30 μ l DEPC handle.
(2) reverse transcription is cDNA (40 μ l): get 2.5mM dNTP 4 μ l, and 5 * first strandbuffer, 8 μ l, DTT 4 μ l, oligodT 2 μ l, water 16.6 μ l add the about 2g of RNA behind the mixing, 65 ℃ of water-baths 5 minutes, quick ice bath 2-3 minute.Add 50u/ μ l RNasin 0.4 μ l, 37 ℃ of water-bath＞1 hour behind Superscript II (200u/ μ l) the 1 μ l mixing.Take out back 70 ℃ of water-baths 10 minutes.-20 ℃ of preservations.
(3) the weight chain variable region gene of pcr amplification anti-CD 19 antibodies
Chain variable region gene pcr amplification reaction system (50 μ l): design quoting general degenerated primer, upstream primer 5 '-GAC ATT CAG CTG ACC CAG WCT SMH-3 '; Downstream primer 5 '-CCGTTA GAT CTC CAR BTT KGT SCS-3 '.With cDNA is template, high-fidelity pyrobest polymeric enzymatic amplification.The PCR cycling program is 94 ℃ of 5min; 94 ℃ of 50s, 55 ℃ of 1min, 72 ℃ of 1min; Last 72 ℃ are extended 10min, totally 33 circulations.Reaction finishes to carry out the extracting of phenol chloroform immediately after 72 ℃ of back adding 1u general T aq enzymes (available from the precious biotech firm in Dalian) extend 10min.
Heavy chain variable region gene pcr amplification reaction system (50 μ l): upstream primer 5 '-CAG GTSMAR CTG CAG SAG TCW GG-3 '; Downstream primer 5 '-TGA GGA GAC GGT GAC CGT GGTCCC TTG GCC CC-3 '.With cDNA is template, high-fidelity pyrobest polymeric enzymatic amplification.The PCR cycling program is 94 ℃ of 5min; 94 ℃, 30s, 55 ℃, 1min, 72 ℃, 1min; Last 72 ℃ are extended 10min, totally 33 circulations.Reaction finishes to carry out the extracting of phenol chloroform immediately after 72 ℃ of back adding 1u general T aq enzymes (available from the precious biotech firm in Dalian) extend 10min.PCR the results are shown in Figure 1.
(4) structure of sequencing vector: the pMD-18T carrier is available from the precious biotech firm in Dalian.Weight chain variable region gene PCR product is reclaimed, be connected with the pMD-18T carrier, ligation is undertaken by the test kit requirement.Get and connect product 5 μ l, conversion is with intestinal bacteria (DH5a) competence of calcium chloride method preparation, select positive colony with indigo plant/hickie method, each picking of weight chain 10 clones send order-checking, order-checking confirms gently, each cloned genes sequence of heavy chain is in full accord, be respectively 324bp and 366bp, and this gene order meets the characteristics of the some conservative framework amino acid that antibody had in the albumen database fully, this sequence is the antibody gene sequence.Difference called after pMD-18T19-VH and pMD-18T19-VL.
2. the structure of single-chain antibody gene expression vector pET28a ScFv
Designed and synthesized according to gene order light, variable region of heavy chain and to be used for VH, the primer of VL gene amplification and splicing.
Primer 1:CCG GAA TTC GAC ATT GTG CTC ACC CAG TCT CCA
Primer 2: GGA GCC GCC GCC GCC AGA ACC ACC ACC ACC CCG TTT TAT TTC CAG
CTT?GGT?CCC
Primer 3:GGC GGC GGC GGC TCC GGT GGT GGT GGT TCT CAG CCG GCC ATG CGC
CAG?GTC?CAG?CTG?CAG?CAG
Primer 4:CCC AAG CTT GTG AGG AGA CTG TGA GAG TGG TGC C
5 ' end at primer 1 adds the EcoRI restriction enzyme site, and 3 ' end of primer 4 adds the HindIII restriction enzyme site.From constructed pMD-18T19-VH and pMD-18T19-VL carrier, use pcr amplification VH, the VL fragment after the recovery, by the anti-CD19ScFv gene fragment of pcr amplification, is seen Fig. 2 through splicing overlap extension.PCR product behind the purifying after EcoRI+HindIII handles again with handle through EcoRI+HindIII after pET28a (+) carrier that carries (His) 6 labels be connected (16 ℃ of connections are spent the night).Form the expression vector of the anti-CD19-ScFv of pET28a, see Fig. 3.Order-checking shows that anti-CD19ScFv gene fragment is 750bp, infers that by aminoacid sequence ScFv is about the albumen of 27KD.The correct clone that checks order is used for protein expression.
3. the expression of anti-CD19ScFv antibody fragment, purifying and renaturation
(1) uses PET28a (+)-CD19ScFv plasmid transformation escherichia coli BL21 that makes up, and go up the screening transformant at the LB flat board (1%agar) that contains 50 μ g/ml kantlex.Picking is after identifying correct mono-clonal activation, and 37 ℃ of concussions are cultured to OD
600=0.7-0.9, adding IPTG is 0.1mmol/L to final concentration, and 37 ℃ of abduction deliverings 5 hours are got 100 μ l bacterium liquid, and centrifugal back is resuspended with the last sample buffer of 100 μ l, 1 * SDS, and 100 ℃ are boiled 5min.Get sample on the 20 μ l, 10%SDS-PAGE electrophoresis detection expressing protein, coomassie brilliant blue staining.Experimental results show that recombinant plasmid pET28a (+) ScFv has induced expression of recombinant proteins through IPTG in intestinal bacteria, the 750bp fragment expression goes out the band (His) of about 29Kd
6Recombinant protein, see Fig. 4.
(2) anti-CD19 single-chain antibody (ScFv) purifying: the bacterial sediment of expressing is resuspended in precooling 50mmol/L Tris-HCl, 100mmol/L NaCL, 1mmol/L EDTA, the pH7.0 dissolving of 1/30 volume of culture.Ultrasonication bacterium behind the multigelation 3 times, 4 ℃, centrifugal 30 minutes of 30000g.The 3M urea and the 50mmol/L Tris-HCl (pH7.0) that add 1/30 volume of culture in the precipitation dissolved back 30000g centrifugal 30 minutes.Collect inclusion body for 4 ℃.With the 6M Guanidinium hydrochloride and the 0.1MTris-HCl of 1/40 volume of culture, pH7.0, in 4 ℃ of shaken over night dissolving inclusion bodys.4 ℃, centrifugal 30 minutes place to go precipitations of 30000g, supernatant is used for purifying.The nickel post is purchased the company in Novagen, Startbuffer (6M Guanidinium hydrochloride with 3 times of column volumes, 0.1M Tris-HCl, pH7.0) go up sample behind the balance nickel post, 20 times of column volumes, pH are that 7.0 6M urea, 50mmol/L Tris-HCl and 50mmol/L imidazoles are washed post, contain 250mmol/L imidazoles, 6M urea and the ScFv of 50mmol/L Tris-HCl elution of bound on the nickel post with 4 times of column volumes respectively.
(3) SDS-PAGE electrophoresis and Western blot identify: purified product is through the 10%SDS-PAGE electrophoresis, and coomassie brilliant blue R250 dyeing confirms to obtain the single band of molecular weight for about 30Kd behind the purifying.Further with Western blot checking, concrete grammar reference molecule clone is (His) on anti-and the anti-CD19-ScFv fusion rotein with anti-His-tag antibody (IgG) wherein
6Western blot reaction is carried out in the label combination.The result confirms that the purifying band is the target protein expression product, sees Fig. 5, Fig. 6.
(4) renaturation of CD19ScFv: with the sample of wash-out the TEA damping fluid (0.4M arginine-HCL, 0.1MTris-HCl, 2mmol/L EDTA, the pH7.0) renaturation of slowly dialysing, 4 ℃ are spent the night.
4.BCA method is measured proteinic concentration:
(1) preparation protein determination reaction solution is got A liquid (BCA disodium, 1%; Na
2CO
3H
2O, 2%; Sodium tartrate, 0.16%; NaOH, 0.4%; NaHCO
3, 0.95%; PH11.25) 1ml adds B liquid (CuSO
45H
2O, 4%) 20 μ l mixings.
(2) answer adding 50 μ l sample protein solution in the liquid, and protein standard liquid (bovine serum albumin of different concns) mixing, reaction is 30 minutes in 37 ℃ of water-baths.
(3) reaction solution of getting each pipe carries out light absorption analysis in the 562nm wavelength, and the drawing standard protein concentration according to the OD-protein concentration relation of standard protein, calculates the concentration of protein sample in the linear graph of absorbance value (OD).Anti-CD19ScFv protein concentration is 85.71 μ g/ml. after the renaturation
5. anti-CD19 ScFv antibody activity is measured---competition experiments
Get CD19 and express male Nalm6 clone, make 1 * 10
6Cell suspension, application of sample is in 40 porocyte culture plates, 5 * 10
5Cells/well.Negative control group adds 100 μ l people AB serum, hatches 1h for 4 ℃, 3000rpm, and 4 ℃ of centrifugal 8min abandon supernatant liquor, and PBS washes cell 2 times, adds mouse source property IgG 20 μ l (1mg/ml); Positive controls adds anti-CD19 monoclonal antibody HI19a working fluid 20 μ l in the sealing of people AB serum after 1 hour; Test group adds the anti-CD19ScFv of 100 μ l earlier after adding the sealing of people AB serum, hatch 1h for 4 ℃, 3000rpm, 4 ℃ of centrifugal 8min, abandon supernatant liquor, PBS washes cell 2 times, adds anti-CD19 monoclonal antibody HI19a 20 μ l (1mg/ml) again, hatches 1h for 4 ℃, 3000rpm, 4 ℃ of centrifugal 8min abandon supernatant liquor, and PBS washes cell 2 times.Three groups of cells are resuspended in respectively among the PBS, add 20 μ l sheep anti-mouse igg-FITC two and resist, and hatch 45min for 4 ℃, the unconjugated fluorescence antibody of PBS flush away, and flow cytometer detects monoclonal antibody HI19a and Nalm6 clone bonded positive rate.
Combine positive rate with monoclonal antibody HI19a be 92.64% to the Nalm6 cell in the positive controls, Nalm6 cell in the experimental group is after anti-CD19 ScFv competition combination, with HI19a bonded positive rate be 55.17%, show that but anti-CD19 ScFv competitive inhibition HI19a combines with Nalm6 clone, promptly anti-CD19 ScFv can combine with Nalm6 clone surface C D19 antigen-specific, sees Fig. 7 a, 7b, 7c, 7d.
6. anti-CD19ScFv antibody activity is measured---and dissociation constant is measured
(1) add 100 μ l Nalm6 cell pyrolysis liquids in 96 hole enzyme plates, 37 ℃ of bags were by 2 hours;
(2) get rid of coating buffer, wash 3 times, add and get rid of deblocking liquid after confining liquid (PBS contains 3% bovine serum albumin, 10% sheep blood serum) seals 2h, PBS washing 3 times with PBS (containing 0.05%Tween-20).
(3) same form two holes add the CD19ScFv of 1: 4 doubling dilution of 100 μ l, and the bovine serum albumin solution with 3% compares, and hatch 2h for 37 ℃.
(4) remove behind the reaction solution with PBS washing three times, add anti-His-tag monoclonal antibody (dilution in 1: 2000,0.1 μ g/ml), hatch 2h for 37 ℃.
(5) get rid of first antibody solution, the PBS back of giving a baby a bath on the third day after its birth time adds horseradish peroxidase sheep anti-mouse igg (dilution in 1: 1000), hatches 2h for 37 ℃.
(6) get rid of dereaction liquid, after PBS gives a baby a bath on the third day after its birth time, add colour developing liquid (OPD, H
2O2) react about 10 minutes with 2N H
2SO
4Termination reaction, (Vamed Engineering Austria) measures each hole OD with microplate reader
492nmLight absorption value.The absorbance value of each ScFv concentration is got average.
(7) dissociation constant is calculated: with data substitution Δ A=Δ A
MAX* L/ (Kd+L) is Δ A=-Kd * Δ A/L-Δ A
MAX(Δ A is OD value poor of experimental group and control group, and L is the concentration of the CD19ScFv of the reorganization that adds.Regression analysis, computational solution is from constant K d value, Kd=1.7 * 10
-8Mol/L (R
2=0.99), sees Fig. 8.
SEQUENCE LISTING (sequence table)
＜110〉Inst. of Hematology, Chinese Academy of Medical Sciences
＜120〉be used for the engineered antibody and uses thereof of target conjugated lymphocyte leukemia cell's anti-CD19
<160>4
<170>PatentIn?version?3.1
<210>1
<211>366
<212>DNA
＜213〉Mus musculus (mouse)
<220>
<221>V_region
<222>(1)..(366)
<400>1
caggtccagc?tgcagcagtc?tggggctgag?ctggtgaggc?ctgggtcctc?agtgaagatt 60
tcctgcaagg?cttctggcta?tgcattcagt?agctactgga?tgaactgggt?gaagcagagg 120
cctggacagg?gtcttgagtg?gattggacag?atttatcctg?gagatggtga?tactaactac 180
aatggaaagt?tcaagggtca?agccacactg?actgcagaca?aatcctccag?cacagcctac 240
atgcagctca?gcggcctgac?atctgaggac?tctgcggtct?atttctgtgc?aagaaagacc 300
attagttcgg?tagtagattt?ctactttgac?tactggggcc?aaggcaccac?tctcacagtc 360
tcctca 366
<210>2
<211>324
<212>DNA
＜213〉Mus musculus (mouse)
<220>
<221>V_region
<222>(1)..(324)
<400>1
gacattgtgc?tcacccagtc?tccaaaattc?atgtccacat?cagtaggaga?cagggtcagc 60
tcacctgcaa?ggccagtcag?aatgtgggta?ctaatgtagc?ctggtatcaa?cagaaaccag 120
gacaatctcc?taaaccactg?atttactcgg?caacctaccg?gaacagtgga?gtccctgatc 180
gcttcacagg?cagtggatct?gggacagatt?tcactctcac?catcactaac?gtgcagtcta 240
aagacttggc?agactatttc?tgtcaacaat?ataacaggta?tccgtacacg?tccggagggg 300
ggaccaagct?ggaaataaaa?cggg 324
<210>3
<211>122
<212>PRT
＜213〉Mus musculus (mouse)
<220>
<221>PEPTIDE
<222>(1)..(122
<400>3
Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Glu?Leu?Val?Arg?Pro?Gly?Ser
1 5 10 15
Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Ala?Phe?Ser?Ser?Tyr
20 25 30
Trp?Met?Asn?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile
35 40 45
Gly?Gln?Ile?Tyr?Pro?Gly?Asp?Gly?Asp?Thr?Asn?Tyr?Asn?Gly?Lys?Phe
50 55 60
Lys?Gly?Gln?Ala?Thr?Leu?Thr?Ala?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr
65 70 75 80
Met?Gln?Leu?Ser?Gly?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Phe?Cys
85 90 95
Ala?Arg?Lys?Thr?Ile?Ser?Ser?Val?Val?Asp?Phe?Tyr?Phe?Asp?Tyr?Trp
100 105 110
Gly?Gln?Gly?Thr?Thr?Leu?Thr?Val?Ser?Ser
115 120
<210>4
<211>108
<212>PRT
＜213〉Mus musculus (mouse)
<220>
<221>PEPTIDE
<222>(1)..(108)
<400>4
Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Lys?Phe?Met?Ser?Thr?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Ser?Val?Thr?Cys?Lys?Ala?Ser?Gln?Asn?Val?Gly?Thr?Asn
20 25 30
Val?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Lys?Pro?Leu?Ile
35 40 45
Tyr?Ser?Ala?Thr?Tyr?Arg?Asn?Ser?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Thr?Asn?Val?Gln?Ser
65 70 75 80
Lys?Asp?Leu?Ala?Asp?Tyr?Phe?Cys?Gln?Gln?Tyr?Asn?Arg?Tyr?Pro?Tyr
85 90 95
Thr?Ser?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
100 105
Claims (8)
1, a kind of engineered antibody that is used for target conjugated lymphocyte leukemia cell's anti-CD19 is characterized in that containing the described anti-CD19 monoclonal antibody HI19a variable region of light chain VL gene nucleotide series of described anti-CD19 monoclonal antibody HI19a variable region of heavy chain VH gene nucleotide series of SEQ ID NO.1 and SEQ ID NO.2.
2, the engineered antibody that is used for target conjugated lymphocyte leukemia cell's anti-CD19 according to claim 1, it is characterized in that the expressed aminoacid sequence of described anti-CD19 monoclonal antibody HI19a variable region of heavy chain VH gene shown in SEQ ID NO.3, comprise and contain the partly or entirely fragment of this sequence C DRs.
3, the engineered antibody that is used for the anti-CD19 of target conjugated lymphocyte according to claim 1, it is characterized in that the expressed aminoacid sequence of described anti-CD19 monoclonal antibody HI19a variable region of light chain VL gene shown in SEQ ID NO.4, comprise and contain the partly or entirely fragment of this sequence C DRs.
4, the pMD-18T carrier that comprises the cDNA of claim 1.
5, constructed pET28a (+) expression vector that comprises the cDNA of claim 1.
6, according to constructed pET28a (+) expression vector of the described cDNA of claim 5, it is characterized in that expressed cDNA aminoacid sequence, comprise and contain the partly or entirely fragment of this sequence C DRs.
7, according to constructed pET28a (+) expression vector of the described cDNA of claim 5, it is characterized in that described expression vector at the aminoacid sequence of expression in escherichia coli as described in SEQ ID NO.3 and the SEQID NO.4.
8, claim 1,2,3,4,5, the 6 or 7 described engineered antibodies that are used for target conjugated lymphocyte leukemia cell's anti-CD19, the application in the leukemic medicine of preparation treatment.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410072713 CN1775808A (en) | 2004-11-15 | 2004-11-15 | Anti CD19 engineered antibody for target conjugated lymphocyte, leuco cyte and its use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410072713 CN1775808A (en) | 2004-11-15 | 2004-11-15 | Anti CD19 engineered antibody for target conjugated lymphocyte, leuco cyte and its use |
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| Publication Number | Publication Date |
|---|---|
| CN1775808A true CN1775808A (en) | 2006-05-24 |
Family
ID=36765543
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410072713 Pending CN1775808A (en) | 2004-11-15 | 2004-11-15 | Anti CD19 engineered antibody for target conjugated lymphocyte, leuco cyte and its use |
Country Status (1)
| Country | Link |
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| CN (1) | CN1775808A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100412193C (en) * | 2006-07-04 | 2008-08-20 | 浙江大学 | Anti human CD19 mouse immune globulin variable zone gene and uses |
| CN100543035C (en) * | 2006-09-14 | 2009-09-23 | 中国医学科学院血液学研究所 | Be used for the treatment of bone-marrow-derived lymphocyte leukemia, lymphadenomatous B7.1-CD19scFv fusion gene engineering albumen and uses thereof |
| RU2476441C2 (en) * | 2007-10-19 | 2013-02-27 | Сиэтл Дженетикс, Инк. | Cd19-binding agents and use thereof |
| CN103694349A (en) * | 2006-09-08 | 2014-04-02 | 米迪缪尼有限公司 | Humanized anti-cd19 antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
| CN105111312A (en) * | 2010-07-19 | 2015-12-02 | 国际药物发展生物技术公司 | Anti-CD19 antibody having ADCC and CDC functions and improved glycosylation profile |
| CN110331134A (en) * | 2019-07-19 | 2019-10-15 | 上海交通大学医学院附属瑞金医院 | A kind of universal cell therapy product and its preparation method and application for expressing antigen recognition region |
| 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 |
| JP2023502190A (en) * | 2019-12-17 | 2023-01-23 | 合源生物科技(天津)有限公司 | CD19-targeted chimeric antigen receptor and uses thereof |
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2004
- 2004-11-15 CN CN 200410072713 patent/CN1775808A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100412193C (en) * | 2006-07-04 | 2008-08-20 | 浙江大学 | Anti human CD19 mouse immune globulin variable zone gene and uses |
| CN103694349A (en) * | 2006-09-08 | 2014-04-02 | 米迪缪尼有限公司 | Humanized anti-cd19 antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
| CN100543035C (en) * | 2006-09-14 | 2009-09-23 | 中国医学科学院血液学研究所 | Be used for the treatment of bone-marrow-derived lymphocyte leukemia, lymphadenomatous B7.1-CD19scFv fusion gene engineering albumen and uses thereof |
| RU2476441C2 (en) * | 2007-10-19 | 2013-02-27 | Сиэтл Дженетикс, Инк. | Cd19-binding agents and use thereof |
| CN105111312A (en) * | 2010-07-19 | 2015-12-02 | 国际药物发展生物技术公司 | Anti-CD19 antibody having ADCC and CDC functions and improved glycosylation profile |
| CN105111312B (en) * | 2010-07-19 | 2018-12-14 | 国际药物发展生物技术公司 | Has the function of the anti-CD19 antibody of the glycosylation feature of ADCC and CDC and improvement |
| CN110331134A (en) * | 2019-07-19 | 2019-10-15 | 上海交通大学医学院附属瑞金医院 | A kind of universal cell therapy product and its preparation method and application for expressing antigen recognition region |
| WO2021012857A1 (en) * | 2019-07-19 | 2021-01-28 | 上海交通大学医学院附属瑞金医院 | Universal cell therapy product for expressed antigen recognition region, and preparation method therefor and application thereof |
| CN110331134B (en) * | 2019-07-19 | 2023-09-08 | 上海交通大学医学院附属瑞金医院 | Universal cell therapy product expressing antigen recognition region and preparation method and application thereof |
| JP2023502190A (en) * | 2019-12-17 | 2023-01-23 | 合源生物科技(天津)有限公司 | CD19-targeted chimeric antigen receptor and uses thereof |
| JP2023502191A (en) * | 2019-12-17 | 2023-01-23 | 合源生物科技(天津)有限公司 | Combination of plasmids and their application in the preparation of engineered immune cells |
| JP7430202B2 (en) | 2019-12-17 | 2024-02-09 | 合源生物科技(天津)有限公司 | Combination of plasmids and their application in the preparation of modified immune cells |
| JP7439128B2 (en) | 2019-12-17 | 2024-02-27 | 合源生物科技(天津)有限公司 | CD19 targeting chimeric antigen receptor and its use |
| 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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