CN1552735A - Engineering antibody against CD44 for inducing leukemia cell differentation and necrosis - Google Patents
Engineering antibody against CD44 for inducing leukemia cell differentation and necrosis Download PDFInfo
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Abstract
An engineering antibody CD44 for inducing the differentiation and wither of leukemia cells, the gene in the heavy chain and light chain variable region of monoclonal antibody HI44a of CD44, the polypeptide coded by said gene, the carrier containing said gene, and the application of said gene and polypeptide in preparing medicines for diagnosing and treating leukemia and disclosed.
Description
Technical field
The present invention relates to the leukemic antibody of a kind of treatment, in particular for the engineered antibody of the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis.
Background technology
Acute leukemia is one of main several diseases that threaten human life's health, and in the U.S., just have 2.7 people to suffer from AML every year among 100,000 adults, and reached 14.1 at the age above this numeral among 65 years old the adult.It is the malignant disease that a class originates from hematopoiesis (or lymph) stem cell.Because stem cell is impaired, the leukemia cell loses the ability of further differentiation and maturation, perhaps breeds and the differentiation capability imbalance, and is stuck in cytocerastic different steps, be embodied in cell infinite multiplication in vivo, and its differentiation and maturation and apoptosis is obstructed.Therefore leukemic treatment can be set about from three aspects: cell death inducing, inducing cell differentiation and stem cell transplantation reconstitute hematopoiesis function.
From the seventies in 20th century, researcher has begun the research of leukemia cell's induction-differential therapy.Since Collins etc. at first reports the induction of differentiation of dimethyl sulfoxide (DMSO) (DMSO) to HL-60 HL-60, obtained very big progress so far about the research of inducing differentiation agent; Wherein some drugs has been used for clinical, and obtain than satisfied curative effect, become the leukemic ordinary method of treatment acute morning of children's graininess as all-trans-retinoic acid (ATRA), but traditional ATRA is an oral preparations, is difficult on molecular level inducer remission or keeps secular hematologic response.Also can cause untoward reactions such as vitamin A acid syndrome; Except that acute promyelocytic leukemia, all-trans-retinoic acid is not all succeeded to AML and other treatment of solid tumors.Therefore leukemic induction-differential therapy is still an important directions of present research.Prepare a kind of polytype leukemia medicament of clinical treatment that can be applied to and also set up the clinical meaning that relevant technology of preparing then has particularly important.
Known CD44 is the I type transmembrane glycoprotein that extensively has the single-gene coding, relevant with cell adhesion, can combine with various kinds of cell epimatrix composition such as hyaluronic acid, fine Saliva Orthana, collagen protein, Laminin ELISA etc., this will produce certain effect to the cell of expressing it.And the raising of CD44 expression level is with the tumour cell growth in vivo and shift relevant.Discover that CD44 all has expression at the leukemia initiating cell of all acute leukemias (AML) hypotype: (1) CD44 relevant with normal medullary system differentiation (the anti-CD44 antibody of function interdiction can significantly suppress the generation of medullary cell in long-term marrow is cultivated); (2) CD44 can be used as signaling molecule active cells function when combining with special antibody or acceptor hyaluronic acid; (3) the CD44 molecule is positioned at cell surface, and is more approaching than being easier to.These factors become CD44 to induce a possible target spot of acute leukemia initiating cell differentiation.Existing bibliographical information is used anti-CD44 antibody A 3D8, and H90 or hyaluronic acid can reverse the medullary system differentiation retardance of each hypotype of AML.
Because leukemia treating mainly adopts control propagation, apoptosis-induced chemotherapeutics, comprises alkylating agent, antimetabolite etc.These medicines also have major injury to normal hematopoietic cell when killing the leukemia cell, so there is serious toxic side effect in body.In addition, leukemia cell regular meeting produces resistance to chemotherapeutics, and recurrence rate is higher after the chemotherapy simultaneously, has a strong impact on the validity of clinical chemotherapy pharmacological agent.
Summary of the invention
Technical problem to be solved by this invention provides a kind of engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis, and the monoclonal antibody HI44a that provides can effectively induce acute myeloid leukaemia differentiation and apoptosis; The anti-CD44 monoclonal antibody chain variable region gene and heavy chain variable region gene and the expression product thereof that provide are expressed the anti-CD44 ScFv segment that produces after both recombinate, and can reduce the immunogenicity of the anti-CD44 antibody of mouse source property.
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 the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis, contain the described anti-CD44 monoclonal antibody HI44a variable region of light chain VL gene nucleotide series of described anti-CD44 monoclonal antibody HI44a variable region of heavy chain VH gene nucleotide series of SEQ ID NO.1 and SEQ ID NO.3.
The expressed aminoacid sequence of anti-CD44 monoclonal antibody HI44a variable region of heavy chain VH gene comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs shown in SEQ ID NO.2.
The expressed aminoacid sequence of anti-CD44 monoclonal antibody HI44a variable region of light chain VL gene comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs shown in SEQ ID NO.4.
The PUC18 carrier that comprises above-mentioned cDNA.
Constructed PET22b (+) expression vector that comprises above-mentioned cDNA.
The cDNA aminoacid sequence that described expression vector is expressed comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs.
The described engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis, the application in the leukemic medicine of preparation treatment.
Anti-CD44 antibody HI44a is all a kind of anti-CD44 mouse monoclonal antibodies of consonance gene stem cell company of Inst. of Hematology, Chinese Academy of Medical Sciences, belong to the IgG2a hypotype, this antibody capable effectively reverses the differentiation retardance of polytype acute myeloid leukaemia initiating cell, impel leukemia cell's function maturation, and the energy induced apoptosis in leukemia cell lines, for leukemic treatment provides a kind of new medicine.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 genetic engineering antibody preferably resolves this problem, it had both kept the special avidity of mouse source monoclonal antibody, greatly reduce the heterology of mouse monoclonal antibody again, thereby have broad application prospects.
We use the RT-PCR technology, the weight chain variable region gene of from hybridoma, being cloned into anti-CD44 antibody of success, and will resist the weight chain variable region gene of CD44 antibody to be cloned into prokaryotic expression carrier, make up the single-chain antibody of anti-CD44 antibody, reduce the immunogenicity of the anti-CD44 antibody of mouse source property, and in intestinal bacteria, carry out efficient secretion expression, thus improve its output, reduce production costs.The advantage of single-chain antibody is: more complete anti-molecular weight is little, and penetrativity is strong, and construction schedule is short, and antigenicity is low, is easy to the genetically engineered operation, lays the foundation for further studying other engineered antibody.Can be easy to mass production at expression in escherichia coli, production cost is low.
Description of drawings
Fig. 1 is the influence of HI44a to the leukemia cellular form.
Fig. 2 is the influence of HI44a to each hypotype leukemia cell NBT reducing power.
Fig. 3 is the expression that HI44a reduces proto-oncogene c-myc, and strengthens the expression of M-CSF.
Fig. 4 is pcr amplification VH, VL gene fragment electrophorogram.A：Marker?B：VL?C：VH。
Fig. 5 is pcr amplification single-chain antibody (ScFv) gene fragment collection of illustrative plates.A：Marker?B：ScFv。
Fig. 6 is anti-CD44 ScFv expression vector PET22b (+) ScFv structural representation.
Fig. 7 is SDS-PAGE (12%) collection of illustrative plates of ScFv expression product.A:Marker; B: do not induce tropina; C: induce back tropina D: ScFv albumen behind the purifying.
Fig. 8 is that the Western blot of anti-CD44 ScFv analyzes.
Fig. 9 is that competitive immunization fluorescence suppresses experiment.(1) the anti-CD44 ScFv of negative control: PBS+; (2): positive control: HI44a+PBS; (3): the anti-CD44ScFv of HI44a+.
Embodiment
Below in conjunction with the drawings and specific embodiments the engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis of the present invention is described in further detail:
The engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis contains the described anti-CD44 monoclonal antibody HI44a variable region of light chain VL gene nucleotide series of described anti-CD44 monoclonal antibody HI44a variable region of heavy chain VH gene nucleotide series of SEQ IDNO.1 and SEQID NO.3.
The expressed aminoacid sequence of anti-CD44 monoclonal antibody HI44a variable region of heavy chain VH gene comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs shown in SEQID NO.2.
The expressed aminoacid sequence of anti-CD44 monoclonal antibody HI44a variable region of light chain VL gene comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs shown in SEQ ID NO.4.
The PUC18 carrier that comprises above-mentioned cDNA.
Constructed PET22b (+) expression vector that comprises above-mentioned cDNA.
The cDNA aminoacid sequence that described expression vector is expressed comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs.
The described engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis, the application in the leukemic medicine of preparation treatment.
Embodiment 1:HI44a is the inducing leukemia cytodifferentiation effectively
Get and just control acute myeloid leukemia people marrow 5ml, the separation and Extraction mononuclearcell is with 2 * 10
5/ ml is inoculated in 24 orifice plates and induces differentiation agent HI44a co-cultivation, and culture condition is RPMI 1640 (contains volume fraction be 10% foetal calf serum), 37 ℃, 5%CO
2, saturated humidity cultivated 4-6 days.After this, collecting cell detects the variation of differentiation index.
(1) cellular form changes.After leukemia cell and HI44a cultivate altogether, can obviously show cellular form and transform to ripe direction, cell shows pyknosis, and karyoplasmic ratio reduces, features such as kernel minimizing.Wherein obvious with the leukemia cell of M3 and M5 hypotype, see Fig. 1.
(2) mensuration of NBT reducing power.HI44a can significantly increase each hypotype leukemia cell's NBT reducing power, wherein the M3 hypotype changes the most remarkable, the NBT positive cell is increased to 55% from 10% of control group, and at M2, M4 and M5 hypotype, the NBT positive rate is respectively 3 1% (contrasts 9%), 25% (contrast 12%), 32% (contrast 11%).The P value all＜0.01 is seen Fig. 2.
(3) variation of HI44a effect back cell surface specificity differentiation antigen.We have detected 31 examples respectively, 24 examples, and 21 routine patient leukemia cell's surface C D11b, CD14, the expression of CD15, after the discovery HI44a effect, leukemia cell's surface C D11b, CD14, CD15 express and all obviously increase, and see Table 1,2,3.
The influence that table 1 HI44a expresses each hypotype leukemia cell differentiation antigen CD11b
The CD11b positive rate
FAB hypotype example number control group HI44a group P
AML2 8 10.42 19.64 0.015
AML3 7 6.60 16.79 0.059
AML4 8 9.25 16.87 0.026
AML5 8 12.40 23.31 0.006
Add up to 31 9.74 19.22 0.000
The influence that table 2 HI44a expresses each hypotype leukemia cell differentiation antigen CD14
The CD14 positive rate
FAB hypotype example number control group HI44a group P
AML2 7 25.43 37.20 0.037
AML3 5 18.15 29.85 0.146
AML4 6 32.52 51.96 0.057
AML5 6 32.28 42.18 0.022
Add up to 24 27.40 40.60 0.000
The influence that table 3 HI44a expresses each hypotype leukemia cell differentiation antigen CD15
The CD15 positive rate
FAB hypotype example number control group HI44a group P
AML2 6 46.47 54.77 0.018
AML3 6 62.92 68.58 0.073
AML4 4 61.00 72.04 0.162
AML5 5 61.00 75.00 0.007
Add up to 21 57.40 66.82 0.000
With isolating leukemia cell and HI44a co-cultivation, culture condition is RPMI 1640 (contains volume fraction be 10% foetal calf serum), 37 ℃, 5%CO
2, saturated humidity cultivated collecting cell, PBS washing, Annexin-V test kit (Annexin-V-FLOUS staining Kit 48-72 hour; ROCHE) detect early apoptosis of cells.After testing, after HI44a acted on 48-72 hour to 10 routine patients' leukemia cell, leukemia cell's early apoptosis rate obviously increased, and sees Table 4.
Table 4 HI44a is to the influence of each hypotype leukemia cell early apoptosis
Annexin-v positive rate (%)
FAB hypotype example number control group HI44a group P
1 8.18 13.13
AML2，n＝2 2 2.45 40.11
1 7.39 9.98
AML3，n＝3 2 2.14 26.84
3 59.54 75.77
1 4.19 6.89
AML4，n＝2 2 37.06 65.67
1 63.93 77.99
AML5，n＝3 2 69.77 83.19
3 7.44 12.25
Add up to 10 26.21 41.18 0.003
RT-PCR detects c-myc, and G-CSF and M-CSF express and change.Leukemia cell and HI44a co-cultivation be after 24 hours, collecting cell.Trizol (GIBCO) single stage method is extracted cell total rna, is primer with Oligod (T), is cDNA through M-MLV reversed transcriptive enzyme reverse transcription.House-keeping gene β-actin is as confidential reference items.The primer of pcr amplification is: c-myc, 5 '-CCC GAC GCG GGG AGG CTA TT-3 ' and 5 '-TCT CCA GCT GGT CGG CCG TG-3 '; G-CSF, 5 '-TTG GAC ACACTG CAG CTG GAC GTC GCC GAC TT-3 ' and 5 '-ATT GCA GAG CCA GGGCTG GGG AGC AGT CAT AGT-3 '; M-CSF, 5 '-CAG TCA GAT GGA GACCTC GT-3 ' and 5 '-GGT GTC TCA TAG AAA GTT CG-3 '.(primer is synthetic by Shanghai Sangon company).The PCR reaction conditions is: c-myc, and 94 ℃ of sex change 45s, 60 ℃ of annealing 1min, 72 ℃ are extended 1.5min, totally 32 circulations.M-CSF, 94 ℃ of sex change 45s, 61 ℃ of annealing 45s, 72 ℃ are extended 1.5min, totally 32 circulations.(M2,3 among the 8 routine patient leukemia cells after the cultivation that is detected; M3,1; M4,1; M5,3), do not detect the expression of G-CSF, and M-CSF expresses in two routine M2 and two routine patients' M5 leukemia cell and increases.In all 8 routine patients that detect, c-myc expresses all and reduces, and sees Fig. 3.
Embodiment 4: the weight chain variable region gene clone of anti-CD44 antibody
Use the RT-PCR method and from anti-CD44 antibody hybridoma cell, clone the weight chain variable region gene of anti-CD44 antibody:
(1) RNA extracts: adopt the Trizol single stage method, 1) to get hybridoma about 106, adds 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 1ml75% is washed 7500rpm, 4 ℃, centrifugal 5 minutes.7) abandon supernatant, precipitation is dried, and adds 30 μ LDEPC water dissolution.
(2) reverse transcription is CDNA (40 μ l): get 2.5mMdNTP4 μ l, and 5 * first strandbuffer, 8 μ l, DTT4 μ l, OligodT2 μ l, water 16.6 μ l add the about 2 μ g of RNA behind the mixing, 65 ℃ of water-baths 5 minutes, ice bath 2-3 minute fast.Add 50u/ μ lRNasin0.4 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 the anti-CD44 antibody of pcr amplification
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 '-CCG TTA GAT CTC CAR BTT KGT SCS-3 '.With CDNA is template, high-fidelity PfuDNA polymeric enzymatic amplification.The PCR cycling program is 94 ℃ of 5min; 94 ℃ of 45S, 64 ℃ of 1min, 72 ℃ of 1min; Last 72 ℃ are extended 10min, totally 35 circulations.
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 PfuDNA polymeric enzymatic amplification.The PCR cycling program is 94 ℃ of 5min; 94 ℃, 45S, 58 ℃, 1min, 72 ℃, 1min; Last 72 ℃ are extended 7min, totally 35 circulations.
(4) structure of sequencing vector: the PUC18 carrier is preserved by this laboratory.With PUC18 carrier Sma I single endonuclease digestion, weight chain variable region gene PCR product reclaims, with cut after the carrier flush end be connected after, calcium chloride transforms according to a conventional method, select positive colony with indigo plant/hickie method, after identifying with EcoR I/SalI double digestion, send order-checking, this gene meets the characteristics of the some conservative framework amino acid that antibody had in the albumen database fully, and this sequence is the antibody gene sequence.Called after PUC18-VH and PUC18-VL see Fig. 4 respectively.
Embodiment 5: the structure of single-chain antibody gene expression vector PET22b (+) ScFv
According to light, the restriction enzyme mapping of variable region of heavy chain and the restriction enzyme site of carrier construction pAYZ, designed and synthesized and be used for VH, the primer of VL gene amplification and splicing.
The VL upstream primer
5’-GACTCG
CCATGGACATTCAGCTGACCCAG-3’；
The VL downstream primer
5’-ACCACCAGATCCACCTCCACCCGAGCCACCGCCACCCCGTTTGATCTCCAGTTTTGT-3’。
The VH upstream primer
5’-GGCTCGGGTGGAGGTGGATCTGGTGGTGGCGGTTCGCAGGTGAAGCTGCAGGAGTCT-3’；
The VH downstream primer
5’-CATGGG
CTCGAGTGAGGAGACGGTGACCGT-3’。
Introduce the Nco I/Xho I restriction enzyme site of clone's usefulness and the linker sequence of single-chain antibody and (adopt (GGGGS) the most widely
3Be linker).Pcr amplification VH from constructed PUC18-VH and PUC18-VL carrier, the VL fragment after the recovery, by the directly synthetic ScFv gene of PCR, is seen Fig. 5 through splicing overlap extension (SOE).The ScFv gene of PET22b (+) carrier and recovery is used Nco I/Xho I double digestion respectively, connect according to a conventional method after the recovery and transform and make up anti-CD44 ScFv expression vector PET22b (+) ScFv.Positive colony identifies that with Nco I/Xho I double digestion the back order-checking is definite.Correct clone is used for expressing.Sequencing result shows correctly, presses aminoacid sequence and infers 732bp, and ScFv is about the albumen of 26.1KD, sees Fig. 6.
Embodiment 6: the expression of anti-CD44ScFv antibody fragment, purifying
(1) expression and the SDS-PAGE of anti-CD44 single-chain antibody (ScFv) in intestinal bacteria analyzes: the LB inoculation of medium one positive single bacterium colony that contains penbritin at 3ml, after 37 ℃ of concussion overnight incubation, inoculating 30 μ l contains in the LB substratum of penbritin in 3ml, after about 4h is cultivated in 28 ℃ of concussions, add IPTG to final concentration be 0.8mmol/L, IPTG induces the bacterium liquid of results 1ml behind the 8h, the last sample buffer of centrifugal adding 50 μ l 2 * SDS, and 100 ℃ are boiled 10min.Get sample on the 5 μ l, 12%SDS-PAGE detects expressing protein, coomassie brilliant blue staining.Experimental results show that and realized the expression of recombinant plasmid pET22b (+) ScFv in intestinal bacteria, the 732bp fragment expression goes out the band (His) of about 28Kd
6Albumen, see Fig. 7.
(2) anti-CD44 single-chain antibody (ScFv) purifying: the bacterial sediment of expressing is resuspended in the ice precooling 20mmol/L Tris-HCl (PH 7.2) of 1/10 volume of culture, the ultrasonication cell, 13000rpm, 4 ℃ of centrifugal 30min get supernatant and are used for purifying.
Albumen adopts the Chelating Sepharose Fast Flow of Pharmacia company to carry out purifying, presses operational manual balance nickel post, hangs nickel, cleans.The sample of roughing out is added the nickel ion affinity chromatographic column, target protein is attached on the pillar, after the washing, contain the Tris-HCl buffer solution elution of 150mM imidazoles with the usefulness of 6 times of column volumes, elutriant was dialysed 48 hours with the PBS damping fluid.
(3) Western blot identifies: the main reference molecular cloning method carries out the Western trace, and the result proves that band is an expression product, sees Fig. 8.
Embodiment 7: anti-CD44 ScFv antibody activity is measured
Competitive immunization fluorescence inhibition test: get THP-1 clone and make 1 * 10
6Cell suspension, application of sample is in 40 porocyte culture plates, 5 * 10
5Cells/well, anti-CD44 monoclonal antibody HI44a20 μ l (1mg/ml), negative control group adds 20 μ l PBS, hatches 1h for 4 ℃, 3000rpm, 4 ℃ of centrifugal 8min abandon supernatant liquor, and PBS washes cell 2 times, add anti-CD44 ScFv 100 μ l (1mg/ml) behind the purifying, positive controls adds PBS, hatches 1h, 3000rpm for 4 ℃, 4 ℃ of centrifugal 8min, abandon supernatant liquor, PBS washes cell 2 times, and cell is resuspended among the PBS, it is anti-to add 20 μ l sheep anti-mouse igg-FITC two, hatch 45min for 4 ℃, the unconjugated fluorescence antibody of PBS flush away, cells were tested by flow cytometry mouse monoclonal antibody HI44a and THP-1 clone bonded positive rate.
Monoclonal antibody HI44a and CD44 express positive THP-1 clone, and to combine positive rate be 51.03%, after anti-CD44 ScFv competition, HI44a and THP-1 clone bonded positive rate are 27.10%, experimental results show that but anti-CD44 ScFv competitive inhibition HI44a combines with THP-1 clone, promptly anti-CD44 ScFv can combine with THP-1 clone surface C D44 antigen-specific, sees Fig. 9.
SEQUENCE LISTING (sequence table)
＜110〉Inst. of Hematology, Chinese Academy of Medical Sciences
＜120〉be used for the engineered antibody of the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis
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<160>4
<170>PatentIn?version?3.2
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ttcctcaaga?tcaccagtgt?ggacactgca?gatactggca?catactactg?tgctcgaaga 300
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20 25 30
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35 40 45
Trp?Leu?Ala?His?Ile?Tyr?Trp?Asp?Asp?Gly?Lys?Arg?Tyr?Asn?Pro?Ser
50 55 60
Leu?Lys?Ser?Arg?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Arg?Asn?Gln?Val
65 70 75 80
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Claims (7)
1, a kind of engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis is characterized in that containing the described anti-CD44 monoclonal antibody HI44a variable region of light chain VL gene nucleotide series of described anti-CD44 monoclonal antibody HI44a variable region of heavy chain VH gene nucleotide series of SEQ ID NO.1 and SEQ ID NO.3.
2, the engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis according to claim 1, it is characterized in that the expressed aminoacid sequence of anti-CD44 monoclonal antibody HI44a variable region of heavy chain VH gene shown in SEQ ID NO.2, comprise and contain partly or entirely fragment or the fusion rotein of this sequence C DRs.
3, the engineered antibody that is used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis according to claim 1, it is characterized in that the expressed aminoacid sequence of anti-CD44 monoclonal antibody HI44a variable region of light chain VL gene shown in SEQ ID NO.4, comprise and contain partly or entirely fragment or the fusion rotein of this sequence C DRs.
4, the PUC18 carrier that comprises the cDNA of claim 1.
5, constructed PET22b (+) expression vector that comprises the cDNA of claim 1.
6, the expressed cDNA aminoacid sequence of expression vector according to claim 5 comprises and contains partly or entirely fragment or the fusion rotein of this sequence C DRs.
7, according to claim 1,2,3,4, the 5 or 6 described engineered antibodies that are used for the anti-CD44 of inducing leukemia cytodifferentiation and apoptosis, the application in the leukemic medicine of preparation treatment.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310107583 CN1279058C (en) | 2003-12-18 | 2003-12-18 | Engineering antibody against CD44 for inducing leukemia cell differentation and necrosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310107583 CN1279058C (en) | 2003-12-18 | 2003-12-18 | Engineering antibody against CD44 for inducing leukemia cell differentation and necrosis |
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| Publication Number | Publication Date |
|---|---|
| CN1552735A true CN1552735A (en) | 2004-12-08 |
| CN1279058C CN1279058C (en) | 2006-10-11 |
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| CN 200310107583 Expired - Fee Related CN1279058C (en) | 2003-12-18 | 2003-12-18 | Engineering antibody against CD44 for inducing leukemia cell differentation and necrosis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288958A (en) * | 2013-03-22 | 2013-09-11 | 暨南大学 | A single chain antibody against a cancer stem cell-specific protein CD44 and applications thereof |
| CN114712381A (en) * | 2022-03-30 | 2022-07-08 | 浙江大学 | Application of AK2 gene in preparation of leukemia induced differentiation treatment drug |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368173B (en) * | 2008-04-09 | 2011-12-28 | 协和干细胞基因工程有限公司 | Antihuman CD44 monoclone antibody hybridoma cell line, monoclone antibody, engineering antibody, carrier, reagent kit and uses thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288958A (en) * | 2013-03-22 | 2013-09-11 | 暨南大学 | A single chain antibody against a cancer stem cell-specific protein CD44 and applications thereof |
| CN103288958B (en) * | 2013-03-22 | 2015-03-04 | 暨南大学 | A single chain antibody against a cancer stem cell-specific protein CD44 and applications thereof |
| CN114712381A (en) * | 2022-03-30 | 2022-07-08 | 浙江大学 | Application of AK2 gene in preparation of leukemia induced differentiation treatment drug |
| CN114712381B (en) * | 2022-03-30 | 2024-04-26 | 浙江大学 | Application of AK2 gene in preparation of leukemia induced differentiation therapeutic drug |
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| CN1279058C (en) | 2006-10-11 |
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