CN103214580B - Anti Her2 immune cytokine and application thereof - Google Patents
Anti Her2 immune cytokine and application thereof Download PDFInfo
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Abstract
The invention discloses an anti Her2 immune cytokine and application thereof, which belongs to the field of gene engineering technology. The amino acid sequence of the immune cytokine is shown in the SEQ ID No. 1. The anti Her2 immune cytokine of the present invention has obviously improved biological activity and output, and can effectively inhibit proliferation of tumour cells with high expression and effectively inhibit proliferation of Herceptin drug resistant breast cancer.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of anti-Her2 immune cell factor and application thereof.
Background technology
Cancer is one of disease that mortality ratio is the highest.China is newly-increased cancer patient about 2,000,000 people every year, and wherein breast cancer incidence rises with the amplitude increasing progressively 4% every year on average in China city.The U.S. has >18 ten thousand women to suffer from breast cancer every year, and the annual morbidity of China there is no definite statistical information, but the size of population huge according to China, year number of patients far should exceed the U.S..
Her2 is the transmembrane glycoprotein with tyrosine kinase activity, belongs to I type growth factor receptors family member.Her2 process LAN in the malignant tumour of the multiple epithelial cell origin such as mammary cancer, ovarian cancer, prostate cancer, cancer of the stomach, lung cancer, and expression level is very low in the normal tissue.Therefore, Her2 is the desirable target molecule of immunotherapy of tumors.Since U.S. FDA in 1998 ratifies the clinical treatment of anti-Her2 antibody (Herceptin) for Her2 process LAN patient with breast cancer, the molecular targeted therapy of mammary cancer has achieved larger achievement, and the sales volume of Herceptin in 2007 reaches about 4,000,000,000 dollars.From the result of Antybody therapy, alone Herceptin is 15-30% as the efficient of a clinical line medication, applies efficiently increase to 50% with chemotherapy drugs in combination.But, and the patient of not all Her2 high expression level can be benefited from Antybody therapy, patient wherein over half is insensitive to Herceptin treatment, and in medication after 1 year, the Herceptin of larger proportion treats effective patient and occurs resistance, causes Endodontic failure.Herceptin fails to respond to any medical treatment and the mechanism of resistance it be unclear that, but research shows, development has the medicine of the novel targeted Her2 of different mechanism of action, will fill up the blank of currently available products treatment spectrum, for more patient provides new medication to select.
Such as, the Lapatinib of U.S. FDA approval listing in 2007 is a kind of small-molecule drug of novel targeted Her2 tyrosine kinase domain, and its mechanism of action is different from Herceptin.Lapatinib apply separately efficient be 28%, but be used for the patient after Herceptin Endodontic failure as Second line Drug, efficiently still have 8%, this result has important practical significance, and the patient for Herceptin resistance or Endodontic failure brings new existence and wishes.Scholarly forecast, the annual sales amount of Lapatinib in 2010 reaches 1,000,000,000 dollars.The therapeutic antibodies Pertuzumab and for example carrying out III clinical trial phase is Her2 specific antibody newly developed, its action site is different from Herceptin, Her2 dimerization functional domain can be incorporated into, Her2 homodimer is stoped to be formed and form heterodimer with other Her family members, thus the Her2 intracellular signaling that block ligand activates.Preclinical study shows, the tumour of Pertuzumab to the low expression of Her2 also has restraining effect.The listing of Pertuzumab by for the low expression of large quantities of Her2, without medicine can patient the chance of therapeutic choice is provided.The targeted drug Antitumor test of different mechanism of action has complementarity.Vigorously advocate in the practice of ebm and individualized treatment current, Development of Novel target Her2 antitumor drug is very necessary, more rational medication will be provided to select for clinicist and extensive patients.
Interleukin-22 (IL-2) is the important reporter molecule in immune response, is also one of the most strong known antitumor cell factor, has biologic activity widely.But the systemic administration of IL-2 is everlasting and is produced high density away from the circulation of tumor locus, and does not reach the optimum concn for the treatment of in the microenvironment of tumor by local.Because the systemic administration of IL-2 can cause fatal side effect, this makes the therapeutic dose of IL-2 restricted greatly.How to improve the effective concentration of cytokine at tumor by local, strengthen its anti-tumor activity, avoid systemic toxicity to react is the direction that investigators make great efforts simultaneously always.
Immune cell factor is the fusion rotein of tumor specific antibody and the cytokine utilizing gene engineering method to build.Its mechanism of action is different from other anti-tumor target tropism biotech drug.It can utilize the target function of antibodies specific, the cytokine with anti-tumor activity is taken to tumor tissues local, to improve the cytokine concentration of tumor by local, at target site recruitment, immune stimulatory cell, produce the specific killing to tumour cell, the treatment tool especially for metastasis (metastases) and MRD has an enormous advantage.External existing 2 kinds of immune cell factors carry out I, II clinical trial phase at present.The result of I clinical trial phase shows, immune cell factor can bring out the immuno-stimulating of body, and therapeutic dose is only 7.5 mg/ square metre/day, decreases tens times than the consumption of common Antybody therapy!
Adopt gene engineering method, successful design, structure are containing anti-Her2 scFv, the Fc section of human IgG1, the fusion rotein (HF) of IL-2.HF remains and identifies the ability of ErbB-2 antigen and the biologic activity of IL-2.But there is following defect in HF: 1, biologic activity is low; 2, expression level is low, can not be used for large-scale production.These defects make the clinical application of HF be limited by very large, and are badly in need of carrying out molecular modification to overcome the bottleneck of large-scale production to HF, make HF can be applied to the clinical treatment of the mammary cancer of the Her2 positive.
Summary of the invention
The object of the present invention is to provide a kind of anti-Her2 immune cell factor.
The present invention also aims to provide the gene of above-mentioned anti-Her2 immune cell factor of encoding.
The present invention also aims to provide a kind of recombinant vectors containing anti-Her2 immune cytokine gene.
The present invention also aims to provide a kind of clone and Host Strains of expressing anti-Her2 immune cell factor.
The present invention also aims to the application providing above-mentioned anti-Her2 immune cell factor in the antibody class medicine of preparation treatment Her2 positive breast cancer.
The present invention also aims to provide above-mentioned and state that anti-Her2 immune cell factor is positive at preparation treatment Her2, Herceptin(Trastuzumab) resistance mammary cancer antibody class medicine in application.
A kind of anti-Her2 immune cell factor, described immune cell factor comprises people CD16 monoclonal antibody heavy signal peptide, Her2 antibody heavy chain variable region, Linker 1, Her2 antibody chain variable region, the Fc section of human IgG1, Linker 2 and IL-2 mature peptide.
The aminoacid sequence of above-mentioned immune cell factor is as shown in SEQ ID No. 1 in sequence table, and the aminoacid sequence of described Linker 1 is as shown in SEQ ID No. 2 in sequence table, and the aminoacid sequence of described Linker 2 is as shown in SEQ ID No. 3 in sequence table.
To encode the gene of above-mentioned anti-Her2 immune cell factor.
Carrier containing Her2 immune cell factor encoding gene anti-described in claim 2, as carrier for expression of eukaryon pCID.
Clone containing Her2 immune cell factor encoding gene anti-described in claim 2, as CHO/dhfr-cell.
Host Strains containing Her2 immune cell factor encoding gene anti-described in claim 2, as E. coli JM109.
The application of above-mentioned anti-Her2 immune cell factor in the antibody class medicine of preparation treatment Her2 positive breast cancer.
The application of above-mentioned anti-Her2 immune cell factor in the antibody class medicine of the mammary cancer of the preparation treatment Her2 positive, Trastuzumab (Herceptin) resistance.
Beneficial effect of the present invention: 1. anti-Her2 immune cell factor biologic activity of the present invention and output significantly improve.2. anti-Her2 immune cell factor of the present invention effectively can suppress the tumor cell proliferation of Her2 high expression level in vivo and in vitro.3. anti-Her2 immune cell factor of the present invention effectively can suppress the Cells Proliferation of Human Breast Cancer of Herceptin resistance in vivo and in vitro.
Accompanying drawing explanation
Fig. 1 is the structural representation of anti-Her2 immune cell factor.
Fig. 2 is the genophore plasmid figure containing anti-Her2 immune cell factor.
Fig. 3 is the expression amount of HFI and HF in engineering cell.
Fig. 4 is the binding activities of HFI and HF albumen and antigen.
Fig. 5 is the biologic activity of HFI and HF albumen.
Fig. 6 HFI is in vitro on the impact of the Cells Proliferation of Human Breast Cancer of high expression level Her2.
Fig. 7 HFI is in vivo on the impact that the breast cancer transplantable tumor of high expression level Her2 is bred.
Fig. 8 HFI is in vitro on the impact of Herceptin resistance breast cancer cell BT474, MDA-453 and SKBR3 propagation.
Fig. 9 HFI is in vivo on the impact of Herceptin resistance breast cancer cell transplanted tumor propagation.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.
Following examples do not limit the present invention, in embodiment, unspecified operation steps is normal experiment method, please refer to " Molecular Cloning: A Laboratory guide " third edition corresponding section (J. Pehanorm Brooker E.F. is Ritchie etc. not, Science Press) or consult the specification sheets of used kit.
The experiment material source that following examples adopt: E. coli JM109 engineering strain is purchased from Tian Gen bio tech ltd; CHO, BT474, MDA-453, SKBR3, MCF-7 cell is purchased from ATCC; T4 DNA ligase enzyme is Gibco BRL Products; Restriction enzyme is Biolab Products; Taq archaeal dna polymerase, cloning vector pGEM-T Easy are Promega Products; Ex Taq archaeal dna polymerase is TaKaRa Products; Plasmid extraction kit, DNA fragmentation reclaim test kit purchased from QIAGEN company; Protein A affinity chromatography medium is purchased from this yuan of Zhenyang company; Foetal calf serum (FCS), DMEM substratum, 1640 substratum are HyClone Products; MTX(methotrexate) and MTT(tetra-tetrazolium bromide) be Sigma Products.
The structure of embodiment 1 anti-Her2 immune cytokine gene expression vector
Anti-Her2 immune cell factor comprises people CD16 monoclonal antibody heavy signal peptide, Her2 antibody heavy chain variable region, Linker 1, Her2 antibody chain variable region, the Fc section of human IgG1, Linker 2 and IL-2 mature peptide, its structure as shown in Figure 1, it on the basis of HF fusion rotein, carries out improvement to Linker 2 form, called after HFI, wherein the aminoacid sequence of Linker 1 is as shown in SEQ ID No. 2 in sequence table, and the aminoacid sequence of Linker 2 is as shown in SEQ ID No. 3 in sequence table.
To contain anti-human CD16 monoclonal antibody heavy signal peptide, anti-Her2 scFv, the Fc section of human IgG1, the antigen-4 fusion protein gene carrier pCID/HF of IL-2 is template (in carrier, the nucleotide sequence of fusion rotein is as shown in SEQ ID No. in sequence table 5), with its nucleotide sequence of primer P1(as shown in SEQ ID No. in sequence table 6) and its nucleotide sequence of P2(as shown in SEQ ID No. in sequence table 7) pcr amplification contains the gene of anti-human CD16 monoclonal antibody heavy signal peptide of encoding, the gene of Her2 antibody heavy chain variable region, Linker1, the fragment of the gene of Her2 antibody chain variable region and the Fc gene of people's IgG antibody 1, condition is: 95 DEG C of sex change 2 min, then 94 DEG C of sex change 1 min, 58 DEG C of annealing 1 min, 72 DEG C of extension 2 min, carry out 30 circulations, last 72 DEG C extend 10 min.Insert in expression vector pCID with NheI and XhoI site, obtain carrier pCID/ScFv-Fc.
With its nucleotide sequence of primer P3(as shown in SEQ ID No. in sequence table 8) and P4 (its nucleotide sequence is as shown in SEQ ID No. in sequence table 9) pcr amplification contain N-terminal of encoding and contain, after optimization, there is the decapeptide Linker2 of good characteristic and the gene fragment of IL-2 mature peptide.Amplification condition is: 95 DEG C of sex change 2 min; Then 94 DEG C of sex change 1 min, 56 DEG C of annealing 1 min, 72 DEG C of extension 1 min, carry out 30 circulations; Last 72 DEG C extend 10 min.Insert in expression vector pCID/ScFv-Fc with XhoI and MluI site, obtain carrier for expression of eukaryon pCID/HFI, as shown in Figure 2.
PCID/HFI is transformed JM109 competence bacteria, screening positive clone, amplification also plasmid DNA purification, concrete steps are:
1) 100 μ l competence bacterias are placed in frozen water, add DNA 0.5 μ l, leave standstill 30 minutes;
2) 42 DEG C of water-baths 2 minutes;
3) ice bath 5 minutes;
4) add not containing antibiotic LB substratum, at 37 DEG C, 150 rpm shaking culture 50 minutes.
5) transformed bacteria is inoculated in the LB agar plate containing penbritin, puts 37 DEG C of incubator overnight incubation.
6) select positive colony 4, be inoculated in LB liquid nutrient medium respectively, put 37 DEG C of shaking table concussions and cultivate.
7) positive colony plasmid DNA is extracted with test kit.
With Nhe I and MluI double digestion qualification plasmid DNA, the gene of the anti-Her2 immune cell factor that obtains encoding, its nucleotide sequence as shown in SEQ ID No. 4 in sequence table, through Invitrogen company sequence verification.
Double digestion system is: H
2o 18 μ l
Buffer?(10×)4?μl
DNA?16?μl
NheⅠ?1?μl
MluⅠ?0.9?μl。
37 DEG C of water-baths 4 hours.
The screening of embodiment 2 engineering cell strain and the expression of HFI
By CHO/dhfr-cell cultures in the IMDM substratum containing 10% FCS, 0.1 mol/L xanthoglobulin, 0.016 mol/L Thymine deoxyriboside.According to Lipofectamine reagent specification sheets by recombinant vectors PCI/HFI transfection CHO/dhfr-cell.After transfectional cell cultivates 48 h, adopt Sandwich ELISA to detect the transient expression of immune cell factor, method is the same.
Adopt limiting dilution assay to carry out colonized culture to the cell of transfection, substratum is for containing 10% dialysis FCS, 10
-6the DMEM substratum of M MTX.In screening process, in different time points, Sandwich ELISA is adopted to detect the antibody expression of each cell clone.Detailed process is as follows: with goat anti-human igg (2 μ g/ml) bag by 96 orifice plates, 4 DEG C are spent the night.After closing 1h with 2% bovine serum albumin, add the Chinese hamster ovary celI culture supernatant of transfection, 37 DEG C of incubation 1h.After washing 5 times, add goat anti-human igg's (1:5000 dilution) of horseradish peroxidase-labeled, 37 DEG C of incubation 1h.With OPD colour developing after washing 5 times, measure OD490.High-expression clone is resuming generation containing MTX selective medium relaying, and adopts Sandwich ELISA to detect, and filters out most high-expression clone, as engineering cell strain.
By HFI engineering cell and HF engineering cell all with 5 × 10
6being inoculated in floorage is 150 cm
2culturing bottle in, under same culture conditions (30 DEG C, 5% CO2), cultivate 6 days.
Results culture supernatant, detects the content of target protein in supernatant by Sandwich ELISA, in HFI culture supernatant that result shows (as shown in Figure 3A) target protein content be that in 0.30 mg/ml, HF culture supernatant, target protein content is 0.19mg/ml.
Results culture supernatant, get 5 μ l HFI and HF culture supernatant detects its target protein content by Diagnosis of Sghistosomiasis notation, in HFI culture supernatant that result shows (as shown in Figure 3 B), object target protein content is that in 0.28 mg/ml, HF culture supernatant, protein content is 0.18 mg/ml.
Result shows, the anti-Her2 immune cell factor (HFI) after optimizing has higher output (* *, p<0.01) compared with HF in Chinese hamster ovary celI.Get culture supernatant by protein A affinitive layer purification, by the sample after purifying and culture supernatant by SDS-PAGE and coomassie brilliant blue staining post analysis.Result shows that the purity of HFI is greater than 90%.
Embodiment 3 HFI and HF antigen-binding activity and biological activity assay experiment
Collect 1 × 10
6individual SKBR3 cell, after PBS buffer solution, add HFI and HF after the purifying of different concns, 30 min are hatched in ice bath concussion, wash 3 times with PBS, add the goat anti-human igg of FITC mark, under ice bath, 30 min are hatched in concussion, wash 3 times with PBS, flow cytometer (Becton Dickinson company) is analyzed, using the cell of not hatching with transfectional cell culture supernatant as negative control.
The CTLL-2 cell of cultivation is washed 3 times, after counting, with 3 × 10 with 1640 substratum
4/ hole is inoculated in 96 orifice plates, adds HFI and HF after purifying simultaneously, using the IL-2 standard substance of doubling dilution as positive control.After cultivating 18 h, add 10 μ l MTT(5 mg/ml), continue cultivation 4 h.With 10% SDS-0.01 mol/L HCl lysing cell, survey A490 value.
Result shows (as shown in Figure 4), and the antigen-binding activity of HFI is a little more than HF(*, p<0.05), the biologic activity of HFI is significantly higher than HF(as shown in Figure 5; * *, p<0.01).
Embodiment 4 HFI kills and wounds the application in the medicine of Her2 high expression level breast cancer cell in preparation
1, the Cells Proliferation of Human Breast Cancer of vitro inhibition high expression level Her2
The MCF-7 cell of MCF-7 cell and MCF-7/Her2(high expression level Her2) with 3 × 10
396 orifice plates are inoculated in/hole, add the HFI(10 μ g/ml after purifying, Fig. 6 B simultaneously), with commercialization Herceptin(20 μ g/ml, Fig. 6 A) medicine in contrast.10 μ l MTT(5 mg/ml are added after cultivating 96 h), continue cultivation 4 h.With 10%SDS-0.01 mol/L HCl lysing cell, survey A490 value.
As shown in Figure 6, HFI specificity can suppress the propagation (* *, p<0.01) of the breast cancer cell MCF-7/Her2 cell of high expression level Her2 to result in vitro, and the propagation of HFI to the MCF-7 cell of low expression Her2 does not make significant difference.
2, the Cells Proliferation of Human Breast Cancer of high expression level Her2 is suppressed in body
4 week age nude mice, dorsal sc implants oestrogenic hormon slow release tablet.Next day, by logarithmic phase BT474 cell by 1 × 10
7/ to be only inoculated in the right armpit of nude mice subcutaneous.Laboratory animal is 3 groups at random, 6/group.After transplanted tumor inoculation the 5th day starts administration.Control group (control): physiological saline, 0.1 ml/ time/only, twice weekly; Herceptin group: commercialization Herceptin, according to 3 mg/kg/ time/only, volume injected is 0.1 ml, twice weekly; HFI group: HFI purification of samples, according to 0.5 mg/kg/ time/only, volume injected is 0.1 ml, twice weekly.From transplanted tumor inoculation, within every two days, measure transplanted tumor volume, calculation formula is: transplanted tumor volume=major diameter × minor axis
2/ 2.
Result shows, HFI effectively can suppress the growth of transplanted tumor in nude mouse, its curative effect similar to Herceptin treatment group (as shown in Figure 7).
The application of embodiment 5 HFI in the medicine of the anti-Herceptin resistance Cells Proliferation of Human Breast Cancer of preparation
1, the propagation of HFI vitro inhibition Herceptin resistance breast cancer cell
Adopt the screening method progressively improving Herceptin concentration in culture system, breast cancer cell BT474, MDA-453 and SKBR3 cell of high expression level Her2 is tamed.Screening through 6 months obtains the cell strain (BT474-HercepR, MDA-453-HercepR and SKBR3-HercepR) of three strain Herceptin resistances.
By above-mentioned three kinds of Herceptin mdr cells with 3 × 10
396 orifice plates are inoculated in/hole, add the HFI after purifying (5 μ g/ml), with commercialization Herceptin(5 μ g/ml simultaneously) in contrast.10 μ l MTT(5 mg/ml are added after cultivating 96 h), continue cultivation 4 h.With 10%SDS-0.01 mol/L HCl lysing cell, survey A490 value.
As shown in Figure 8, the propagation of Herceptin to 3 kinds of mdr cells does not make significant difference result, and HFI has significant restraining effect (* *, p<0.01) to the propagation of resistance breast cancer cell BT474, MDA-453 and SKBR3 in vitro.
2, the propagation of Herceptin resistance breast cancer cell is suppressed in HFI body
4 week age nude mice, dorsal sc implants oestrogenic hormon slow release tablet.Next day, by logarithmic phase BT474-Herceptin mdr cell by 1 × 10
7/ to be only inoculated in the right armpit of nude mice subcutaneous.Laboratory animal is 3 groups at random, 6/group.After transplanted tumor inoculation the 5th day starts administration.Control group: physiological saline, 0.1 ml/ time/only, twice weekly; Herceptin group: commercialization Herceptin, according to 3 mg/kg/ time/only, volume injected is 0.1 ml, twice weekly; HFI group: HFI purification of samples, according to 0.5 mg/kg/ time/only, volume injected is 0.1 ml, twice weekly.From transplanted tumor inoculation, within every two days, measure transplanted tumor volume, calculation formula is: transplanted tumor volume=major diameter × minor axis
2/ 2.
Result shows, HFI has stronger restraining effect (as shown in Figure 9) to the propagation of breast carcinoma resistance cell strain BT474 transplanted tumor in vivo.
Claims (7)
1. an anti-Her2 immune cell factor, it is characterized in that, described immune cell factor comprises people CD16 monoclonal antibody heavy signal peptide, Her2 antibody heavy chain variable region, Linker 1, Her2 antibody chain variable region, the Fc section of human IgG1, Linker 2 and IL-2 mature peptide; The aminoacid sequence of described immune cell factor is as shown in SEQ ID No.1 in sequence table, and the aminoacid sequence of described Linker 1 is as shown in SEQ ID No.2 in sequence table, and the aminoacid sequence of described Linker 2 is as shown in SEQ ID No.3 in sequence table.
2. the gene of anti-Her2 immune cell factor described in coding claim 1.
3. the carrier containing anti-Her2 immune cell factor encoding gene described in claim 2.
4. the clone containing anti-Her2 immune cell factor encoding gene described in claim 2.
5. the Host Strains containing anti-Her2 immune cell factor encoding gene described in claim 2.
6. the application of anti-Her2 immune cell factor described in claim 1 in the antibody class medicine of preparation treatment Her2 positive breast cancer.
7. the application of anti-Her2 immune cell factor described in claim 1 in the antibody class medicine of the mammary cancer of the preparation treatment Her2 positive, Trastuzumab resistance.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002102855A2 (en) * | 2000-11-17 | 2002-12-27 | University Of Rochester | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
| CN102199218A (en) * | 2011-04-29 | 2011-09-28 | 中国人民解放军军事医学科学院基础医学研究所 | Fusion protein of Her2 antibody and interleukin 2 and application thereof |
-
2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002102855A2 (en) * | 2000-11-17 | 2002-12-27 | University Of Rochester | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
| CN102199218A (en) * | 2011-04-29 | 2011-09-28 | 中国人民解放军军事医学科学院基础医学研究所 | Fusion protein of Her2 antibody and interleukin 2 and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Design and assembly of anti-CD16 ScFv antibody with two different linker peptides;Jiannan Feng等;《Journal of Immunological Methods》;20031130;第282卷;摘要 * |
| Hirsch,B等.GenBank: CAP05188.1.《GenBank》.2008, * |
| Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor;Wei-Yun Zhang等;《Acta Biochimica et Biophysica Sinica》;20041015;第36卷(第10期);第707-712页 * |
| Welsh,W.等.GenBank: AAB08080.《GenBank》.2002, * |
| 人源化抗P185 HER-2单区抗体与IL-2融合基因的构建表达和生物学活性;刘乐等;《中国肿瘤生物治疗杂志》;19970815;第4卷(第3期);第210页 * |
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