CN104592395B - The Preparation method and use of VEGFR2 single-chain antibodies and MICA fusion proteins - Google Patents
The Preparation method and use of VEGFR2 single-chain antibodies and MICA fusion proteins Download PDFInfo
- Publication number
- CN104592395B CN104592395B CN201510038297.5A CN201510038297A CN104592395B CN 104592395 B CN104592395 B CN 104592395B CN 201510038297 A CN201510038297 A CN 201510038297A CN 104592395 B CN104592395 B CN 104592395B
- Authority
- CN
- China
- Prior art keywords
- mica
- antibody
- fusion antibody
- ak404r
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to genetic engineering antibody technical field, the Preparation method and use for the fusion antibody that the single-chain antibody (AK404R) of anti-human vascular endothelial cell growth factor R-2 a kind of is connected with MICA is specifically disclosed.The invention discloses the structure of fusion antibody expression plasmid, the transfection of recombinant plasmid.The method that the present invention also provides expression and purified fusion antibody, fusion antibody is obtained by eukaryotic secreting, expressing, affinitive layer purification.The fusion antibody AK404R MICA of the present invention can specifically bind to human vascular endothelial growth factor receptor 3 body 2 (VEGFR2), can significantly inhibit Human umbilical vein endothelial cells (HUVEC) propagation, migration, cell invasion and tubule and be formed.The fusion antibody AK404R MICA of the present invention can also significantly inhibit tumour cell K562 growth, can be applied to prepare diagnosticum and therapeutic agent.
Description
Technical field
The invention belongs to bioengineering field, and in particular to it is a kind of it is new can be with human vascular endothelial growth factor receptor 3 body
The fusion antibody (AK404R-MICA) being connected with MICA of the high-affinity of 2 specific bonds, can suppress the increasing of endothelial cell
Grow, migrate, cell invasion and tubule are formed, and can also significantly inhibit tumour cell K562 growth, be it is a kind of have it is antitumor
The genetic engineering fusion antibody of activity.
Background technology
Tumour cell can penetrate into lymphatic vessel and blood vessel and be invaded by blood circulation in normal tissue, turn into cancer
The disease of potential threat life.One necessary requirement of the transfer of tumour cell is exactly the new life of blood vessel.However, new vessels
One of key of formation is exactly vascular endothelial growth factor (VEGF) family and associated receptor, and it with blood vessel endothelium by giving birth to
Growth factor receptor body (VEGFR) combines signal path in active cell, stimulates vascular endothelial cell proliferation, migration, promotes new green blood
The generation of pipe, the transfer of morbidity and tumour with a variety of kinds of tumor of body have close relationship.VEGFR-2, in human body
Also known as KDR (Kinase Insert Domain Receptor), has higher affinity with VEGF, and it is in mediation VEGF
Played an important role in the biological activity such as stimulating endothelial cell propagation and vasopermeability..KDR belongs to tyrosine kinase receptor
(Receptor protein-tyrosine kinase), 2nd~3 area are relevant with VEGF combination, 2 and 4 areas mainly influence its with
VEGF separation, 1st area are mainly responsible for the combination of regulation acceptor and part, the position for suppressing to be combined with VEGF are there may be in 4th~7 area
Point, wherein combination contribution of 3rd area (i.e. KDR3, containing 97 amino acid) to VEGF is the most prominent.
MHC work chain molecule A (Major Histocompatibility Complex class
I-related chain molecules A, MICA), be a kind of high glycosylation by mhc gene encode transmembrane glycopeptide
In vain, being played an important role in immunosurveillance, MICA is natural killer (NK) cell activation receptors NKG2D part, and research shows,
The tumour cell for expressing MICA is generally more sensitive to NK cell killings.NK surface receptor 2D (NKG2D), is C
A member in type agglutinin superfamily, all NK cells are not only expressed in, and it is thin to be expressed in CD8+T cells, the macrophage of activation
Cellular surface.The crosslinking of NKG2D and its part can trigger the cytotoxic activity of NK cells, be played in cancer immunosurveillance very heavy
The effect wanted, the expression of NKG2D parts almost determines the power of body NK cellullar immunologic responses on target cell.
This laboratory obtains the single-chain antibody using extracellular 3rd areas of KDR as antigen using display technique of bacteriophage before
AK404R, it is contemplated that the relatively small molecular weight (30KDa) of single-chain antibody and lack Fc fragments, our construction expressions
One fusion antibody AK404R-MICA goes to strengthen the validity of its oncotherapy.
Bevacizumab (Bevacizumab, trade name Avastin) is to be most commonly used to be directed to VEGF- in the market
The antibody drug of VEGFR2 signal paths.It is the Humanized monoclonal antibodies of restructuring, the approval for obtaining FDA in 2004, is the U.S.
The medicine of the suppression Tumor Angiongesis of first listing that gets the Green Light.IgGl antibody energy and people are confirmed by examination of the inside with outside system
VEGF (VEGF) combines and blocks its bioactivity.And Arastin contain human antibody structural area and
Can be with reference to the complementary determining region of VEGF mouse source monoclonal antibody.
The content of the invention
Goal of the invention
The present invention provides a kind of fusion antibody AK404R-MICA with potential medical science and pharmacy value.The present invention's melts
Close antibody and be characterized in that VEGFR2 single-chain antibody is connected with MICA, the increasing of Leukemia K562 cell can be suppressed in vitro
Grow.
Technical scheme
A kind of VEGFR2 single-chain antibody is connected the preparation of fusion antibody with MICA, it is characterised in that：
The fusion antibody that VEGFR2 single-chain antibody is connected with MICA includes the single-chain antibody using extracellular 3rd areas of KDR as antigen
AK404R and MICA, connected by a Linker (G4S).
A kind of nucleic acid of separation, it is characterised in that the nucleic acid encodes above-mentioned antibody.
A kind of expression vector, contain above-mentioned nucleic acid.
A kind of recombinant host cell, containing above-mentioned expression vector.
A kind of Pichia pastoris secreting, expressing method, the fusion antibody for secreting, expressing.
The fusion antibody of any of the above-described or the application of fusion antibody fragment, optionally combined with KDR3 or NKG2D is tied
Close.
The fusion antibody fragment of any of the above-described, selected from single-chain antibody, Fab, scFv, double antibody and three antibody.
The fusion antibody of any of the above-described or the conjugate of fusion antibody fragment.
Described conjugate, contain antineoplastic, target part or reporter moiety.
Invention further illustrates：
1596 nucleotides of fusion antibody gene order total length in the present invention, wherein 735 nucleotides of single-chain antibody,
MICA828 nucleotides, pass through a Linker (G4S) connect.
Expression vector and Host Strains containing the present inventor's vascular endothelial growth factor receptor single-chain antibody gene, MICA bases
The expression vector and Host Strains of cause and the expression vector and host cell of fusion antibody belong to protection scope of the present invention.Expand
Increase the primer pair of any fragment of fusion antibody gene of the present invention also within protection scope of the present invention.
It is a further object to provide a kind of side that can express and purify above-mentioned fusion antibody AK404R-MICA
Method.
The present invention passes through a G4S connection single-chain antibodies and MICA and importeding into Pichia pastoris screens that fermentation obtains can be special
The opposite sex combines KDR3 and NKG2D fusion antibody.After culture 5 days, low-temperature centrifugation removes cell fragment, and supernatant is crossed into the affine layer of nickel
Analysis post is isolated and purified；Western Blot identifications isolate and purify obtained AK404R-MICA；Elisa assay fusion antibody
With KDR3 and NKG2D combination；Utilize the affine of surface plasma body resonant vibration analysis fusioning protein and KDR3 and NKG2D
Power；Examine inhibitory action of the fusion protein to Leukemia K562 cell.
The fusion antibody AK404R-MICA that the present invention obtains and Leukemia K562 cell and Human umbilical vein endothelial cells
HUVEC has high specific combination, and extracorporeal suppression tumor cell growth experiment result shows, the fusion antibody that the present invention obtains can
Substantially to suppress the growth of tumour cell.The present invention is treatment and diagnoses with the expression of people's interior cutaneous vessel growth factor receptors, special
Be not that disease that overexpression is characterized provides the therapy based on antibody, relevant disease include but is not limited to autoimmunity disease and
Cancer.
Brief description of the drawings
Fig. 1.Figure 1A is the structure of expression plasmid.L1：Linker1, L2：Linker2, restriction site：EcoR1 and
XbaI.Figure 1B is the product figure after MICA and AKA404R PCR.Fig. 1 C are product figures of the AK404R-MICA after PCR.
Fig. 2.Fig. 2A shows SDS-PAGE protein electrophorese figures, and the fusion antibody for describing fermentation expression is divided by nickel post
From the result of purifying, the various concentrations imidazole buffer of swimming lane 1~9 washing nickel post result, swimming lane 2~9 is imidazole buffer containing 100mM
The schematic diagram of fusion protein is shown in destination protein (70KD) Fig. 2 B that liquid eluting nickel post obtains；Fig. 2 C show SDS-PAGE warps
MICA after purification renaturation, swimming lane 1 are irreducibility MICA, and swimming lane 2 is reproducibility MICA.Fig. 2 D show that SDS-PAGE is purified
NKG2D after renaturation, swimming lane 1 are irreducibility NKG2D, and swimming lane 2 is reproducibility NKG2D.Fig. 2 E, which are shown, utilizes Western
Blot fusion antibodies and the combination for recombinating KDR3.Fig. 2 F, which are shown, utilizes Western Blot fusion antibodies and the knot for recombinating NKG2D
Close.
Fig. 3.Fig. 3 A show etc. that surface plasma resonance analysis fusion antibody and the affinity recombinated between NKG2D are normal
Number, Ka=ka=6.77 × 109/ MS, kd=267.4/S and KD=3.95 × 10-8M.Fig. 3 B shows are to wait surface plasma to be total to
Affinity constant between vibration analysis fusion antibody and restructuring KDR3, Ka=4.26 × 106/ MS, kd=261/S and KD=6.13
×10-7M.Fig. 3 C show fusion antibody with recombinating NKG2D ELISA binding activity.Fig. 3 D show fusion antibody with
Recombinate KDR3 ELISA binding activity.
Fig. 4.Fig. 4 A, D Flow cytometries fusion antibody, AK404R and HUVEC cells combination, Percentage bound are respectively
62.7% and 67.0%, control group MICA and PBS are respectively 2.1% and 1.5%.Figure B, D Flow cytometry fusion antibody,
MICA and U937 combination, Percentage bound are respectively 69.7% and 58.0%, and control group A K404R and PBS and U937 is without conspicuousness knot
Close.C, D Flow cytometry fusion antibody, AK404R, MICA and PBS are schemed with HEK293 (human embryonic kidney cells) cells without aobvious
Write and combine.
Fig. 5.Fig. 5 A show that dosage and time dependence is presented to the inhibitory action of HUVEC cells in fusion antibody.Fig. 5 B exhibitions
What is shown is that fusion antibody is acted on the Inhibit proliferaton of HUVEC cells, the half inhibiting rate IC of 48 hours50：~333.186nM,
HEK293 is as negative cells.
Fig. 6.Fig. 6 A displayings are Transwell Matrigels, and fusion antibody can effectively suppress the invasion and attack of endothelial cell.Figure
6B quantitative analysis Transwell Matrigels, fusion antibody can be with the invasion and attack of dose-dependent inhibition endothelial cell.
Fig. 7.Fig. 7 A show K562 cellulotoxic experiment, as the ratio of effector cell/target cell raises and raise.Fig. 7 B
MDA-MB-435 cellulotoxic experiment is shown, as the ratio of effector cell/target cell raises and is raised.Fig. 7 C displayings
B16F1 cellulotoxic experiments, as the ratio of effector cell/target cell raises and are raised.Fig. 7 D displaying 3T3-L1's (control)
Cellulotoxic experiment.
Lifted below by non-limiting example and the present invention is described in detail.To those skilled in the art, without departing substantially from
To any obvious change that it is done on the premise of spirit of the invention, if being particularly the equivalent substitution of pair dry part, all
Form to infringement of patent right of the present invention, corresponding legal liabilities will be undertaken.
Embodiment
The percentage being related in embodiment, wherein fixating reagent are percent weight in volume, and liquid reagent is volume basis
Than.
The structure of embodiment 1AK404R-MICA plasmids and transfection
The single-chain antibody gene of human vessel endothelium growth factor resisting acceptor 2 is expanded with pHEN2-AKA404R (laboratory preservation)
Increase and obtain, devise pair of primers, sense primer P1 accordingly：5’-GGAATTCGAGGTGCAGCTGGTG-3 ' and anti-sense primer
P2：5 '-GGTTCTGAACCACCACCACCACCTAGGACGGTCAG-3 ', MICA gene are expanded with pET28a-MICA and obtained, on
Swim primer P3：5 '-TAGGTGGTGGTGGTGGTTCAGAACCTCACAGCCTG-3 ' and anti-sense primer：5’-
GCTCTAGAGCTCAATGATGATGATGATGATGCTTGCCGCTTGGGAC3’.Lower end line base-pair be EcoR1 and
Xba1 restriction enzyme site.The C-terminal of single-chain antibody links thing (linker) G with MICA using one4S (G- glycine,
S- serines) merged by overlap PCR, use sense primer P1 and anti-sense primer P4.The purpose expanded
Gene (AK404R-MICA) and carrier pPICZ α are attached with restriction enzyme EcoR1 and Xba1 and are transferred to DH5 α.Sun
Property is cloned in picking in the LB flat boards containing 25 μ g/ml bleomycins and comes out and be sequenced.
The recombinant vector pPICZ α-AK404R-MICA of acquisition carry out electricity using electroporation method and are transferred to Pichia pastoris (X-
33), positive colony screens in the yeast extract powder peptone glucose culture plate (YPD) containing 100 μ g/ml bleomycins,
Schematic diagram is as shown in Figure 1 with result.
The expression and purifying of the fusion antibody of embodiment 2
The clone for being picked out high expression using SDS-PAGE and western blot from 50 positive colonies is fermented.It is high
Expression cloning is incubated overnight to OD in 30 DEG C of 10mlYPD fluid nutrient mediums (containing 100 μ g/ml bleomycins), 220rpm600：2-
4, with 1: 1000 be inoculated into 200ml BMGY (2% peptone, 1% yeast extract, 100mM kaliumphosphate buffers (pH6.0),
1.34%YNB, 4 × 10-5Biotin, 1% glycerine) 30 DEG C, 220rpm is incubated overnight to OD600：1-2,3000g centrifugation 5min are gone
Except after supernatant use 200ml BMMY (2% peptone, 1% yeast extract, 100mM kaliumphosphate buffers (pH6.0), 1.34%
YNB, 4 × 10-5Biotin) and 0.5% 30 DEG C of methanol, 220rpm cultures is added, 0.5% methanol hair was added every 24 hours
Ferment 4 days.6th day, collect the fusion antibody that supernatant is secreted and purified with nickel post.After purification as shown in Figure 2 A.
The Western Blot identifications of the fusion antibody of embodiment 3
Obtained KDR3 and NKG2D is purified to carry out being denatured SDS-PAGE electrophoresis, separation gel with AK404R (as control)
Concentration is 15%；4 DEG C, 20mA constant currents transfer 1.5h, albumen is transferred to pvdf membrane (being purchased from Millipore)；Transfer terminates, will
Film 37 DEG C of closing 2h in 5%MTBS (TBS containing 5% skim milk)；Fusion antibody is diluted with 5%MTBS, 4 DEG C incubate overnight
Educate, TBST (containing 0.05%Tween-20 in TBS) is washed 3 times, each 10min；2h is carried out with anti-MICA 37 DEG C of antibody
After incubation, with 5%MTBS by 1: 5000 dilution HRP-anti-Mouse secondary antibodies (being purchased from Millipore), 37 DEG C are incubated 1.5h,
TBS is washed 3 times, each 10min；Exposed with ECL solution, as a result such as Fig. 2 E, shown in 2F.
The surface plasma body resonant vibration of embodiment 4 analyzes part and receptor interaction
The dynamics of KDR3, NKG2D and fusion antibody is studied using BiacoreX100 (GE), fusion antibody
It is sequestered in detection chip GM5 (GE Healthcare, BR-1000-12), KDR3 (250,125,62.5,31.5,15.6,
7.8nM) and NKG2D (250,125,62.5,31.5,15.6,7.8nM) sample detection is combined and dissociation curve, and BIA is assessed
Software analysis, calculate and obtain kinetic constant.Experimental result such as Fig. 3 A, shown in 3B.
The EUSA of embodiment 5 (ELISA)
4 DEG C of 100 μ 1NKG2D or KDR3 (10000nm, being dissolved in CBS) coatings overnight are added per hole.Added after cleaning per hole
200 μ l 5% 37 DEG C of closing 2h of skim milk.250 μ l TPBS (PBS containing 0.05%Tween are added per hole
20) micro concussion instrument cleaning 5min, is repeated 3 times, with the fusion antibodies of various concentrations (0.5,1,2,3.9,7.8,15.6,31.3,
62.5,125,250,500,1000nM) room temperature coating 1.5h, TPBS and PBS respectively wash three times, add anti-MICA
Monoclonal antibody are incubated at room temperature 1.5h, (are purchased from by 1: 5000 dilution HRP-anti-Mouse secondary antibodies with 5%MTBS
Millipore 1.5h) is incubated at room temperature, TBS is washed 3 times, each 10min.100 μ l nitrite ions (0.03%H are added per hole2O2With
2mg/ml TMB are dissolved in 0.1M NaAc buffer, pH 6.0, now with the current), 37 DEG C of incubation 15min.50 μ l are added per hole
Terminate liquid (1M H2SO4), ELIASA detection OD450-OD650Nm light absorption values.As a result such as Fig. 3 C, shown in 3D.
The Flow cytometry fusion antibody of embodiment 6 and HUVEC, U937 and HEK293 Percentage bound
Adjust HUVEC, U937 and HEK293 cell concentrations to 2 × 106Individual/mL, in each 1.5mL EP pipes respectively plus 250 μ L
Single cell suspension.Negative control group：First closed with 5% skim milk, after 2%FBS-PBS is washed 3 times, only add 500 μ L
The fluorescence secondary antibody ice bath of FITC marks is incubated 1h；Sample sets：First closed with 5% skim milk, after 2%FBS-PBS is washed 3 times,
2000nM fusion antibody ice bath is incubated 1h, after 2%FBS-PBS is washed 3 times, adds the fluorescence secondary antibody of 500 μ L FITC marks
Ice bath is incubated 1h；After each group sample finally is washed into 3 times with 2%FBS-PBS, cell, BD is resuspended with 500 μ L PBS solutions
FACS flow cytomeries.Experimental result such as Fig. 4 A, 4B, 4C, shown in 4D.
The cell proliferation experiment of embodiment 7
4x103Individual HUVEC, U937 and HEK293 cell are inoculated on 96 orifice plates at 37 DEG C, 5%CO2Incubator in cultivate
24h, various concentrations gradient (0nM, 3.9nM, 7.8nM, 15.6nM, 31.2nM, 62.5nM, 125nM, 250nM, 500nM,
Fusion antibody 1000nM) adds culture 24,48 and 72 hours, and AK404R, MICA and PBS are as a control group.11 μ are added per hole
L MTT, 37 DEG C of incubators are incubated 4h.Discard the culture medium containing MTT, add DMSO, ELIASA (Thermo) in 570nm and
630nm detects absorbance, analyzes experimental result, calculates IC50Value.Experimental result such as Fig. 5 A, shown in 5B.
Embodiment 8Transwell Matrigels
1×104Individual HUVEC cells and the fusion protein (20nM, 100nM and 500nM) of various concentrations gradient use serum-free
ECM culture mediums are resuspended cell and are laid on Matrigel matrix (being purchased from Millipore) glue by 1: 2 dilution.AK404R
(20nM, 100nM and 500nM), MICA (500nM) and PBS (500nM) are as control.It is incubated in 37 DEG C, 5%C02 incubators
After 30min, ECM complete mediums of the 600 μ L containing 5%FBS and 20ng/mLECGS is added per hole in lower room, finally, will
Transwell cells are transferred in the lower room containing nutrient solution, are subsequently placed in 37 DEG C, culture effect 12h in 5%C02 incubators.Training
20 minutes poststainings are fixed after supporting with the μ L/ holes of 4% paraformaldehyde 600 of 4 DEG C of precoolings to take pictures.Experimental result such as Fig. 6 A, shown in 6B.
The cellulotoxic experiment of embodiment 9
The Nonradioactive Cytotoxicity assay (being purchased from Promega) of CytoTox 96, target cell difference
For K562, MDA-MB-435, B16F10 and 3T3-L1 (control group).Leucocyte is isolated thin as effect from Healthy People
Born of the same parents.Effector cell and target cell with 5: 1,30: 1 and 100: 1 co-incubations and add fusion protein, and AK404R and MICA are at 37 DEG C
Cultivate 5h.50 μ L of supernatant are drawn to indicate to carry out LDH activity detection according to kit.Killing activity (%)=[(OD experimental groups-OD
Total Spontaneous release)/(the total Spontaneous releases of OD maximum release group-OD)] × 100%.Experimental result is as shown in Figure 7.
Claims (6)
1. the single-chain antibody of VEGFR2 a kind of and MICA fusion protein, it is characterised in that the amino acid sequence of described fusion protein
It is classified as SEQ NO.1.
2. a kind of nucleic acid, it is characterised in that it encodes the fusion protein described in claim 1.
3. a kind of expression vector, contain the nucleic acid described in claim 2.
A kind of 4. recombinant host cell, containing the expression vector described in claim 3.
5. the application of the fusion protein described in claim 1, it is characterised in that described fusion protein combined with KDR3 or with
NKG2D is combined.
6. application of the fusion protein in tumor is prepared described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510038297.5A CN104592395B (en) | 2015-01-27 | 2015-01-27 | The Preparation method and use of VEGFR2 single-chain antibodies and MICA fusion proteins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510038297.5A CN104592395B (en) | 2015-01-27 | 2015-01-27 | The Preparation method and use of VEGFR2 single-chain antibodies and MICA fusion proteins |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104592395A CN104592395A (en) | 2015-05-06 |
| CN104592395B true CN104592395B (en) | 2017-11-14 |
Family
ID=53118484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510038297.5A Expired - Fee Related CN104592395B (en) | 2015-01-27 | 2015-01-27 | The Preparation method and use of VEGFR2 single-chain antibodies and MICA fusion proteins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104592395B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3927750A4 (en) * | 2019-02-20 | 2022-11-02 | Research Institute at Nationwide Children's Hospital | Cancer-targeted, virus-encoded, regulatable t (catvert) or nk cell (catvern) linkers |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2016410294A1 (en) * | 2016-06-24 | 2019-01-03 | Xyphos Biosciences Inc. | Insertable variable fragments of antibodies and modified a1-a2 domains of NKG2D ligands |
| CN109762785B (en) * | 2018-12-10 | 2022-06-24 | 苏州近岸蛋白质科技股份有限公司 | Method for efficiently applying to in-vitro culture of human NK cell non-trophoblast |
| CN109678963A (en) * | 2018-12-18 | 2019-04-26 | 中国药科大学 | A kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell |
| CN110903402B (en) * | 2019-11-27 | 2023-02-28 | 中国药科大学 | Bispecific fusion protein and construction method and application thereof |
| CN111333732B (en) * | 2019-12-17 | 2023-02-28 | 中国药科大学 | Preparation and application of bispecific antibody targeting human BCMA and activating NK cells |
| CN117980328A (en) * | 2021-07-02 | 2024-05-03 | 来凯医药科技(上海)有限公司 | Depletion of activated Hepatic Stellate Cells (HSCs) and uses thereof |
| CN114292337B (en) * | 2021-12-17 | 2023-07-14 | 中南大学 | Soluble NK-CAR fusion protein, preparation method and application thereof in mediated immune cell targeted tumor cell killing medicament |
| WO2024055995A1 (en) * | 2022-09-14 | 2024-03-21 | 寻济生物科技（北京）有限公司 | Anti-vegfa fusion protein, and preparation method therefor and use thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101863980B (en) * | 2009-12-31 | 2013-05-08 | 中国药科大学 | Whole human source anti-vascular endothelial cell growth factor receptor 2 single chain antibody |
| BR112014015018A2 (en) * | 2011-12-19 | 2020-10-27 | Synimmune Gmbh | bispecific antibody molecules and their method of production, as well as pharmaceutical composition and nucleic acid molecule |
-
2015
- 2015-01-27 CN CN201510038297.5A patent/CN104592395B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3927750A4 (en) * | 2019-02-20 | 2022-11-02 | Research Institute at Nationwide Children's Hospital | Cancer-targeted, virus-encoded, regulatable t (catvert) or nk cell (catvern) linkers |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104592395A (en) | 2015-05-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104592395B (en) | The Preparation method and use of VEGFR2 single-chain antibodies and MICA fusion proteins | |
| WO2018050039A1 (en) | Novel anti-pd-1 nano-antibody and application thereof | |
| CN107217042A (en) | A kind of genetically engineered cell system for producing no fucosylation albumen and its method for building up | |
| CN106659779A (en) | Liquid separator for removing a liquid from a sample of a breathing gas and airway adapter | |
| CN103965363B (en) | Fusion protein efficiently combined with PD-1 and VEGF, coding sequence and application thereof | |
| CN107312093A (en) | VEGF fusion protein | |
| CN109485730A (en) | Anti- CD22 Chimeric antigen receptor | |
| US20200062818A1 (en) | Bifunctional fusion proteins to inhibit angiogenesis in tumor microenvironment and to activate adaptive immune responses and the genes and uses thereof | |
| CN104974262B (en) | Recombination double functions fusion protein and its preparation method and purposes | |
| CN106589131B (en) | Fusion protein 4D5Fv-PE25, and preparation method and application thereof | |
| CN104031148B (en) | Monoclonal antibody and its application for people PrPc | |
| CN103910799B (en) | A kind of fusion rotein based on anti-EGFR single-chain antibody and arginine nine aggressiveness and application | |
| EP3387014A1 (en) | Single chain fusionconstructs comprising multimeric antibody fragments fused to collagen trimerization domains | |
| CN105916883B (en) | Bifunctional fusion proteins and its preparation method and application | |
| CN101486758B (en) | Mda-7/IL-24 mutant polypeptide, and preparation and use thereof | |
| CN104193828B (en) | The recombination fusion protein of HER2 and VEGFR signal paths is blocked simultaneously | |
| CN101987873A (en) | P53 fusion protein and application thereof | |
| CN103360497A (en) | Novel antitumor fusion protein vaccine, and preparation method and application thereof | |
| CN107164412A (en) | A kind of safety-type anti-CEA Chimeric antigen receptors modify the preparation method and applications of T cell | |
| CN107236046A (en) | A kind of recombinant human endostatin fusion protein and its preparation method and application | |
| CN106854248A (en) | A kind of preparation and its application of cell factor mutant fusion antibody | |
| CN108129569B (en) | Preparation and application of double-targeting anti-tumor recombinant protein based on antibody and megalocytosis | |
| CN105504057B (en) | Anti- hantaan virus mouse/people's chimeric antibody and its application | |
| Khodabakhsh et al. | New proline, alanine, serine repeat sequence for pharmacokinetic enhancement of anti-VEGF single-domain antibody | |
| CN109400711A (en) | PDGFR β targeting tumor necrosis factor related apoptosis inducing ligand variant and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Juan Inventor after: Wang Min Inventor after: DESMOND OMANE ACHEAMPONG Inventor after: Tang Mingying Inventor after: Wang Youfu Inventor after: Zhao Xin Inventor after: Yuan Xibin Inventor before: Wang Min Inventor before: DESMOND OMANE ACHEAMPONG Inventor before: Tang Mingying Inventor before: Zhang Juan Inventor before: Wang Youfu Inventor before: Zhao Xin Inventor before: Yuan Xibin |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171114 Termination date: 20220127 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |