CN101450215A - Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside - Google Patents
Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside Download PDFInfo
- Publication number
- CN101450215A CN101450215A CNA2008101779370A CN200810177937A CN101450215A CN 101450215 A CN101450215 A CN 101450215A CN A2008101779370 A CNA2008101779370 A CN A2008101779370A CN 200810177937 A CN200810177937 A CN 200810177937A CN 101450215 A CN101450215 A CN 101450215A
- Authority
- CN
- China
- Prior art keywords
- nano
- particle
- antibody
- immunotoxin
- parcel
- 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.)
- Granted
Links
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the biological medicine field, in particularly, the invention discloses a PLGA nano grain that toxin is coated inside and antibody is connected outside, a preparing method and usage. The PLGA that is coated with PE38KDEL I type mutant and is connected with humanization SM5-1 antibody has advantages of high antitumor activity, small non specific toxicity and immunogenicity, low sensitivity for fighting against PE neutrality antibody, and can be used for preparing antitumor drug.
Description
Technical field:
The invention belongs to biomedicine field, more specifically, the invention discloses polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) nano-particle, Preparation Method And The Use that a kind of interior packaged toxin connects antibody outward.
Background technology:
Immunotoxin is to carry out chemical crosslinking or gene fusion and the complex that forms by antibody or antibody fragment and proteotoxin (as bacillus pyocyaneus extracellular toxin PE).At present, the immunotoxin that makes up of PE carries out gene fusion to the PE that removes the cell bound fraction and antibody fragment and forms.Although the immunotoxin that PE makes up has been obtained the achievement that attracts people's attention in the clinical treatment of tumor, yet, its clinical practice still is subject to the serious non-special toxicity of immunotoxin, these non-special toxicity mainly show as liver toxicity one because a kind of non-special toxicity that immunotoxin and normal structure non-specific binding cause, and the toxin moiety in the immunotoxin that PE makes up plays an important role in non-special toxicity.It is found that recently certain methods (as reducing the isoelectric point, IP of immunotoxin) can be used as the non-special toxicity that reduces immunotoxin.In addition, the immunoreation of the toxin moiety in the immunotoxin that makes up at PE is the major obstacle of treatment solid tumor, and the immunogenic method that overcomes immunotoxin is generally to unite uses immunosuppressant (CTLA4Ig or Rituximab).If can reduce the immunogenicity and the non-special toxicity of immunotoxin,, thereby obtain better clinical effectiveness just we can strengthen the clinical using dosage of immunotoxin.But except polyethyleneglycol modified immunotoxin, there be limited evidence currently of has the method that can solve these two problems simultaneously.
Summary of the invention:
The present inventor is through a large amount of experiments, to be applied to wrap up small-molecule drug usually, the polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) that reduces these poisonous side effect of medicine wraps up the PE toxin, here the I type mutant of preferred PE38KDEL: PE38KDEL I, in order to increase its targeting, the present inventor connects the Fab ' fragment of humanization SM5-1 monoclonal antibody by the carbodiimides technology on polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) surface of having wrapped up PE38KDEL I, obtain a kind of interior parcel PE38KDEL I type mutant, connected the polylactic-co-glycolic acid copolymer nano granule of humanization SM5-1 antibody outward.
The interior parcel PE38KDEL I type mutant that the present inventor utilizes the present invention to obtain connects humanization SM5-1 monoclonal antibody outward ' segmental polylactic acid-glycolic guanidine-acetic acid copolymer nano granule (hereinafter to be referred as PE-NP-S) carried out a series of experiment, experimental result shows that PE-NP-S demonstrates better anti-tumor activity, and have littler non-special toxicity and an immunogenicity, the sensitivity of antagonism PE neutrality antibody is lower, has reached purpose of the present invention.
The present inventor also discloses the method for preparing above-mentioned nano-particle, comprises the at first nano-particle of preparation parcel immunotoxin, connects two steps of antibody at nano grain surface then.
Similarly, utilize method disclosed by the invention, can also connect other complete antibody or other humanized antibody fragment (for example Fab ' fragment or F (ab ')
2) obtain outside above-mentioned antibody or the segmental polylactic acid-glycolic guanidine-acetic acid copolymer nano granule that wraps up PE38KDEL I type mutant of connecting, these complete antibodies or humanized antibody fragment can be by the antibody of tumor cell endocytosis or its fragment, as recombinant humanized anti-HER 2 monoclonal antibody (rhuMAbHER2) and the anti-CD22 monoclonal antibody of recombinant humanized.
Correspondingly, utilize method disclosed by the invention, can also utilize the toxin of polylactic acid-glycolic guanidine-acetic acid copolymer parcel other types and then obtain various nano-particle at above-mentioned antibody or the antibody fragment of its outside connection, these toxin can be one of ricin and diphtheria toxin, diphtherotoxin.
Nano-particle disclosed by the invention can be formed pharmaceutical preparation together with other acceptable accessories.These pharmaceutical preparatioies can be used for the treatment of tumor.Tumor can be the male tumor of the conjugated protein antigen presentation of SM5-1, or HER2, CD22 express male tumor.
The male tumour medicine of the described conjugated protein antigen presentation of treatment SM5-1 includes but not limited to alleviate or/and alleviate the medicine of the various symptoms of the male tumor of the conjugated protein antigen presentation of SM5-1.The male tumor of the conjugated protein antigen presentation of SM5-1 described here comprises hepatocarcinoma, breast carcinoma and melanoma etc., preferred hepatocarcinoma.
More specifically, the invention discloses:
1. the parcel immunotoxin connects the polylactic acid-glycolic guanidine-acetic acid copolymer nano granule of antibody outward in one kind.
2. as 1 described nano-particle, wherein immunotoxin is one of ricin, diphtheria toxin, diphtherotoxin, PE38KDELI type mutant, and antibody is the Fab ' fragment of humanized antibody or humanized antibody.
3. be that interior parcel PE38KDEL I type mutant connects humanization SM5-1 monoclonal antibody outward as 2 described nano-particle ' segmental polylactic acid-glycolic guanidine-acetic acid copolymer nano granule.
4. as 3 described nano-particle, its particle diameter is 70～140nm, and parcel efficient is 35～45%, and the antibody joint efficiency is 15～25 μ g/mg nano-particle.
5. a method for preparing as 1 to 4 arbitrary described nano-particle comprises the at first nano-particle of preparation parcel immunotoxin, connects two steps of antibody at nano grain surface then.
6. as 5 described methods, wherein the nano-particle step of preparation parcel immunotoxin comprises:
A) immunotoxin is dissolved in the aqueous solution and joins oil-based solvent together with polylactic acid-glycolic guanidine-acetic acid copolymer then, ultrasonicly obtain water in oil colostrum;
B) utilize the colostrum that a) obtains to prepare emulsion;
C) after emulsion joins in the poly-vinyl alcohol solution again, stir and make the organic solvent volatilization, solution is through centrifugal, washing, and low-temperature freeze drying promptly makes the finished product nano-particle.
7. as 6 described methods, wherein the aqueous solution in the step a) is PBS, and oil-based solvent is an ethyl acetate.
8. as 6 described methods, wherein step a) also is included in the aqueous solution that has dissolved immunotoxin and adds trehalose.
9. as 5 method, wherein connect the antibody step and hatch jointly under comprising the nano-particle of parcel immunotoxin and EDC, NHS condition, will be dissolved in the antibody of HEPES (pH7.4) or antibody fragment then and join in the sedimentary nanosphere lucifuge reaction and get in lucifuge at nano grain surface.
10. as the purposes of the arbitrary described nano-particle of 1-4 in preparation medicine for treating tumor thing.
11. as 10 described purposes, wherein tumor is that conjugated protein antigen of SMS-1 or Her2 express male tumor.
12. as 11 described purposes, wherein the male tumor of the conjugated protein antigen presentation of SM5-1 is breast carcinoma, melanoma and hepatocarcinoma.
13. as 12 described purposes, wherein the male tumor of the conjugated protein antigen presentation of SM5-1 is a hepatocarcinoma.
In the present invention, as not specifying, PE-NP-S refers to interior parcel PE38KDEL I type mutant disclosed by the invention and connects the segmental polylactic acid-glycolic guanidine-acetic acid of humanization SM5-1Fab ' copolymer nano granule outward, Mut-I is that the immunotoxin that carries out after the gene fusion of SM5-1 single-chain antibody and PE38KDEL I mutant is (if not otherwise specified following, PE38KDEL I all refers to PE38KDEL I mutant, in each accompanying drawing also all with), PE-NP is the PLGA nano-particle of parcel PE38KDEL I, FITC-PE-NP-S is the PE-NP-S of FITC labelling, and PE-NP-CD25 is for connecting the segmental PE-NP of Fab ' of anti-CD25.NP-S is the Fab ' fragment of SM5-1 in PLGA nano-particle (NP) connection that does not contain immunotoxin.
Description of drawings:
Fig. 1: the morphology of PE-NP-S nano-particle and particle size distribution figure, wherein Fig. 1-1:PE-NP-S throws the morphology photo of Electronic Speculum (TEM), and resolution is 0.2 micron, Figure 12: the particle size distribution figure that PE NP-S measures by the particle diameter instrument.
Fig. 2: competition is in conjunction with experimental result, and wherein 1 * 10
5The Mus source SM5-1 (mSM5-1) of the FITC labelling of Ch-hep-3 cell and sub-saturated concentration, Mut-I, CD25-PE38KDEL or the nano-particle of increased concentrations were hatched 1 hour at 4 ℃ gradually, analyzed with streaming behind the cell washing.Maximum fluorescence intensity is defined as: when not competing antibody, and the average fluorescent strength that the Mus source SM5-1 of FITC labelling dyes and obtains the Ch-hep-3 cell.All data are the meansigma methods of three independent experiments.
Fig. 3: FITC-PE-NP-S in conjunction with and by the process analysis of Ch-hep-3 cell endocytic, the Ch-hep-3 cell was colored in the following time: 0,1 and 30 minute.Fig. 3-1:Ch-hep-3 cell, incubation time are 1 minute, and Fig. 3-2:Ch-hep-3 cell, incubation time are 30 minutes, Fig. 3-3:QGY7701 cell, incubation time are 30min, and wherein Fig. 3-1-1 is for differing, Fig. 3-1-2 is the green fluorescence of FITC, Fig. 3-1-3 is 4 ' and, the blue fluorescence of 6 diamidinos-2-benzene indole hydrochloride (pair cell nuclear dyes), Fig. 3-1-4 is a superimposed image, Fig. 3-2-1,3-2-2,3-2-3,3-2-4 and 3-3-1,3-3-2,3-3-3,3-3-4 all with, resolution is 10 μ m.
Fig. 4: anti-PE TPPA result, Fig. 4-1: Mus is to the IgG of PE38KDELI reaction, 5 groups of mices time of arrow indication respectively immunity the PE-NP-S of 1mg/kg, PE-NP, PE-NP-CD25, Mut-I, with PE38KDEL I, anti-PE IgG level is detected at the 21st day in the serum; Matched group immunity PBS; *, P＜0.05; Fig. 4-2: anti-PE neutrality measurement result.
Fig. 5: Mut-I and PE-NP-S are to the Graft Versus Tumor of BALB/c nude mice, 5 * 10
6The Ch-hep-3 cell was subcutaneously injected in the mice at the 0th day, in case tumor length has arrived 50mm
3, at the 5th, 7 and 9 day (arrow indication), mice was accepted twice vein treatment every day, every group of 6 mices.Data show is mean tumour volume ± standard deviation (n=6).Fig. 5 has eight treatment groups, be respectively PBS, PE38KDEL I (5mg/kg), PE-NP (5mg/kg, the PE38KDEL I that presses parcel calculates)+(wrap up efficient by actual antibody joint efficiency and toxin converts SM5-1Fab ', the antibody equivalent that is connected on the PE-NP-CD25 of antibody amount and 5mg/kg dosage), PE-NP-CD25 (5mg/kg, the PE38KDEL of parcel be I's and connect the segmental PLGA nano-particle of anti-CD25 monoclonal antibody Fab ', PE38KDEL I by parcel calculates), PE-NP-S (0.5mg/kg, parcel PE38KDEL I also connects the segmental PLGA nano-particle of SM5-1Fab ', PE38KDELI by parcel calculates), PE-NP-S (2.5mg/kg is by the PE38KDEL I calculating of parcel), Mut-I (0.5mg/kg), Mut-I (5mg/kg).
The specific embodiment:
Following examples, experimental example only are further detailed the present invention, should not be construed as limitation of the present invention.
The preparation of PE38KDEL I, purification and analytical method are seen (Wang H, Song S, Kou G, et al.Treatment of hepatocellular carcinoma in a mouse xenograft model withan immunotoxin which is engine ered to elimi nate vascular leak syndrome.Cancer Immunol Immunother 2007; 56 (11): 1775-1783.Song S, Xue J, Fan K, et al.Preparation and characterization of fusion protein truncatedPseudomonas Exotoxin A (PE38KDEL-I) in Escherichia coli.Protein ExprPurif 2005; 44:52-7.); Humanization SM5-1 monoclonal antibody is by Li B, Wang H, Zhang D, et al.Construction and characterizati on of a high affinity humanizedSM5-1 monoclonal antibody.Biochem Biophys Res Commun 2007; 357 (4): the method that provides among the 951-6, carry out obtaining behind the affinity chromatograph with the cell conditioned medium of ProteinA post to the Chinese hamster ovary celI (Chinese hamster ovary cancerous cell) of expressing human source SM5-1 monoclonal antibody; In addition, unless mention especially, SM5-1 herein refers to humanization SM5-1, the segmental method of Fab ' of humanization SM5-1 monoclonal antibody and humanization anti-CD25 monoclonal antibody is prepared into referring to Martin FJ, Hubbell WL, Papahadjopoulos D.Immunospeci fictargeting of liposomes to cells:anovel and efficient method for covalent attachment of Fab ' fragmentsvi adisulfide bonds.Biochemi stry 1981; 20:4229-38.; Three strain hepatoma cell strain Ch-hep-1, Ch-hep-3 and QGY7701 open source information are referring to Preparation andCharacterization of Paclitaxel-loaded PlGA Nanoparticles Coated withCationic SM5-1 Single-chain Antibody, Journal of Biochemistry andMolecular Biology, Vo.40, No.5, September 2007, page 731-739; (mol ratio of lactic acid and ethanolamine is 50:50 to PLGA, and RG503, molecular weight 40～75kDa) buy the company from German Boehringer Ingelheim; The preparation method of Mut-I is referring to Wang H, Song S, Kou G, et al.Treatment of hepatocellular carcinoma in a mouse xenograft modelwith an immunotoxin which is engineered to eliminate vascular leaksyndrome.Cancer Immunol Immunother 2007; 56 (11): 1775-1783; All the other chemical reagent are AG purity all available from sigma.
Embodiment 1: the preparation of the nano-particle PE-NP of parcel PE38KDEL I
PE38KDEL I is dissolved in PBS (pH7.4) by the concentration of 2mg/ml, the trehalose that takes by weighing 15 milligrams adds the above-mentioned protein solution of 150 μ l, put upside down mixing to fully the dissolving after, join in 1.5 milliliters the ethyl acetate ultrasonic 15 seconds (power is 14 watts) (Branson in ice bath jointly with 40 milligrams PLGA again
450) obtain colostrum.Then, (molecular weight: 25-35kDa), aqueous solution (mass volume ratio is 1%) joins in the colostrum (Water-In-Oil) for preparing 2 milliliters polyvinyl alcohol, and ultrasonic again 15 seconds (power is 14 watts) obtains emulsion [water (Water-In-Oil)].The emulsion that obtains joins 50 milliliters polyvinyl alcohol (molecular weight: 25-35kDa) (mass volume ratio is 0.3%) in the aqueous solution again.Final solution restir made organic solvent volatilization in 8 hours, the nano-particle that makes at last through centrifugal (30000g * 30min), wash three times after, (freeze-drying process: the sample branch is filled in the lyophilizing bottle low-temperature freeze drying again, 2～5ml/ bottle, pre-freeze is 4 hours under-20 ℃ of conditions, puts in the freeze dryer, the laminate temperature is set at-40 ℃, treat sample temperature after-30 ℃, unlatching vacuum pump, lyophilizing 24 hours, aseptic condition discharges down vacuum, and takes out sample) promptly make finished product nano-particle PE-NP.The preparation of empty nano-particle NP also is to prepare (the PE38KDEL I that does not contain 2mg/kg) according to the method described above.
The preparation of the PLGA nano-particle of embodiment 2:FITC (Fluorescein isothiocyanate) labelling
The PLGA of 100mg is dissolved in 5 milliliters the anhydrous methylene chloride, uses 1,3-dicyclohexylcarbodiimide (0.7 milligram) and N-N-Hydroxysuccinimide (0.4 milligram) to activate then.React after 4 hours, side-product dicyclohexyliosurea removes with the method for dialysis that (dialysis process is referring to Ataman-Onal, Y., Munier, S., Ganee, A., Terrat, C., Durand, P.Y., Battail, N., Martinon, F., Le, G.R., Charles, M.H., Delair, T.and Verrier, B. (2006) Surfactant-free anionic PLA nanoparticles coated with HIV-1 p24protein induced enhanced cellular and humeral immune responses in variousanimal models.J.Control.Release 112,175-185.).After the dialysis, in above-mentioned reactant liquor, add 0.7 milligram of aminophylline, reaction made the succinimidyl ester group of PLGA change primary amine group in 4 hours, reaction finishes, join 500 milliliters-20 ℃ of pre-cold diethyl ethers carry out purification with the sedimentation method, carry out vacuum drying (vacuum 20Pa, baking temperature are 25 ℃, 12 hours drying times) then.To be dissolved in the FITC (1.2mg) of dimethyl sulfoxine (DMSO) and 50 milligrams the PLGA with primary amine group (product behind the vacuum drying) reaction.Product to deionized water dialysis (described method is identical above the dialysis process) after, lyophilizing.Behind the PLGA polymer and the mixed of PLGA with the FITC labelling, get the PLGA nano-particle FITC-PE-NP of FITC labelling according to the multi-emulsion method metric system of embodiment 1 according to 1:9.Embodiment 3: the particulate preparation of targeted nano
(5 μ g/ μ l, PE-NP is dissolved in the 10mM NaH of pH 6.3 to 1 milliliter PLGA nano-particle (embodiment 1 product P E-NP) solution
2PO
4Aqueous solution) and the 50mg/ml EDC of 10 μ l (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide) aqueous solution, after the common lucifuge of 50mg/ml NHS (N-hydroxysuccinimide) aqueous solution of 10 μ l is hatched 20 minutes, 30000g removes supernatant after centrifugal 10 minutes, to be dissolved in 50mM HEPES buffer (4-hydroxyethyl piperazine ethanesulfonic acid then, 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid) SM5-1 (pH7.4) or anti-CD25 Fab ' (1 μ g/ μ l) joined in the sedimentary nano-particle lucifuge reaction after 2 hours, centrifugal, make the nano-particle PE-NPS or the PE-NP-CD25 that connect antibody fragment after the washing.
The step that the NP that the preparation of NP-S can utilize embodiment 1 to obtain passes through embodiment 3 obtains.
The preparation of FITC-PE-NP-S can utilize FITC-PE-NP that embodiment 2 obtains to obtain by the step of embodiment 3.
Experimental example
Unless stated otherwise, the following experimental example product that all utilizes the foregoing description the to obtain experimental example 1 that experimentizes: parcel efficient, mean diameter and particle size distribution measuring
The percentage ratio of the PE38KDEL I that is wrapped up into by the PLGA nano-particle is parcel efficient, the content of dispersion of PE38KDEL I is the drug loading of PE38KDEL I in every milligram of nano-particle, the antibody fragment joint efficiency is the quality of the antibody fragment that connects of every milligram PLGA nanosphere, parcel efficient, the drug loading of PE38KDEL I and antibody fragment joint efficiency all are by MicroBCA proteinassay kit (Pierce, Rockford, IL) the by specification method is measured (Ofra Benny, Maayan Duvshani-Eshet, TheresaCargioli, Lorenzo Bello, Andreas Bikfalvi, Rona S.Carroll, and MarcelleMachluf, Continuous Delivery of Endogenous Inhibitors from Poly (Lactic-Co-Glycolic Acid) PolymericMi crospheres Inhibits Glioma TumorGrowth, Clinical Cancer Research 2005; 768 (11): 768-76).The mean diameter of nano-particle and particle size distribution are that (Malvern UK) measures by particle diameter instrument Zeta Sizer 3000HS.The configuration of surface of nano-particle and particle diameter are measured by projection Electronic Speculum JEOL 2010 Transmission ElectronicMicroscopy.
The parcel efficient of result: PE-NP-S is 41.8 ± 2.3%, and the drug loading of PE38KDEL I is 8.5 ± 0.2 μ g/mg, and the antibody joint efficiency is Fab '/mg nano-particle (mean ± SD of 19.4 ± 2.4 μ g SM5-1; N=4).Measure by Zeta Sizer 3000HS particle diameter instrument, the average diameter of PE-NP-S is 107.7 ± 30.9nm (mean ± SD; N=10).By the observation of transmission electron microscope TEM, we find the circle that is shaped as of PE-NP-S, and particle size distribution narrow be 107.67 ± 27.6nm, see Fig. 1.
Carry out above-mentioned experiment for PE-NP-CD25 and also obtained identical result.
Experimental example 2: competitive bonded combination and endocytosis activity experiment
Competitive binding experiment: 1 * 10
5(preparation method is referring to U.Trefzer for the Mus source SM5-1 of the FITC labelling of Ch-hep-3 cell and sub-saturated concentration, N.Rietz, Y.Chen, H.Audring, G.Herberth, P.Siegel, S.Reinke, P.Koniger, S.Wu, J.Ma, Y.Liu, H.Wang, W.Sterry, Y.Guo, SM5-1:a new monoclonal antibody which is highly sensitive andspecific for melanocytic lesions, Arch.Dermatol.Res.292 (2000) 583-589.) and gradually the Mus source SM5-1 of increased concentrations, Mut-I or PE-NP-S were hatched 1 hour at 4 ℃, simultaneously with CD25-PE38KDEL, PE NP CD25 is as negative control.Cell is analyzed with flow cytometer by after the centrifuge washing.(BectonDickinson, San Jose CA) analyzes the flow cytometer result with FACScan flow cytometer.Competitor's IC
50(half-inhibition concentration) calculates with four parameter algorithms, the results are shown in Figure 2, and the result shows that PE-NP-S and Mut-I can competitively block Mus source SM5-1 in conjunction with the Ch-hep-3 cell, mean that they have all kept specificity in conjunction with the conjugated protein antigenic ability of SM51.The affinity of PE-NP-S (average IC
50± SD, n=3) be 0.40 ± 0.02 μ g/ml, the affinity (16.47 ± 2.69 μ g/ml) that is much higher than Mut-I, and it is similar with the affinity (0.32 ± 0.03 μ g/ml) of Mus source SM5-1, mean the Fab ' antibody fragment of SM5-1 is connected to the combination activity that does not change it on the nano-particle, maximum fluorescence intensity is defined as: when not competing antibody, and the average fluorescent strength that the Mus source SM5-1 of FITC labelling dyes and obtains the Ch-hep-3 cell.
In conjunction with and endocytosis experiment measure by Laser Scanning Confocal Microscope: Ch-hep-3 cell and PE-NP-S were hatched 0,1 or 30 minute at 37 ℃, used the PBS washed twice then, fixed 30 minutes with 10 milliliters 4%PFA (paraformaldehyde).At last, cell is taken pictures with Leica TCS SP2 Confocal Spectral Microscope (UV-VIS), the photo burnt software analysis of Leica copolymerization, the results are shown in Figure 3, the result shows by 1 minute hatch, the fluorescence of FITC only is detected (Fig. 3-1) at the Ch-hep-3 cell surface, after hatching 30 minutes, FITC fluorescence can clearly be detected (Fig. 3-2) in the Ch-hep-3 cell cytoplasm.Yet,, do not detect any FITC fluorescence (Fig. 3-3) at the cell strain QGY7701 of the conjugated protein antigen presentation feminine gender of SM5-1.
In addition, the present inventor utilizes PE-NP and PE-NP-CD25 to carry out same experiment, experimental result shows that PE-NP is opposite with PE-NP-S result with PE-NP-CD25, thereby showing that it all not can be incorporated into cell surface antigen can not be by Ch hep-3 cell endocytic.
The present inventor utilizes the conjugated protein antigen presentation positive cells of other strain SM5-1 strain Ch-hep-1 also to obtain the result similar to above-mentioned experiment.
Experimental example 3: vitro cytotoxicity experiment
Three strain hepatoma carcinoma cell in 96 orifice plates with 1 * 10
4The density in/hole is at 37 ℃, 7.5%CO
2In cultivate after 24 hours and PE-NP-S, Mut-I or PE38KDEL I, PE-NP+SM5-1Fab ', PE-NP-CD25 were hatched two days altogether at 37 ℃, the concentration of PE38KDEL I is 1 to 10 in the medicine, 000pM.The cells survival rate with cell tire the non-radioactive cell proliferation test kit (Cell Titer 96 non-radioactive cellproliferation assay kit, Promega, Madison, WI USA) measures.In brief, 20 μ l of MTS/PMS solution are added in each hole, 37 ℃ hatch two hours after, carry out reading with microplate reader at the 490nm wavelength, the results are shown in Table 1.
Table 1 medicine is to the IC of hepatoma cell strain
50(pM)
aData show is mean+SD (n=3).
Table 1 shows that the conjugated protein antigenic Ch-hep-3 cell of high expressed SM5-1 is to the most responsive (IC of PE-NP-S
50=32.39pM).Moderate is expressed the conjugated protein antigenic Ch-hep-1 cell of SM5-1 to the also relatively responsive (IC of PE-NP-S
50=82.26pM).Yet the QGY7701 cell of expressing the conjugated protein antigen negative of SM5-1 is to the quite insensitive (IC of PE-NP-S
502000pM).Concerning Mut-I, we have obtained similar result, this means that PE-NP-S and Mut-I play key effect to the specific binding activity pair cell toxicity of expressing the conjugated protein antigenic breast carcinoma cell strain of SM5-1.Compare with Mut-I, PE-NP-S is to the bigger (IC of PE-NP-S of the cytotoxicity of Ch-hep-3 cell
50Be 32.39 ± 5.36pM, and the IC of Mut-I
50Be 82.26 ± 9.58pM (mean ± SD); Both have remarkable statistical significance, P＜0.05) (table 1).In the Ch-hep-1 cell, also obtained similar result.Yet, the mixture of the Fab ' of PE-NP and SM5-1 (Fab ' of SM5-1 measure be attached to PE-NP-S on the amount of Fab ' of SM5-1 the same), PE-NP-CD25 has very high IC to this three strains cell strain
50Value, the cytotoxicity that means PE-NP-S are to rely on the Fab ' that is combined in the SM5-1 on the nano-particle, rather than the Fab ' of free SM5-1.
The present inventor also utilizes simultaneously the NP and the NP-HER that do not wrap up PE38KDEL I to carry out above-mentioned cytotoxicity experiment, and the result shows and do not wrap up PE38KDEL I that these nano-particle are no cytotoxicity almost.
Experimental example 4: non-special toxicity and liver enzyme experiment
8 medicines that female BALB/c mouse intravenous injection 200 μ l dosage improve gradually, the mortality rate of observation animal in fortnight.LD
50Being defined as the dosage that kills the PE38KDEL I that 50% animal needs, is to draw by trimmed Spearman-Karbar statistical method.Liver injury is then by carrying out quantitative analysis (the ALT detection kit that Shanghai Vaccine and Serum Institute provides detects) to the enzymatic activity of ALT in the blood plasma.The medicine of various dose is injected into BALB/c mouse by intravenous mode, and nearly all death all occurs in injects within 3 days the LD of Mut-I and PE38KDEL I
50Value is 11.56mg/kg, and opposite is that the PE38KDEL I of PLGA parcel then can be good at being tolerated by mice: PE-NP-S, the LD of PE-NP and PE-NP-CD25
50Value is 52.79mg/kg.In order to assess the hepatotoxicity of PLGA parcel PE38KDEL I, mice is after the intravenous injection of accepting various medicines (dosage is 5mg/kg PE38KDEL I), we collected blood sample 3 hours and 24 hours, the dosage of the PE38KDEL I of PLGA parcel refers to PE-NP discharged PE38KDEL I in 24 hours in 37 ℃ cumulant, after injection Mut-I and PE38KDEL I, ALT level in the blood plasma significantly increases, yet, PE-NP-S in injection equivalent, after PE-NP and the PE-NP-CD25, the blood plasma level of ALT does not significantly change.
Experimental example 5: anti-PE TPPA experiment
Mice is to the IgG reaction experiment of PE38KDEL I: 24 female BALB/c mouse (～20g) be divided into 6 groups, the PE38KDEL I of every group of peritoneal immunity 1mg/kg in the time of the 1st, 14 day, blood sample is collected at the 21st day the 1st day immunity back.ELISA (enzyme-linked immunosorbent assay) dull and stereotyped with PE38KDEL I wrap by after, measure in the serum specific IgG antibodies by ELISA to PE38KDEL I.Measuring antibody is that (CA), DAB (3,3 '-diaminobenzidine) is as reaction substrate for zymed Lab, San Francisco for connection HRP (horseradish peroxidase) sheep anti-mouse igg antibody.Blood serum sample was collected in immunity in the 1st day in later the 21st day, measure mouse-anti PE38KDEL I IgG antibody horizontal in the serum with ELISA, the mensuration of PE-NP-S, Mut-I and relevant reference substance also together, the results are shown in Figure 4-1, the result shows, PE-NP-S compares its absorbance with Mut-I have significant difference, the generation of PE38KDEL I and Mut-I the anti-PE38KDEL I IgG of high titre.Opposite is that the immunogenicity of PE-NP-S is then very low.
Anti-PE neutrality experiment is to carry out with the male breast carcinoma cell strain of the conjugated protein antigen presentation of SM5-1: every hole inoculation 1 * 10 in 96 orifice plates
5The Ch-hep-3 cell was hatched 24 hours at 37 ℃.Second day, the 42nd day the plasma sample that mouse immune 1mg/kg PE38KDEL I (in the immunity of the 1st, 14 and 28 day difference once) collects the back after 30 minutes, was used culture fluid ten doubling dilutions 56 ℃ of hot deactivations.After 200pM PE-NP-S or Mut-I (PE38KDEL I dosage) are added to and hatch 30 minutes behind each diluting plasma sample, mixture and various contrast are all joined in the Ch-hep-3 cell, continued to hatch 48 hours at 37 ℃, the cells survival rate is with the cell non-radioactive cell proliferation test kit (Promega that tires, Madison, WI USA) measures, the results are shown in Figure 4-2, the result show Mut-I to the cell toxicant performance of Ch-hep-3 by anti-PE neutrality serum dose-dependent inhibition.Especially and the cells survival rate of 200pM Mut-I, the undiluted neutrality serum Ch-hep-3 of hatching altogether be 98%, this means that the cytoactive of Mut-I has been eliminated fully.Yet (data show is not mean+SD (n=4) .*** to PE-NP-S, P＜0.001 by the appreciable impact of anti-PE neutrality serum to the cytotoxicity of Ch-hep-3; The cells survival rate of PE-NP-S and Mut-I group relatively demonstrate significant difference (two-sample individual t test)).
We have also obtained similar result in the Ch-hep-1 cell.
Simultaneously, we also with ELISA confirmed anti-PE neutrality serum can not in and the Fab ' of SM5-1 or SM5-1.
Experimental example 6: anti-tumor activity experiment
5 * 10
6The Ch-hep-3 cell be subcutaneously injected in the 0th day the BALB/c nude mice (～20g) in, reached 50mm at the 5th day gross tumor volume
3Since the 5th day, mice began to accept intravenous drug, carried out twice of every day at the 5th, 7 and 9 day.Have eight treatment groups, be respectively PBS, PE38KDEL 1 (5mg/kg), PE-NP (5mg/kg) associating SM5-1 Fab ' (5.25mg/kg), PE-NP-CD25 (5mg/kg), PE-NP-S (0.5mg/kg), PE-NP-S (2.5mg/kg), Mut-I (0.5mg/kg), Mut-I (5mg/kg), every group all has 6 mouse, tumor with vernier caliper measurement was once observed 40 days in per 5 days altogether.Gross tumor volume=(wide
2* long)/2.All mouse are all from the Shanghai Chinese Academy of Sciences, and mouse is placed in the environment of no pathogenic microorganism and raises, raise a week after reuse do the experiment mice.
Experimental result is seen Fig. 5, and experimental result shows that Mut-I and PE-NP-S demonstrate dose dependent to the inhibition of tumor.For tumor regression completely, be defined as completely tumor and disappear and reach 50 days.We find that the Mut-I of 0.5mg/kg * 6 can make 1 complete tumor regression of mice.In the Mut-I of 5mg/kg * 6 treatment group, we have observed tumor regression completely in 6 mices.Yet the anti-tumor activity of PE-NP-S is greatly improved; In the PE-NP-S of 0.5mg/kg * 6 treatment group, 2 complete tumor regressions of mice are arranged; And in the PE-NP-S of 2.5mg/kg * 6 treatment group, all mices have reached tumor regression completely; These data have clearly shown the effectiveness that improves the immunotoxin therapeutic dose.On the contrary, treat without any anti-tumor activity up to the control drug of 5mg/kgPE38KDEL I.These data show give the effectiveness of higher immunotoxin therapeutic dose.
Comprehensive above-mentioned experimental example, as can be seen, PE-NP-S and Mut-I are to the LD of the toxicity of mice: PE-NP-S
50Be 52.79mg/kg, and the LD of Mut-I
50Be 11.56mg/kg, the PE-NP-S of injection 5mg/kg does not cause that liver significantly damages, and on the contrary, the Mut-I of 5mg/kg has severe impairment to liver, and after these results suggest PE38KDEL I wrapped up by PLGA, its toxicity reduced greatly.In addition, PE-NP-S has littler immunogenicity than Mut-I, and it is more insensitive at external antagonism PE neutrality antibody, after anti-PE TPPA has confirmed to hatch altogether with anti-PE neutrality serum, the cytotoxicity to the hepatoma cell strain of the conjugated protein antigen positive of SM5-1 of PE-NP-S is not obviously influenced, on the contrary, Mut-I has then completely lost its cytotoxicity.In addition, in pre-immune PE38KDEL I, the long anti-tumor activity that PE-NP-S in the conjugated protein antigen liver cancer mouse of high expressed SM5-1 and Mut-I arranged, although there is anti-PE neutrality antibody in blood circulation, PE-NP-S still demonstrates powerful anti-tumor activity, and Mut-I does not then have.The sensitivity that PE-NP-S reduces may be because the parcel of PLGA and its quick endocytosis.PE-NP-S has specific combination to the conjugated protein antigen of SM5-1, and almost completely by the conjugated protein antigen positive tumor cell of SM5-1 endocytosis.Because endocytosis optionally, PE-NP-S can effectively cause the conjugated protein antigen-positive cell death of SM5-1, and less to the conjugated protein antigen negative cytotoxicity of SM5-1.The reason of the conjugated protein antigen-positive cell of PE-NP-S selective killing SM5-1 is that the SM5-1 antibody fragment of nano grain surface has mediated endocytosis, then the killer cell that comes out of the drug release in the nano-particle.Bigger than Mut-I to the cytotoxicity of the conjugated protein antigen positive hepatoma cell strain of SM5-1 is because the targeted nano granule has higher transhipment, binding ability than immunotoxin.The anti-tumor in vivo experiment confirm anti-tumor activity of PE-NP-S be the twice of Mut-I, in addition, the toxicity in vivo of PE-NP-S is 1/5 of Mut-I, so the therapeutic efficiency of PE-NP-S has improved 10 times.The mechanism of PE-NP-S effective antitumour effect may be because the particulate two-phase transhipment of targeted nano: in first transports mutually, in tumor tissues, slowly assembling of nano-particle, and because enhanced infiltration and retention effect, nano-particle can be further enhanced in the concentration of tumor tissues; In second phase, interact by ligand-receptor, the targeted nano granule in conjunction with and by the tumor cell endocytosis.Further research about the tissue distribution of PE-NP-S is then underway.In addition, the particle diameter of nano-particle plays an important role to its distribution in vivo.The nano-particle of 100～200nm can be escaped engulfing of reticuloendothelial system, and circulation time in vivo reaches the longest.By the multiple emulsion solvent evaporation method, having obtained mean diameter is the PE-NP-S of 107.67 ± 27.6nm, has macrocyclic feature.What is more important, the present inventor has selected the conjugated protein antigenic Fab ' fragment of rhuMAbSM5-1, rather than complete antibody is used as the targeted molecular of PE-NP-S, this also can reduce tumor-associated macrophages to the engulfing of nano-particle, and nano-particle is also more extensive to the infiltration of solid tumor.
The present inventor has also carried out extracorporeal releasing experiment to disclosed PE-NP-S among the present invention, and (extracorporeal releasing experiment is with reference to Gaspar MM, Blanco D, Cruz ME, Alonso MJ.Formulation ofL-asparaginase-loaded poly (lactide-co-glycolide) nanoparticles:influence of polymer properties on enzyme loading, activity and in vitrorelease.J Control Release 1998; 52:53-62. carry out), experimental result shows: after 1 hour, only 2.9% PE38KDEL I discharges from PE-NP, make during neutrality antibody unlikely fully and PE, because behind the immunotoxin that inoculation PE makes up, animal can cause anti-PE immunoreation, and the sensitivity that PE-NP-S reduces neutrality antibody can increase the effectiveness of repeated application medicine, and these researchs provide good try for the neutrality antibody that overcomes at PE38KDEL I.PE-NP-S has littler immunogenicity, and reason may be less for the particle diameter of nano-particle, and the nano-particle of 100～200nm is still less by macrophage phagocytic, and macrophage phagocytic is playing an important role aspect the activate immunity reaction.
In a word, PE-NP-S demonstrates better anti-tumor activity, and has littler non-special toxicity and immunogenicity, and the sensitivity of antagonism PE neutrality antibody is lower.
Reference examples: wrap up proteic biologic activity experiment
Utilize the method identical (just not add 10% trehalose or add other additive (as 1% glycerol with embodiment 1,1%PEG400) substituting trehalose) PE-NP that obtains of PE-NP that obtains and the preparation method of utilizing embodiment 1 carried out the proteic biologic activity experiment of parcel (experimental technique referring to: with the FlexiRabbit Reticulocyte proteic biologic activity of the external protein translation kit measurement of Lysate System (Promega company)), the not IC of Bao Guo PE38KDEL
50Value (half-inhibition concentration) be 4.76 ± 2.25pmol (mean ± SD, n=3), and the IC of PE38KDEL behind the parcel
50Value is 4.99 ± 2.96pmol (mean ± SD, n=3), P〉0.05, both relatively do not have significant difference, show that PE38KDEL I that PE-NP that the preparation method of utilizing embodiment 1 obtains extracts has kept and the similar activity of PE38KDEL I of parcel not.And for not adding 10% trehalose or adding other additive (as 1% glycerol, 1%PEG400) substitute the PE-NP that trehalose utilizes embodiment 1 identical method to obtain, the activity of the PE38KDEL I that extraction obtains is compared with the PE38KDEL I that does not wrap up, and its activity all reduces greatly.
The present inventor has obtained the polylactic acid-glycolic guanidine-acetic acid copolymer nano granule that interior parcel Ricin connects humanization SM5-1 antibody outward according to disclosed method among the embodiment, has obtained and the consistent antitumous effect of above-mentioned test example.
In addition, Fab ' the fragment that the present inventor also utilizes the polylactic acid-glycolic guanidine-acetic acid copolymer of parcel PE38KDEL I type mutant to connect the Humanized monoclonal antibodies of anti-Her2 is used for the treatment of Her2 and expresses male tumor, has also obtained the excellent curative consistent with the object of the invention.
Claims (13)
1. the parcel immunotoxin connects the polylactic acid-glycolic guanidine-acetic acid copolymer nano granule of antibody outward in one kind.
2. the described nano-particle of claim 1, wherein immunotoxin is one of ricin, diphtheria toxin, diphtherotoxin, PE38KDELI type mutant, antibody is the Fab ' fragment of humanized antibody or humanized antibody.
3. the described nano-particle of claim 2 is that interior parcel PE38KDEL 1 type mutant connects humanization SM5-1 monoclonal antibody outward ' segmental polylactic acid-glycolic guanidine-acetic acid copolymer nano granule.
4. the described nano-particle of claim 3, its particle diameter is 70～140nm, and parcel efficient is 35～45%, and the antibody joint efficiency is 15～25 μ g/mg nano-particle.
5. a method for preparing the arbitrary described nano-particle of claim 1 to 4 comprises the at first nano-particle of preparation parcel immunotoxin, connects two steps of antibody at nano grain surface then.
6. the described method of claim 5, wherein the nano-particle step of preparation parcel immunotoxin comprises:
A) immunotoxin is dissolved in the aqueous solution and joins oil-based solvent together with polylactic acid-glycolic guanidine-acetic acid copolymer then, ultrasonicly obtain water in oil colostrum;
B) utilize the colostrum that a) obtains to prepare emulsion;
C) after emulsion joins in the poly-vinyl alcohol solution again, stir and make the organic solvent volatilization, solution is through centrifugal, washing, and low-temperature freeze drying promptly makes the finished product nano-particle.
7. the described method of claim 6, wherein the aqueous solution in the step a) is PBS, oil-based solvent is an ethyl acetate.
8. the described method of claim 6, wherein step a) also is included in the aqueous solution that has dissolved immunotoxin and adds trehalose.
9. the method for claim 5, wherein connect the antibody step and hatch jointly under comprising the nano-particle of parcel immunotoxin and EDC, NHS condition, will be dissolved in the antibody of HEPES (pH 7.4) or antibody fragment then and join in the sedimentary nanosphere lucifuge reaction and get in lucifuge at nano grain surface.
10. the purposes of the arbitrary described nano-particle of claim 1-4 in preparation medicine for treating tumor thing.
11. the described purposes of claim 10, wherein tumor is that conjugated protein antigen of SM5-1 or Her2 express male tumor.
12. the described purposes of claim 11, wherein the male tumor of the conjugated protein antigen presentation of SM5-1 is breast carcinoma, melanoma and hepatocarcinoma.
13. the described purposes of claim 12, wherein the male tumor of the conjugated protein antigen presentation of SM5-1 is a hepatocarcinoma.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101779370A CN101450215B (en) | 2007-12-04 | 2008-11-19 | Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside |
| CN201210167046.3A CN102697733B (en) | 2007-12-04 | 2008-11-19 | A kind of interior parcel immunotoxin connects the Poly(D,L-lactide-co-glycolide nano-particle of antibody outward |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710094374 | 2007-12-04 | ||
| CN200710094374.4 | 2007-12-04 | ||
| CN2008101779370A CN101450215B (en) | 2007-12-04 | 2008-11-19 | Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210167046.3A Division CN102697733B (en) | 2007-12-04 | 2008-11-19 | A kind of interior parcel immunotoxin connects the Poly(D,L-lactide-co-glycolide nano-particle of antibody outward |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101450215A true CN101450215A (en) | 2009-06-10 |
| CN101450215B CN101450215B (en) | 2013-08-07 |
Family
ID=40732812
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210167046.3A Active CN102697733B (en) | 2007-12-04 | 2008-11-19 | A kind of interior parcel immunotoxin connects the Poly(D,L-lactide-co-glycolide nano-particle of antibody outward |
| CN2008101779370A Active CN101450215B (en) | 2007-12-04 | 2008-11-19 | Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210167046.3A Active CN102697733B (en) | 2007-12-04 | 2008-11-19 | A kind of interior parcel immunotoxin connects the Poly(D,L-lactide-co-glycolide nano-particle of antibody outward |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN102697733B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103308698A (en) * | 2013-06-17 | 2013-09-18 | 北京北检·新创源生物技术有限公司 | Method for covalently coupling amino-containing molecules to microspheres |
| CN107670028A (en) * | 2017-11-23 | 2018-02-09 | 重庆更尚科技有限公司 | A kind of compound oil emulsion vaccine of Riemerellosis Anatipestifer inactivated vaccine and antibody and preparation method thereof |
| CN107714648A (en) * | 2017-11-23 | 2018-02-23 | 重庆更尚科技有限公司 | A kind of combination vaccine of immunogene and antibody and preparation method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100569288C (en) * | 2005-04-06 | 2009-12-16 | 山东蓝金生物工程有限公司 | A kind of entity-tumor-resistant medicine composition |
| WO2007021970A2 (en) * | 2005-08-15 | 2007-02-22 | Praecis Pharmaceuticals, Inc. | Stable pharmaceutical formulations and methods of use thereof |
| CN101077888A (en) * | 2006-05-24 | 2007-11-28 | 上海医药工业研究院 | Polyethylene glycol modified neurotoxin, preparation method and use thereof |
| CN101249262B (en) * | 2008-04-08 | 2011-02-16 | 中国人民解放军第二军医大学 | Targeted nano granule of humanization monoclonal antibody trastuzumab modified packaged toxin protein, and preparation and applications thereof |
-
2008
- 2008-11-19 CN CN201210167046.3A patent/CN102697733B/en active Active
- 2008-11-19 CN CN2008101779370A patent/CN101450215B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103308698A (en) * | 2013-06-17 | 2013-09-18 | 北京北检·新创源生物技术有限公司 | Method for covalently coupling amino-containing molecules to microspheres |
| CN103308698B (en) * | 2013-06-17 | 2015-04-29 | 北京北检·新创源生物技术有限公司 | Method for covalently coupling amino-containing molecules to microspheres |
| CN107670028A (en) * | 2017-11-23 | 2018-02-09 | 重庆更尚科技有限公司 | A kind of compound oil emulsion vaccine of Riemerellosis Anatipestifer inactivated vaccine and antibody and preparation method thereof |
| CN107714648A (en) * | 2017-11-23 | 2018-02-23 | 重庆更尚科技有限公司 | A kind of combination vaccine of immunogene and antibody and preparation method thereof |
| CN107670028B (en) * | 2017-11-23 | 2020-01-07 | 重庆更尚科技有限公司 | Composite oil emulsion vaccine of riemerella anatipestifer inactivated vaccine and antibody and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102697733A (en) | 2012-10-03 |
| CN102697733B (en) | 2015-08-12 |
| CN101450215B (en) | 2013-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Roncato et al. | Improvement and extension of anti-EGFR targeting in breast cancer therapy by integration with the Avidin-Nucleic-Acid-Nano-Assemblies | |
| US20230037294A1 (en) | Nanoparticle immunoconjugates | |
| US11883505B2 (en) | Anticancer drug-containing plant virus particles | |
| US20210220494A1 (en) | Compositions and methods for targeted particle penetration, distribution, and response in malignant brain tumors | |
| Bigini et al. | In vivo fate of avidin-nucleic acid nanoassemblies as multifunctional diagnostic tools | |
| CN101450215B (en) | Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside | |
| Zhang et al. | Investigation of an antitumor drug-delivery system based on anti-HER2 antibody-conjugated BSA nanoparticles | |
| JP2016534062A (en) | New stable formulation | |
| CN101249262B (en) | Targeted nano granule of humanization monoclonal antibody trastuzumab modified packaged toxin protein, and preparation and applications thereof | |
| Canato et al. | Anti‐HER2 Super Stealth Immunoliposomes for Targeted‐Chemotherapy | |
| Jeswani et al. | Recent advances in the delivery of chemotherapeutic agents | |
| JP2020523383A5 (en) | ||
| US20150071989A1 (en) | Liposomes for hematological tumors | |
| Gao et al. | Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly (lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F (ab′) fragments | |
| Liu et al. | Prostate‐Cancer‐Targeted N‐(2‐Hydroxypropyl) methacrylamide Copolymer/Docetaxel Conjugates | |
| Subasic et al. | Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats | |
| Jung et al. | Clathrin light chain‐conjugated drug delivery for cancer | |
| Wang et al. | Small Molecule–Drug Conjugates Emerge as a New Promising Approach for Cancer Treatment | |
| Collyer et al. | Selective Delivery of Clinically Approved Tubulin Binding Agents through Covalent Conjugation to an Active Targeting Moiety | |
| Kulkarni et al. | Theoretical Exploration of Nanoparticles Targeting Bacterial Prostatitis | |
| Valsalakumari et al. | Preclinical Efficacy of Cabazitaxel Loaded Poly (2-alkyl cyanoacrylate) Nanoparticle Variants | |
| Solidoro et al. | Fluorescent imaging probes for in vivo ovarian cancer targeted detection and surgery | |
| Duwa et al. | Strengthened Binding Affinity of Bispecific Antibody Nanoplatforms Improved the Anti-Tumor Efficacy | |
| Detampel | Targeting of hepatocytes Using vector-conjugated liposomes: evaluation of targeting strategies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: ANTIBODIES NATIONAL ENGINEERING RESEARCH CENTER Free format text: FORMER OWNER: SHANGHAI CP GUOJIAN PHARMACEUTICAL CO., LTD. Effective date: 20110401 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 201203 NO. 399, LIBING ROAD, ZHANGJIANG HIGH-TECH. PARK, PUDONG NEW DISTRICT, SHANGHAI TO: 201203 BUILDING 3, NO. 399, LIBING ROAD, ZHANGJIANG HIGH-TECH. PARK, PUDONG NEW DISTRICT, SHANGHAI |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20110401 Address after: Shanghai city 201203 libing road Zhangjiang High Tech Park of Pudong New Area No. 399 building 3 Applicant after: Antibodies National Engineering Research Center Address before: Shanghai city 201203 libing road Pudong New Area Zhangjiang hi tech Park No. 399 Applicant before: Shanghai CP Guojian Pharmaceutical Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |