WO2011053065A2 - 카테콜 폴리에틸렌글리콜 유도체와 단백질 또는 펩타이드의 접합체 및 이의 제조방법 - Google Patents
카테콜 폴리에틸렌글리콜 유도체와 단백질 또는 펩타이드의 접합체 및 이의 제조방법 Download PDFInfo
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- WO2011053065A2 WO2011053065A2 PCT/KR2010/007567 KR2010007567W WO2011053065A2 WO 2011053065 A2 WO2011053065 A2 WO 2011053065A2 KR 2010007567 W KR2010007567 W KR 2010007567W WO 2011053065 A2 WO2011053065 A2 WO 2011053065A2
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- WIPO (PCT)
- Prior art keywords
- polyethylene glycol
- methoxy
- protein
- catechol
- group
- Prior art date
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- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 179
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 103
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 102
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 title claims abstract description 90
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- 238000000034 method Methods 0.000 title claims abstract description 38
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- 125000003277 amino group Chemical group 0.000 claims abstract description 28
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- 239000000243 solution Substances 0.000 claims description 28
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- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 21
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
Definitions
- the present invention relates to a conjugate of a protein or peptide-catechol polyethylene derivative having a single conjugated polyethylene glycol derivative at the N-terminus of a protein or peptide using a polyethylene glycol derivative having a catechol, and a method for producing the same.
- proteins or peptides related to the growth and differentiation of our body are expressed. These proteins or peptides play a role in maintaining homeostasis by binding to receptors on the cell wall and inducing the expression of various signaling molecules.
- the amount of such proteins or peptides does not maintain an appropriate level in vivo, a problem occurs in the homeostasis due to a lack or overexpression of a signal transducing material in vivo, and various problems appear. For example, when the concentration of growth hormone related to human growth or epidermal growth factor related to wound healing is low, the height may not grow or the wound may not heal well.
- R. Canfield in 1962, F. Esch in 1985, S. Cohen in 1962, and D. Metcalf in 1985 were Lysozyme, a protein factor consisting of amino acids, and fiber.
- Subcellular growth hormone (bFGF), epidermal growth factor (EGF), granulocyte colony stimulating factor (G-CSF), etc. were found and their respective amino acid sequences were also identified (R. Canfield, J. Biol. Chem. 238, 2698). 1963; F. Esch, Proc. Natl. Acad. Sci . USA 82, 6507., 1985; S. Cohen, J. Biol. Chem . 237, 1555., 1962; D. Metcalf, Science 229, 16, 1985).
- PEG polyethylene glycol
- Polyethylene glycol is one of the synthetic polymers approved by the US Food and Drug Administration (FDA).
- FDA US Food and Drug Administration
- a protein polyethyleneglycol conjugate having an increased molecular weight can be obtained, thereby suppressing the permeation of the protein due to the filtration effect in the kidney.
- it exhibits the effect of inhibiting degradation by protein enzymes in vivo through the stealth effect of polyethylene glycol, thereby ultimately increasing the half-life and stability in vivo.
- Proteins that enhance the efficacy in this way are a variety of human growth hormone, erythropotin, interferon, insulin, interleukin, calcitonin and the like.
- polyethylene glycol when polyethylene glycol is used to improve the stability and persistence of protein drugs, polyethylene glycol reacts with a plurality of binding sites of protein drugs, resulting in a heterogeneous mixture of multi-conjugates.
- PEGylated species There has been a problem of being a PEGylated species.
- heterogeneous multiconjugate mixtures many problems arise in measuring the sustained effect and stability of the drug.
- Polyethylene glycol which has been used in the past, has a N-hydroxysuccinimide or N-succinimidyl propionate, which is a substance that reacts specifically with a primary amine, at one end.
- Methoxypolyethylene glycol derivatives TH Kim, Biomaterials, 23, 2311, 2002; H. Lee, Pharm. Res., 19, 845, 2002.
- Mono-pegylated conjugates prepared using succinimide polyethyleneglycol derivatives are known to have very low changes in activity in the body, but N-hydroxysuccine is not present during conjugation with proteins or peptides. Hydrolysis of the mead or N-succinimidylpropionate occurs rapidly, and there is a problem in the process of making a protein or peptide conjugate, resulting in a low yield of obtaining the conjugate.
- the inventors of the present invention when preparing a conjugate of a protein or peptide and the PEG derivative by using a PEG derivative having a catechol-linked compound, the process is simple, and the result is hydrolyzed, in particular, the protein or peptide
- the present invention was completed by confirming that site-specific mono-PEG (mono-PEGylated) conjugated conjugates in N-terminal primary amines can be produced in high yield.
- An object of the present invention is to provide a conjugate of a protein or peptide and a polyethylene glycol derivative in which a polyethylene glycol derivative is mono-bonded to the N-terminus of a specific portion of a protein or peptide, and a method for preparing the same.
- the present invention provides a polyethylene glycol derivative to which a compound having a catechol is bound, the derivative is mono-pegylated at the N-terminus of a specific region of a protein or peptide, and a protein or peptide. It provides a conjugate of the polyethylene glycol derivative and a method for producing the same.
- 1 is a schematic diagram of reaction between methoxy polyethylene glycol and 3,4-dihydroxycinnamic acid.
- 2 is the amino acid sequence of the hinge-3 peptide.
- Figure 3 is the RP-HPLC results of the result of mixing mPEG-CT and hinge-3.
- FIG. 5 is a schematic diagram showing a candidate candidate present in hinge-3.
- FIG. 6 is a graph of RP-HPLC results for fragments after trypsin digestion of PEG-hinge-3, wherein the peak at 13 minutes in black line is the T1 fragment, the peak at 13 minutes in red line is undiluted, and the peak at 23.5 minutes is Pegylated fragments.
- Figure 7 is a graph of MALDI-TOF results after trypsin digestion of PEG-hinge-3, mPEG (MW: 5000) or the peak of mPEG plus T1 fragment (MW: 5883).
- Figure 8 is a schematic diagram of the binding form of the catechol group and succinimidyl succinic acid.
- FIG. 10 shows SDS-PAGE results of succinimidyl succinic acid-PEG-bFGF (column 2) and catechol-PEG-bFGF (column 3) (10A), MALDI-TOF results of trypsin digestion of bFGF (MW: 2495, 10B). ) And MALDI-TOF result (MW: 7468, 10C) of trypsin digestion of catechol-PEG-bFGF.
- Figure 11 SDS-PAGE results of succinimidyl succinic acid-PEG-G-CSF (column 2) and catechol-PEG-G-CSF (column 3) (11A), MALDI-TOF results of trypsin digestion of G-CSF. (MW: 1792, 11B) and MALDI-TOF results of trypsin digestion of catechol-PEG-G-CSF (MW: 6792, 11C).
- Figure 12 is a SDS-PAGE gel photograph of the result of the addition of NalO4 in the ratio of 0, 0.5, 1, 1.5, 2: 1 for catechol at pH 6.0 in the lysozyme protein PEGylation reaction.
- Figure 13 is a graph showing the relative value of the PEGylation yield when NalO4 is added in a ratio of 0, 0.5, 1, 1.5, 2: 1 for catechol at pH 6.0 in the lysozyme protein PEGylation reaction.
- FIG. 14 shows SDS-PAGE results of mPEG-CT and EPO reaction mixtures (2 rows, 2 bands) and purified mPEG-CT-EPO (PEG-EPO, 3 rows, 1 band) using FPLC system.
- FIG. 15 is a graph of the RP-HPLC results, with black line peaks representing mPEG-CT-EPO (98 minutes), EPO mixture (140 minutes), red line peaks for each single-PEG (105 minutes), double-PEG (98 minutes), means multi-PEG (85 minutes).
- 16 is a graph showing in vivo half-life of single-PEG-EPO, multi-PEG-EPO, EPO.
- 17 is a graph measuring the relative volume ratio (hematocrit) of erythrocytes of single-PEG-EPO and EPO.
- the present invention is a protein or peptide-polyethylene characterized in that the polyethyleneglycol derivative to which the compound having the catechol of formula 1 is bound and the derivative are selectively conjugated as a single unit to the N-terminal amine group of the protein or peptide.
- the inventors of the present invention while studying to solve the problems of the known technology, found a functional group that specifically binds to the N-terminal amine group of the protein, which is a waveguide (3,4-dihydroxy-L-phenylalanine group) As an amino acid, a lot of mussel adhesives are known.
- Dopamine or, more broadly, catechol studies have shown that catechol groups can be adsorbed on various surfaces through research papers published by the inventors of the present invention. It has been demonstrated that it can be coated (H. Lee, PNAS, 103, 12999, 2006; H. Lee, Science, 318, 426, 2007; H. Lee, Adv. Mater. 21, 431, 2009).
- the present invention uses a polyethylene glycol derivative having reactivity by binding a compound having a catechol to polyethylene glycol.
- polyethylene glycol derivatives may be selectively bonded to N-terminal amine groups such as proteins or peptides to form conjugates.
- the polyethylene glycol derivatives may be conjugated to a specific site of a protein or peptide as a single molecule (site-specific). mono-PEGylation), while maintaining the physiological activity of the protein or peptide, it is possible to prepare a protein or peptide preparation with increased persistence and stability in vivo.
- there is an advantage that the reaction efficiency can be maintained for a long time by using a catechol group that is very stable to hydrolysis generated when preparing derivatives such as proteins / peptides.
- polyethylene glycol used in the present invention is a term generally used in the art in combination with PEG and Polyethyleneglycol, and also includes the concept of derivatives such as methoxyaldehyde polyethylene glycol.
- the polyethylene glycol used in the present invention is methoxyaldehyde polyethylene glycol, succinimide propionate polyethylene glycol, methoxy succinimide butyrate polyethylene glycol, methoxy succinimidyl succinate polyethylene glycol, methoxy benzotriazole carboxylic acid polyethylene glycol, Methoxy epoxide polyethylene glycol, methoxycarbonylimidazole polyethylene glycol, methoxy p-nitrophenyl carboxylic acid polyethylene glycol, methoxy isocyanate polyethylene glycol, methoxyamine polyethylene glycol including primary amine, methoxy high It is preferably one selected from the group consisting of dragged polyethylene glycol and methoxy carboxypolyethylene glycol including a carboxyl group, but is not limited thereto.
- the polyethylene glycol may react with an amine group or a carboxyl group, and may combine with a catechol group to form a derivative.
- polyethylene glycol may be any kind of polyethylene glycol, such as straight, branched, brushed, or star type.
- the compound having a catechol used in the present invention is a dihydroxyphenylalanine (3,4-dihydroxy-L-phenylalanine) containing a carboxyl group and an amine group, 3-hydroxy-tyramine (3-hydroxy-tyramine containing an amine group ), 3,4-dihydroxyhydrocinnamic acid containing a carboxyl group, 3,4-dihydroxybenzaldehyde containing a aldehyde group and norepinephrine It is preferably one selected from the group consisting of, but is not limited thereto. Furthermore, it is preferable that the molecular weight of the compound which has a catechol used by this invention is 1,000 or less.
- the molecular weight of the polyethyleneglycol derivative which concerns on this invention is 500-100,000, and it is more preferable that it is 2,000-40,000. It is difficult to secure a polyethylene glycol derivative having a molecular weight of more than 100,000, there is a manufacturing problem, if less than 500 polyethylene glycol is in a liquid form, there is a problem due to the physical properties, there is a problem of low reaction efficiency.
- the polyethylene glycol derivative When the polyethylene glycol derivative is conjugated to the protein, it is possible to obtain a conjugate of the protein-polyethylene glycol derivative having an increased molecular weight, and thus has the advantage of inhibiting the permeation of the protein by the filtration effect in the kidney. . In addition, it exhibits the effect of inhibiting degradation by protein enzymes in vivo through the stealth effect of polyethylene glycol, which means that the advantage of increasing half-life and stability in vivo.
- the conjugate of the protein or peptide-polyethylene glycol derivative according to the present invention is conjugated to a specific site as a single molecule as described above, there is an advantage that can prevent the problem that the heterogeneous conjugate mixture is produced. Through this, it is possible to obtain reliable data when measuring the sustaining effect and stability of the drug, and there is an advantage to easily prepare a drug having a desired biological activity or body activity. In addition, because it is conjugated to a specific region called the N-terminal, there is an advantage that can maximize the pharmacological effect without any chemical modification of protein drugs.
- the protein used in the present invention is lysozyme, fibroblast growth hormone (bFGF), granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), epidermal cell growth factor (EGF), human growth hormone (hGH), Interferon (IFN), interleukin-2 (IL-2), vascular epidermal cell growth hormone (VEGF), lutein hormone-expressing hormone (LHRH), growth hormone-expressing hormone (GHRH), mammalian urate oxidase (uricase) And arginine deiminase (arginine deiminase, ADI) may be one selected from the group consisting of, but is not limited thereto, the protein such as lysozyme, bFGF, EGF, GCSF, etc.
- prokaryotic host used above includes Escherichia coli
- the host of the eukaryotes includes Hanshenula polymorpha, Saccharomyces, and cerevisiae.
- Each protein can be obtained from mice, dogs, pigs and the like.
- the peptide used in the present invention is hinge-7 (Hinge-7), hinge-3 (Hinge-3), buforin (hiforin), histonein (histonin), protegrin (protegrin), indolicidin (indolicidin) ), Hisstatin, BIP, magainin 2, glucagon-like peptide (GLP-1), GNRH / LHRH competitor (GNRH / LHRHagonist), somatostatin analogue, immunity Regulatory Peptides Glatiramer, Salmon Calcitonin, Desmopressin, Platelet Coagulation Inhibiting Peptides, Eptifibatide, and Enfuvirtide, an HIV Cell Inhibitor It may be one selected from the group consisting of, but is not limited thereto.
- step (d) adding and reacting the solution of step (c) to the solution of step (a),
- It provides a method for producing a polyethylene glycol derivative is a compound having a catechol (Catechol) of the formula (1).
- the polyethyleneglycol derivative prepared by the above production method has a feature that can be conjugated to the N-terminal amine group of a protein or peptide as a single body.
- Steps (a) and (b) according to the invention are steps of preparing a solution of the reactants, respectively.
- a polar organic solvent may be used as a solvent for dissolving the substance.
- alcohol, dimethyl sulfoxide (DMSO), dimethylformaldehyde (DMF), acetone, and N-methylpyrrolidone (NMP) may be used.
- DMF can be used.
- DMF can prevent oxidation of the catechol group.
- the crosslinking agent added to the solution of the compound with the catechol is benzotriazolyloxytris (dimethylamino) phosphonium hexafluorophosphate
- hydroxybenzotriazole (HBdr) Ethyl- (N ', N'-dimethylamino) propylcarboiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N-hydroxysuccini Mead (N-Hydroxysuccinimide (NHS))
- sodium cyanoborohydride may be at least one selected from the group consisting of.
- crosslinking agent used for bonding an amine group and a carboxyl group HOBt and BOP; EDC and NHS; Or DCC and NHS.
- crosslinking agent used for bonding the amine group and the aldehyde group sodium cyanoborohydride may be used.
- the molar ratio of the compound having BOP: HOBt: catechol is preferably in the range of 1: 1: 1 to 10: 10: 1. This is because when the molar ratio is out of the above range, there may be a problem of inferior reactivity.
- Step (c) is a step of reacting by adding N, N-diisopropylethylamine (DIPEA) to the compound solution having a catechol prepared in step (b).
- DIPEA N, N-diisopropylethylamine
- Step (b) is a step of reacting by adding N, N-diisopropylethylamine (DIPEA) to the compound solution having a catechol prepared in step (b).
- DIPEA N, N-diisopropylethylamine
- Step (d) according to the present invention is a step of reacting by adding the solution prepared in step (c) to the solution prepared in step (a).
- the reaction of this step is preferably carried out at room temperature for about 10 to 14 hours. According to one embodiment of the present invention, it was confirmed that the compound having methoxy polyethylene glycol and catechol is conjugated to form a polyethylene glycol derivative by this step.
- Step (e) dialysis of the solution of step (d) according to the invention.
- Step (e) according to the present invention is a step of dialysis after removal of unreacted material.
- the dialysis is preferably performed for 10 to 14 hours using a dialysis membrane having a molecular weight removal amount of 2,000 to 10,000.
- the dialysis solution used at this time is preferably used distilled water having a pH of 1 to 6. If the pH in the dialysis process is out of the above range, there is a problem that the catechol may be oxidized, which is not preferable.
- Step (f) may further comprise the step of (f) freeze drying the solution dialyzed in step (e) according to the present invention.
- Step (f) according to the present invention is a step of lyophilizing the dialysis complete solution, to obtain a polyethylene glycol derivative in the form of a white powder. This can be carried out by a lyophilization method known to those skilled in the art, through which the solvent can be sublimed, to obtain a polyethylene glycol derivative in the form of a white powder. At this time, further drying may be performed for complete removal of the solvent.
- the present invention also provides
- step (c) adding the solution of step (a) to the solution of step (b) and reacting it;
- a method for preparing a conjugate of a protein or peptide and a polyethylene glycol derivative in which the polyethylene glycol derivative is conjugated to the N-terminal amine group of the protein or peptide as a single unit.
- Step (a) is a step of dissolving a protein or peptide having an N-terminal amine group in a reactor.
- the buffer solution used is phosphate buffer solution, imidazole buffer solution, trimethylfildine buffer solution, triethanolamine buffer solution, sodium diethylbarbiturate buffer solution, bicine buffer solution, Any one selected from aminomethylpropanediol buffers can be used.
- the pH of the buffer solution is preferably 5.5 to 10. If the pH of the buffer solution is less than 5.5, there is a problem that the reactivity is lowered, if it exceeds 10 there is a problem that the stability of the protein or peptide drug is lowered.
- Step (b) according to the present invention is a step of dissolving the polyethylene glycol derivative provided in the present invention in another reactor. Preferably it may be dissolved in a buffer solution as in step (a).
- Step (c) according to the present invention is a step of reacting by adding the solution of step (a) according to the present invention to the solution of step (b), and performing a PEGylation reaction.
- the PEGylation reaction is preferably performed at 4 to 35 ° C. for 2 to 100 hours.
- the oxidizing agent which can increase the activity of the polyethylene glycol derivative is preferably a group consisting of NaIO 4 , MnCl 2 , FeCl 2 , FeCl 3 , KMnO 4 , H 2 O 2 , Na 2 Cr 2 O 7 , Na 3 VO 4 It may be one or more selected from.
- the oxidizing agent may be added at the time of dissolving the polyethylene glycol derivative to proceed with the reaction.
- the oxidizing agent to be added preferably has a mole ratio of 1: 1 to 1:10 with the catechol group.
- the reaction may proceed by adding NaIO 4 in a molar ratio of 1.5: 1.
- reaction time is less than 2 hours, there is a problem that the conjugation efficiency of polyethylene glycol is lowered, and if it exceeds 100 hours, there is a problem that the stability of the protein or peptide is lowered.
- reaction temperature is less than 4 ° C there is a problem that the reaction rate is too slow, and if it exceeds 35 ° C there is a problem that the protein or peptide can be modified.
- step (d) is a step of dialysis or separation using liquid chromatography (HPLC or FPLC) after completion of the PEGylation reaction to remove unreacted material.
- the step may be carried out using a dialysis method, or liquid chromatography method that is apparent to those skilled in the art.
- PEG polyethylene glycol
- MPEG-CT was prepared in the same manner as in Example 1 to conjugate the polyethylene glycol derivative and the peptide (FIG. 1). That is, mPEG-amine (MW: 5k) and HCA were reacted with HOBt, BOP, and DIPEA in a DMF solution to obtain mPEG-CT.
- Example 2- (1) In order to confirm whether the form of the PEG-hinge-3 conjugate prepared in Example 2- (1) is a form in which PEG is conjugated to the hinge-3 peptide as a monolith, RP-HPLC (Reverse phase-High performance liquid chromatography and MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time of Flight) analyzes were performed.
- RP-HPLC Reverse phase-High performance liquid chromatography
- MALDI-TOF Microx-Assisted Laser Desorption Ionization Time of Flight
- HPLC system was used Agilent 1200 series and column was supelco Discovery® BIO Wide Pore C18 5cm * 4.6mm, 3 ⁇ m.
- Acetonitrile solution (0.1% trifluoroacetic acid (TFA)
- water (0.1% trifluoroacetic acid (TFA)
- TFA trifluoroacetic acid
- MALDI-ToF analysis instrument used a Voyager DE-STR instrument from Applied Biosystems, Inc.
- MALDI-TOF analysis also detected only PEG (MW: 5000) and single PEG-hinge-3 (MW: 7238).
- PEG MW: 5000
- PEG-hinge-3 MW: 7238
- hinge-3 has 20 amino acids (see Figure 2).
- the hinge-3 sequence contains only one tryptophan amino acid (see FIG. 2), it was possible to determine whether PEG was conjugated only at the N-terminus when the hinge-3 protein was degraded by trypsin digestion.
- the hinge-3 peptide does not have cysteine, it is dissolved in 4M urea, and after addition of a small amount of calcium chloride (CaCl 2 ), the amount of trypsin is added at a rate of 1/20 of protein. Dissolved and trypsin digestion was performed at 37 ° C. for 12 hours. Thereafter, RP-HPLC analysis was performed.
- CaCl 2 calcium chloride
- proteins can also be N-terminal pegylation.
- succinimidyl succinate which can form covalent bonds with surface exposed primary amine groups.
- this method has a problem that is not location specific.
- susinimidyl succinic acid reacts with random primary amine groups (FIG. 8).
- lysozyme as an example of protein, PEG-lysozyme conjugates were prepared using mPEG-SS in order to compare catechol group and susinimidyl succinic acid.
- a PEG-lysozyme conjugate was prepared by adding 20 mg of mPEG-SS and 5 mg of lysozyme at pH 8.5, which are generally known conditions, and reacting at 4 ° C. for 12 hours.
- SDS PAGE Gel having an acrylamide fraction of 15% was prepared and subjected to voltage to separate lysozyme and PEG-lysozyme conjugates according to size.
- the first column shows only lysozyme as a control
- the second column shows the PEG-lysozyme conjugate of Example 3- (1)
- the third column shows the PEG-lysozyme conjugate of Example 3- (2).
- the result is.
- the conjugate prepared using the methoxy-polyethylene glycol susinimidyl succinic acid (mPEG-SS) in the second row reacted with three amine groups (three bands appeared) (K33, K97, K116).
- the PEG-lysozyme conjugate prepared using mPEG-CT showed only one band, indicating that it reacted with only one amine group.
- the susinimidyl succinic acid-PEG derivative in the case of catechol-PEG derivatives conjugated with the protein, it was confirmed that the conjugate in the form of a single body.
- bFGF (MW: 17.1 kDa) is a basic Fibroblast Growth Factor and is a type of FGF. In adult organs, it functions for wound response and tissue regeneration.
- a PEG-bFGF conjugate was prepared using mPEG-CT in the same manner as in Example 2- (1), and as a result, a PEG-bFGF conjugate was confirmed.
- PEG-bFGF conjugates were prepared using mPEG-SS in the same manner as in Example 3- (1).
- Example 2- (2) PEG-bFGF was confirmed to be PEGylated in a single body through SDS-PAGE analysis and MALDI-ToF analysis. Only pH values were tested differently. This is because all environments of N-terminal amines have different pKa values. bFGF was observed whether PEGylation at pH 6.5.
- the protein loaded in column 2 of 10A was PEGylated at pH 6.5 using mPEG-CT, and two bands of the existing bFGF band and the band of mono-pegylated PEG-bFGF conjugate were observed.
- the loaded protein is a monopegylated PEG-bFGF conjugate that has been purified by FPLC separation.
- the protein-polyethylene glycol conjugate was prepared using the catechol-polyethylene glycol derivative, it was confirmed that the polyethylene glycol was conjugated to the protein as a single body.
- the PEGylation position was confirmed by trypsin digestion of PEG-bFGF in the same manner as in Example 2- (3).
- the T1 fragment peak was observed at 7468. Since the molecular weight of the T1 fragment of unmodified bFGF is 2495 (FIG. 10B), it is clear that the 7468 peak is the addition of mPEG-CT (MW: 5000) and the T1 segment of bFGF (MW: 2495). -Proof of terminal pegylation. As a result, the PEG-bFGF conjugate prepared using mPEG-CT was confirmed that PEG was conjugated to the N-terminus of the bFGF protein in the form of a monomer.
- G-CSF Granulocyte Colony-Stimulating Factor
- PEG-G-CSF conjugate was prepared using mPEG-SS in the same manner as in Example 3- (1).
- PEGylation using mPEG-CT showed only the mechanism G-CSF and mono-pegylated G-CSF bands (column 2), which was purified by FPLC and mono-pegylated. Only G-CSF could be isolated (column 3). This confirmed that PEGylation was monolithic for the G-CSF protein.
- the MALDI-TOF result of trypsin digestion of G-CSF showed a molecular weight of 1792.
- the molecular weight of T1 was 6792 as a result of MALDI-TOF of trypsin digestion of catechol-PEG-G-CSF. Therefore, the above results are thought to be the result of the addition of the T1 fragment and PEG (MW: 5000). Therefore, unlike the susinimidyl succinic acid-PEG derivative, the catechol-PEG derivative is conjugated with the protein in the form of a monolith. Confirmed to be bonded.
- Examples 1 to 5 successfully synthesized a polyethylene derivative (PEG-CT) to which a compound having a catechol is bound, and when the PEG-CT is conjugated to a protein or peptide specifically N- terminal specific It was confirmed that binding is possible.
- PEG-CT polyethylene derivative
- alpha amines N-terminal amines
- epsilon amines lysine residue amines
- Example 7 Check in vivo duration of PEG-EPO conjugates (in vivo)
- Erythropoietin (MW: 30kDa) is a hormone produced by the kidney, which promotes the production of red blood cells in the bone marrow. The most important function is to promote the differentiation and development of red blood cells.
- mPEG-CT (MW: 30kDa) was used because PEGylation of at least 20kDa PEG could be effective in an in vivo environment.
- FIGS. 14 and 15 The results are shown in FIGS. 14 and 15. As can be seen from the SDS-PAGE gel results, after the FPLC process, the EPO mixture (FIG. 15 black line, 140 minutes) and mPEG-CT-EPO (PEG-EPO, FIG. 14 second line or FIG. 15 black line, 98). Min) was completely purified (third line of Figure 14). Pegylation yield was 77%. Single-pegylated EPO was identified from the MALDI-ToF results (data not shown).
- mPEG-SS (MW: 20 kDa) was used as a protein multi-peglation control. Single PEGylated EPO was detected at 105 minutes and double PEGylated EPO was detected at 95 minutes and purified by FPLC (FIG. 15, red line). Peaks were detected at 85 minutes, indicating conjugated and multi-pegylated proteins. Pegylation was much lower yield of mPEG-SS than mPEG-CT, but for convenience of comparison, the height is indicated in the figure.
- a multi-pegylated EPO i.e., multiple PEG-EPO prepared using mPEG-SS prepared in Example 7- (1), was used as a positive control. .
- the amount of 100ug protein / kg (weight of the rat) was injected into the rat by intravenous injection of EPO, and the time of circulating in vivo was measured.
- Five rats were used for each protein, and each rat was injected with ⁇ 2.5 ug of protein. This is an amount corresponding to an EPO of 6.4 * 10 4 mIU, assuming a conversion factor of 2.56 * 10 4 mIU.
- Plasma proteins were quantified by ELISA, and the quantitative values were adjusted according to the affinity of different ELISA antibodies of each protein sample, PEG-EPO, multiple PEG-EPO and EPO.
- hematocrit the relative volume ratio of red blood cells (hematocrit) to the volume of blood in the blood sample was measured.
- a catechol-PEG derivative can be linked to a N-terminal amine group, which is a specific site of a protein or peptide, in a positional and single molecule to obtain a homogeneous polyethylene glycol protein- or peptide-conjugate in high yield.
- the obtained conjugate can minimize the inhibition of protein activity without chemical modification of the protein itself compared to the prior art, the pharmaceutical effect of the conjugate is excellent, the homogeneity can simplify the manufacturing process, and also the biological in vivo Since the drug efficacy is uniformly predicted and resistant to hydrolysis, and the in vivo duration is long, there is an effect of enhancing the drug efficacy and stability of the protein drug in the body.
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Abstract
Description
Claims (30)
- 제1항에 있어서, 상기 유도체는 단백질 또는 펩타이드의 N-말단 아민기에 단일체로 접합할 수 있는 것을 특징으로 하는 폴리에틸렌글리콜 유도체.
- 제1항에 있어서, 상기 폴리에틸렌글리콜은 메톡시알데히드폴리에틸렌글리콜, 숙신이미드프로피온산폴리에틸렌글리콜, 메톡시숙신이미드부탄산폴리에틸렌글리콜, 메톡시숙신이미딜숙신산폴리에틸렌글리콜, 메톡시벤조트리아졸카르본산폴리에틸렌글리콜, 메톡시에폭시드폴리에틸렌글리콜, 메톡시카르보닐이미다졸폴리에틸렌글리콜, 메톡시p-니트로페닐카르본산폴리에틸렌글리콜, 메톡시이소시안산폴리에틸렌글리콜, 1차아민을 포함하는 메톡시아민폴리에틸렌글리콜, 메톡시하이드라지드폴레에틸렌글리콜 및 카르복실기를 포함하는 메톡시카르복실폴리에틸렌글리콜로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 폴리에틸렌글리콜 유도체.
- 제1항에 있어서, 상기 폴리에틸렌글리콜은 직선형, 가지형, 브러시형 및 스타형으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 폴리에틸렌글리콜 유도체.
- 제1항에 있어서, 상기 카테콜을 갖는 화합물은 카복실기와 아민기를 포함하는 다이하이드록실페닐알라닌 (3,4-dihydroxy-L-phenylalanine), 아민기를 포함하는 3-하이드록시-타이라민 (3-hydroxy-tyramine), 카복실기를 포함하는 3,4-다이하이드록시하이드로시나믹산 (3,4-dihydroxyhydrocinnamic acid), 알데히드기를 포함하는 3,4 다이하이드록시벤즈알데히드(3,4-dihydroxybenzaldehyde) 및 노어에피네프린 (Norepinephrine)으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 폴리에틸렌글리콜 유도체.
- 제1항에 있어서, 상기 카테콜을 갖는 화합물의 분자량은 1,000 이하인 것을 특징으로 하는 폴리에틸렌글리콜 유도체.
- 제1항에 있어서, 상기 유도체의 분자량은 500 내지 100,000인 것을 특징으로 하는 폴리에틸렌글리콜 유도체.
- 제8항에 있어서, 상기 폴리에틸렌글리콜은 메톡시알데히드폴리에틸렌글리콜, 숙신이미드프로피온산폴리에틸렌글리콜, 메톡시숙신이미드부탄산폴리에틸렌글리콜, 메톡시숙신이미딜숙신산폴리에틸렌글리콜, 메톡시벤조트리아졸카르본산폴리에틸렌글리콜, 메톡시에폭시드폴리에틸렌글리콜, 메톡시카르보닐이미다졸폴리에틸렌글리콜, 메톡시p-니트로페닐카르본산폴리에틸렌글리콜, 메톡시이소시안산폴리에틸렌글리콜, 1차아민을 포함하는 메톡시아민폴리에틸렌글리콜, 메톡시하이드라지드폴리에틸렌글리콜 및 카르복실기를 포함하는 메톡시카르복실폴리에틸렌글리콜로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 제8항에 있어서, 상기 폴리에틸렌글리콜은 직선형, 가지형, 브러시형 및 스타형으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 제10항에 있어서, 상기 카테콜을 갖는 화합물은 카복실기와 아민기를 포함하는 다이하이드록실페닐알라닌 (3,4-dihydroxy-L-phenylalanine), 아민기를 포함하는 3-하이드록시-타이라민 (3-hydroxy-tyramine), 카복실기를 포함하는 3,4-다이하이드록시하이드로시나믹산 (3,4-dihydroxyhydrocinnamic acid), 알데히드기를 포함하는 3,4 다이하이드록시벤즈알데히드(3,4-dihydroxybenzaldehyde) 및 노어에피네프린 (Norepinephrine)으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 제8항에 있어서, 상기 카테콜을 갖는 화합물의 분자량은 1,000 이하인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 제8항에 있어서, 상기 폴리에틸렌글리콜 유도체의 분자량은 500 내지 100,000인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 제8항에 있어서, 상기 단백질은 라이소자임(lysozyme), 섬유아세포성장호르몬(bFGF), 과립구콜리니자극인자(GCSF), 에리스로포이에틴(EPO), 상피세포성장인자(EGF), 인간성장호르몬(hGH), 인터페론(IFN), 인터루킨-2(IL-2), 혈관표피세포성장호르몬(VEGF), 루트르이틴호르몬발현호르몬(LHRH), 성장호르몬발현호르몬(GHRH), 유리케이즈(mammalian urate oxidase, uricase), 및 아르기닌 디이미네이즈(arginine deiminase, ADI)로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 제8항에 있어서, 상기 펩타이드는 힌지-7(Hinge-7), 힌지-3(Hinge-3), 부포린(buforin), 히스토닌(histonin), 프로테그린(protegrin), 인돌리시딘(indolicidin), 히스타틴(histatin), BIP, 마가이닌 2(magainin 2), 글루카곤 유사 펩타이드(glucagon-like peptide: GLP-1), GNRH/LHRH 경쟁자(GNRH/LHRHagonist), 소마토스타틴 유사체(somatostatin analogue), 면역조절 펩타이드인 글라티라머(glatiramer), 살몬 칼시토닌(salmon calcitonin), 데스모프레신(desmopressin), 혈소판 응고 억제 펩타이드, 엡티피바타이드(eptifibatide), 및 HIV 세포 유입억제제인 엔푸버타이드(enfuvirtide)로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체.
- 다음의 단계를 포함하는, 하기 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법:화학식 1(a) 반응조 내의 극성유기용매에 폴리에틸렌글리콜을 용해시키는 단계;(b) 다른 반응조 내의 극성유기용매에 가교제 및 화학식 1의 카테콜을 갖는 화합물을 용해시키는 단계;(c) 상기 (b) 단계에서 제조된 용액에 N,N-디이소프로필에틸아민 (N,N-Diisopropylethylamine; DIPEA)를 첨가하여 반응시키는 단계; 및(d) 상기 (a) 단계의 용액에 상기 (c) 단계의 용액을 첨가하여 반응시키는 단계.
- 제16항에 있어서, 상기 극성유기용매는 알콜, DMSO(Dimethyl sulfoxide), DMF(Dimethylformaldehyde), 아세톤(Acetone) 및 NMP(N-Methylpyrrolidone)로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법.
- 제16항에 있어서, 상기 폴리에틸렌글리콜은 메톡시알데히드폴리에틸렌글리콜, 숙신이미드프로피온산폴리에틸렌글리콜, 메톡시숙신이미드부탄산폴리에틸렌글리콜, 메톡시숙신이미딜숙신산폴리에틸렌글리콜, 메톡시벤조트리아졸카르본산폴리에틸렌글리콜, 메톡시에폭시드폴리에틸렌글리콜, 메톡시카르보닐이미다졸폴리에틸렌글리콜, 메톡시p-니트로페닐카르본산폴리에틸렌글리콜, 메톡시이소시안산폴리에틸렌글리콜, 1차아민을 포함하는 메톡시아민폴리에틸렌글리콜, 메톡시하이드라지드폴리에틸렌글리콜 및 카르복실기를 포함하는 메톡시카르복실폴리에틸렌글리콜로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법.
- 제16항에 있어서, 상기 가교제는 벤조트리아졸릴옥시트리스 (다이메틸아미노) 포스포니움 헥사플루오로포스페이트 (Benzotriazolyloxytris (dimethylamino) phosphonium Hexafluorophosphate; BOP), 하이드록시벤조트리아졸 (Hydroxybenzotriazole; HOBt), 에틸-(다이메틸아미노)프로필카르보이미드 하이드로클로라이드 (ethyl-(N', N'-dimethylamino)propylcarboiimide hydrochloride; EDC), 다이싸이클로헥실카르보이미드 (Dicyclohexylcarbodiimide; DCC), N-하이드록시숙시니미드(N-Hydroxysuccinimide; NHS) 및 소듐 사이아노보로하이드라이드(Sodium Cyanoborohydride)로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법.
- 제16항에 있어서, 상기 카테콜을 갖는 화합물은 카복실기와 아민기를 포함하는 다이하이드록실페닐알라닌 (3,4-dihydroxy-L-phenylalanine), 아민기를 포함하는 3-하이드록시-타이라민 (3-hydroxy-tyramine), 카복실기를 포함하는 3,4-다이하이드록시하이드로시나믹산 (3,4-dihydroxyhydrocinnamic acid), 알데히드기를 포함하는 3,4 다이하이드록시벤즈알데히드(3,4-dihydroxybenzaldehyde) 및 노어에피네프린 (Norepinephrine)으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법.
- 제16항에 있어서, 상기 (d) 단계의 용액을 투석하는 (e) 단계를 추가로 포함하는 것을 특징으로 하는 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법.
- 제21항에 있어서, 상기 (e) 단계에서 투석된 용액을 동결건조하는 (f) 단계를 추가로 포함하는 것을 특징으로 하는 화학식 1의 카테콜(catechol)을 갖는 화합물이 결합되어 있는 폴리에틸렌글리콜 유도체의 제조방법.
- 다음의 단계를 포함하는, 제1항 내지 제6항 중 어느 한 항의 폴리에틸렌글리콜 유도체가 단백질 또는 펩타이드의 N-말단 아민기에 단일체로 접합되어 있는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법:(a) 반응조 내에 단백질 또는 펩타이드를 용해시키는 단계;(b) 다른 반응조 내에 상기 폴리에틸렌글리콜 유도체를 용해시키는 단계; 및(c) 상기 (a) 단계의 용액을 (b) 단계의 용액에 첨가하여 반응시키는 단계.
- 제23항에 있어서, 상기 폴리에틸렌글리콜은 메톡시알데히드폴리에틸렌글리콜, 숙신이미드프로피온산폴리에틸렌글리콜, 메톡시숙신이미드부탄산폴리에틸렌글리콜, 메톡시숙신이미딜숙신산폴리에틸렌글리콜, 메톡시벤조트리아졸카르본산폴리에틸렌글리콜, 메톡시에폭시드폴리에틸렌글리콜, 메톡시카르보닐이미다졸폴리에틸렌글리콜, 메톡시p-니트로페닐카르본산폴리에틸렌글리콜, 메톡시이소시안산폴리에틸렌글리콜, 1차아민을 포함하는 메톡시아민폴리에틸렌글리콜, 메톡시하이드라지드폴리에틸렌글리콜 및 카르복실기를 포함하는 메톡시카르복실폴리에틸렌글리콜로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
- 제23항에 있어서, 상기 카테콜을 갖는 화합물은 카복실기와 아민기를 포함하는 다이하이드록실페닐알라닌 (3,4-dihydroxy-L-phenylalanine), 아민기를 포함하는 3-하이드록시-타이라민 (3-hydroxy-tyramine), 카복실기를 포함하는 3,4-다이하이드록시하이드로시나믹산 (3,4-dihydroxyhydrocinnamic acid), 알데히드기를 포함하는 3,4 다이하이드록시벤즈알데히드(3,4-dihydroxybenzaldehyde) 및 노어에피네프린 (Norepinephrine)으로 이루어진 군에서 선택되는 하나인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
- 제23항에 있어서, 상기 (c) 단계의 용액을 투석한 다음 접합체를 분리하는 (d) 단계를 추가로 포함하는 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
- 제26항에 있어서, 상기 (d) 단계에서 접합체를 분리하는 방법은 액상크로마토그래피를 이용하는 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
- 제23항에 있어서, 상기 (b) 단계는 폴리에틸렌글리콜 유도체와 산화제를 1:1 내지 1:10의 몰비로 하여 반응조 내에서 용해시키는 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
- 제28항에 있어서, 상기 산화제는 NaIO4, MnCl2, FeCl2, FeCl3, KMnO4, H2O2, Na2Cr2O7, Na3VO4로 구성된 군에서 선택되는 하나 이상인 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
- 제23항에 있어서, 상기 (b) 단계의 반응은 2 내지 100 시간동안 4 내지 25도의 온도에서 수행되는 것을 특징으로 하는 단백질 또는 펩타이드와 폴리에틸렌글리콜 유도체의 접합체의 제조방법.
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US11077195B2 (en) | 2019-02-06 | 2021-08-03 | Synthorx, Inc. | IL-2 conjugates and methods of use thereof |
US11834689B2 (en) | 2017-07-11 | 2023-12-05 | The Scripps Research Institute | Incorporation of unnatural nucleotides and methods thereof |
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US10610571B2 (en) | 2017-08-03 | 2020-04-07 | Synthorx, Inc. | Cytokine conjugates for the treatment of proliferative and infectious diseases |
US11622993B2 (en) | 2017-08-03 | 2023-04-11 | Synthorx, Inc. | Cytokine conjugates for the treatment of autoimmune diseases |
US11701407B2 (en) | 2017-08-03 | 2023-07-18 | Synthorx, Inc. | Cytokine conjugates for the treatment of proliferative and infectious diseases |
US11077195B2 (en) | 2019-02-06 | 2021-08-03 | Synthorx, Inc. | IL-2 conjugates and methods of use thereof |
Also Published As
Publication number | Publication date |
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WO2011053065A9 (ko) | 2011-08-25 |
ES2617713T3 (es) | 2017-06-19 |
JP5657011B2 (ja) | 2015-01-21 |
JP2013509402A (ja) | 2013-03-14 |
EP2495258A4 (en) | 2015-10-07 |
EP2495258A2 (en) | 2012-09-05 |
US20120208258A1 (en) | 2012-08-16 |
US8604127B2 (en) | 2013-12-10 |
EP2495258B1 (en) | 2016-12-07 |
WO2011053065A3 (ko) | 2011-10-27 |
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