CN104288777A - Antibody-polymer combined body and fluorescent derivative thereof and preparation method of antibody-polymer combined body and fluorescent derivative - Google Patents

Antibody-polymer combined body and fluorescent derivative thereof and preparation method of antibody-polymer combined body and fluorescent derivative Download PDF

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CN104288777A
CN104288777A CN201410480011.4A CN201410480011A CN104288777A CN 104288777 A CN104288777 A CN 104288777A CN 201410480011 A CN201410480011 A CN 201410480011A CN 104288777 A CN104288777 A CN 104288777A
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macromolecule
antibody
trastuzumab
fluorescent derivative
coalition
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CN104288777B (en
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高卫平
张立彬
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Tsinghua University
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Abstract

The invention discloses an antibody-polymer combined body and a fluorescent derivative thereof and a preparation method of the antibody-polymer combined body and the fluorescent derivative. The antibody comprises at least one disulfide bond or free sulfydryl; the polymer is combined to the antibody through connection to the disulfide bond or free sulfydryl of the antibody; according to the method, a specific site of the antibody is modified; a polymer and a fluorescent derivative thereof grow in situ; or a polymer and a fluorescent derivative thereof are prepared in advance; and the antibody-polymer combined body and the fluorescent derivative thereof are prepared in a manner of specific site coupling of the antibody. The method disclosed by the invention is simple, convenient and efficient; the prepared product not only reserves the original biological activity of the antibody, but also improves the water solubility, stability, pharmacokinetics, distribution of organisms and treatment efficacy; the immunogenicity is reduced; in addition, compared with an antibody-fluorescence indicator prepared by the traditional method, the antibody-polymer fluorescent derivative prepared by the method has stronger fluorescence signal and detection signal strength.

Description

Antibody-macromolecule coalition, its fluorescent derivative and their preparation method
Technical field
The present invention relates to biomedicine field, relate to the preparation method of antibody-macromolecule coalition and fluorescent derivative thereof particularly.
Background technology
Antibody, as a kind of immune protein most important in organism, has been widely used in multiple fields such as biological medicine development, targeted therapy and clinical diagnosis.Be used alone antibody and there is the problems such as the half-life is short, poorly soluble.Be connected macromolecule with antibody Dispersal risk-macromolecule coalition, effectively can improve the dissolubility of antibody, stability, pharmacokinetics and therapeutic efficiency and reduce its immunogenicity.The synthetic method of traditional antibody-macromolecule coalition is connected with antibody by previously prepared good macromolecule, often also exists that conjugation sites is uncertain, efficiency is low, productive rate is poor, product is difficult to be separated, quality control is poor, activity is difficult to many difficult problems such as maintenance (reaction site is near antibody activity site).Therefore, in the urgent need to designing a kind of general method, effectively to solve an above-mentioned difficult problem.
Summary of the invention
The object of this invention is to provide efficient, the synthesis site-specific antibody-macromolecule coalition of high yield and the method for fluorescent derivative thereof, in order to improve the dissolubility of antibody, stability, pharmacokinetics and therapeutic efficiency and to reduce its immunogenicity.
The invention provides the preparation method of site-specific antibody-macromolecule coalition and fluorescent derivative thereof, described antibody comprises at least one disulfide bond or sulfydryl freely, and described macromolecule is by being connected to disulfide bond or the sulfydryl and being attached on described antibody freely of described antibody.
Described preparation method can comprise two kinds: the first is situ aggregation method, and described method comprises: a1) coalition of Dispersal risk-initiator, wherein said initiator be attached to described antibody disulfide bond or freely on sulfydryl; B1) described antibody-initiator coalition mixes with high polymer monomer or with high polymer monomer and fluorescent monomer in buffer, causes the polymerization of described high polymer monomer and prepare the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition under catalyst action.Preferably, in step a1) in, the disulfide bond of described antibody is reduced into sulfydryl freely, and then described initiator is connected on described sulfydryl.Situ aggregation method, by carrying out modification at the specific site of antibody and growth in situ macromolecule, has efficient, high yield, high activity conservation rate feature.
The second is Graft Method, Graft Method is previously prepared macromolecule, again with specific site coupling Dispersal risk-macromolecule coalition and the fluorescent derivative thereof of antibody, described method comprises: a2) initiator causes high polymer monomer or high polymer monomer and fluorescent monomer polymerization under catalyst action, generates described macromolecule or high molecular fluorescent derivative; B2) described macromolecule is by described initiator and the disulfide bond of described antibody or sulfydryl coupling freely, prepares the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition.Preferably, in step b2) in, the disulfide bond of described antibody is reduced into sulfydryl freely, and then described macromolecule is connected on described sulfydryl by described initiator, prepares the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition.
Described disulfide bond is selected from least one in the disulfide bond of disulfide bond in chain, interchain.Described disulfide bond or sulfydryl are by being selected from gene mutation, introducing the introducing of alpha-non-natural amino acid, oxidoreduction, enzymatic chemistry or biological modification method.
Described polymerization can be selected from atom transition free radical polymerization reaction (ATRP), reversible addion-fragmentation chain transfer polymerization (RAFT), ring opening metathesis polymerization (ROMP), ring opening polyaddition and their combination.Can by adding catalyst regulation and control polymerization.Such as, the catalyst for causing ATRP polymerization reaction includes but not limited to copper ion and part (part comprises: second bipyridine and derivative ligand, π receptor derivative part, nitrogen-atoms cheland and some fat polyamine class parts) thereof; The catalyst causing RAFT polyreaction includes but not limited to water miscible radical initiator, as 4,4 '-azo (4-cyanopentanoic acid), 2,2 '-azo [2-(2-imidazoline-2-base) propane] dihydrochloride, 2,2 '-azo [2-(2-imidazoline-2-base) propane]-anhydrous pyrosulfate, 2,2 '-azo (2-ethyl third amidine) dihydrochloride etc.The catalyst causing ROMP polyreaction includes but not limited to water miscible Grubbs catalyst etc.
Described be aggregated in hypoxia or atmosphere of inert gases under carry out, the response time is 5 to 48 hours, and reaction temperature is 0 to 80 DEG C.
Described initiator can be selected from ATRP initiator, RAFT initiator, ROMP initiator or and the initiator of ring opening polyaddition.Described ATRP initiator can be 2-(2-(2-(3,4-bis-bromo maleimide-N-ethyoxyl) ethyoxyl) ethyoxyl) ethyl 2-bromo-2 Methylpropionic acid ester (DBEB).
Described initiator molecule can be at least one of chemical formula 1, chemical formula 2 or chemical formula 3:
In described chemical formula 1, R 1for functional group and the functional group derivant thereof of the polyreactions such as ATRP, RAFT or ROMP can be caused.The functional group such as can causing ATRP reaction includes but not limited to: N-(2-aminoethyl)-2-bromo-2-methyl propanamide, N-(2-aminoethyl)-2-chloro-2-methyl propanamide, 2-bromo-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide, 2-chloro-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide etc.The functional group can causing RAFT reaction includes but not limited to: R ' C (=S) SR, and wherein R group can be cysteine, hydrazine, azanol; R ' group can be phenyl, alkyl, phthalimidomethyl; And the functional group can causing ROMP reaction includes but not limited to A-B type functional group simultaneously, A can be cysteine, hydrazine, azanol, and B can be alkene.R 2and R 3can be but be not limited to H, I, Br, Cl, C 6h 5s, CH 3c 6h 5s, Ts etc. are easily by the functional group of nucleophilic displacement of fluorine and functional group derivant thereof; R 2and R 3can be the same or different; X and Y can be but be not limited to the atoms such as NH, O, S, Se, X and Y can be the same or different, and Z includes but not limited to N or CH.
In described chemical formula 2, R 1for functional group and the functional group derivant thereof of the polyreactions such as ATRP, RAFT or ROMP can be caused.The functional group such as can causing ATRP reaction includes but not limited to: N-(2-aminoethyl)-2-bromo-2-methyl propanamide, N-(2-aminoethyl)-2-chloro-2-methyl propanamide, 2-bromo-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide, 2-chloro-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide etc.The functional group can causing RAFT reaction includes but not limited to: R ' C (=S) SR, and wherein R group can be cysteine, hydrazine, azanol; R ' group can be phenyl, alkyl, phthalimidomethyl; And the functional group can causing ROMP reaction includes but not limited to A-B type functional group simultaneously, A can be cysteine, hydrazine, azanol, and B can be alkene.R 2and R 3can be but be not limited to I, Br, Cl, C 6h 5s, CH 3c 6h 5s, at least one to toluene ring sulfonyl, R 2and R 3can not be H simultaneously; X can be but be not limited to the atoms such as NH, O, S, Se.
In described chemical formula 3, R 1for functional group and the derivant thereof of the polyreactions such as ATRP, RAFT or ROMP can be caused.The functional group such as can causing ATRP reaction includes but not limited to: N-(2-aminoethyl)-2-bromo-2-methyl propanamide, N-(2-aminoethyl)-2-chloro-2-methyl propanamide, 2-bromo-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide, 2-chloro-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide etc.The functional group can causing RAFT reaction includes but not limited to: R ' C (=S) SR, and wherein R group can be cysteine, hydrazine, azanol; R ' group can be phenyl, alkyl, phthalimidomethyl; And the functional group can causing ROMP reaction includes but not limited to A-B type functional group simultaneously, A can be cysteine, hydrazine, azanol, and B can be alkene.R 2can be but be not limited to H, I, Br, Cl, C 6h 5s, CH 3c 6h 5s, at least one to toluene ring sulfonyl; X can be but be not limited to the atoms such as NH, O, S, Se.
Macromolecule in described antibody-macromolecule coalition is selected from least one of homopolymer, many heteropolymers, block polymer, copolymer, terpolymer.
Described high polymer monomer is selected from least one in lactic acid, chloropropylene oxide, acrylate, methacrylate, acrylamide, Methacrylamide, norborene and oxanorbornene.Described high polymer monomer can be the monomer of PEG class.
Any one structure represented for the had chemical formula 4 to 7 of the high polymer monomer of the polyreactions such as ATRP, RAFT and ROMP:
Wherein, the R group in chemical formula 4 ~ 7 be selected from alkyl, phenyl, benzyl, carboxylic Radix Betae base, sulphonic acid betaine base, oligomeric ethylene glycol, Polyethylene Glycol, preferably, described alkyl is selected from methyl, ethyl, propyl group, isopropyl, the tert-butyl group.
Described high polymer monomer can comprise two reactive groups, and described two reactive groups react each other and form described macromolecule.Described high polymer monomer can comprise one or more reactive group be embedded into when polymerization reaction take place in high molecular skeleton further.Described high polymer monomer can be water solublity or biodegradable.
Described macromolecule can be selected from least one of POEGMA and PMPC.Described macromolecule can have side chain, and described side chain is selected from least one of betaine side chain, carboxyl betaine side chain, sulfuryl betaine side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol.
Polymer fluorescent monomer used in the present invention can be the fluorescent derivative of above-mentioned polymer monomer, and this fluorescent derivative ray structure can comprise the structures such as rhodamine structure, fluorescein structure, tonyred.
The kind of described antibody includes, but are not limited to medicine, agricultural, the monoclonal antibody that scientific research and other industrial circles are correlated with, the immune proteins such as polyclonal antibody, such as: infliximab (infliximab), Rituximab (rituximab), bevacizumab (bevacizumab), adalimumab (adalimumab), Cetuximab (cetuximab), palivizumab (palivizumab), Gemtuzumab ozogamicin (Mylotarg), alemtuzumab (Alemtuzumab), ibritumomab tiuxetan (Zevalin), Trastuzumab (Herceptin) etc.
In addition, if needed, biodegradable material (polymer monomer, oligomer or polymer) also can be incorporated in the structure of antibody-macromolecule coalition and fluorescent derivative thereof.
Preparation method of the present invention has following advantage: first, the method of Dispersal risk in the past-macromolecule coalition often selects the surface lysines of antibody as conjugation sites, and then it is uncontrollable to result in reaction site, have impact on the activity (antibody-antigene binding ability) of antibody greatly, the specific site of antagonist of the present invention is modified, and remains the activity of antibody to greatest extent; Secondly, product (antibody-macromolecule coalition and fluorescent derivative thereof) can be separated with raw material (unreacted catalyst, polymer monomer and fluorescent monomer) by preparation method of the present invention simple and effective.In addition, situ aggregation method step of the present invention is easy.
Accompanying drawing explanation
Fig. 1 respectively illustrates the step of situ aggregation method and grafting method Dispersal risk-macromolecule coalition and fluorescent derivative (Trastuzumab-POEGMA, Trastuzumab-PMPC, Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-PMPC-fluorescent derivative) thereof.
Fig. 2 shows antibody Herceptin, be reduced after antibody Herceptin-SH and the photo of antibody-initiator coalition Trastuzumab-Br.
Fig. 3,4 shows the peptide hydrolysis mass spectrum of antibody-initiator coalition Trastuzumab-Br.
Fig. 5 shows the cartoon figure of the peptide hydrolysis place antibody location of antibody-initiator coalition Trastuzumab-Br.
Fig. 6 shows the peptide hydrolysis mass spectrum of antibody-initiator coalition Trastuzumab-Br.
Fig. 7 shows the cartoon figure of the peptide hydrolysis place antibody location of antibody-initiator coalition Trastuzumab-Br.
Fig. 8 shows gel permeation chromatography (GPC) detection curve of Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, Trastuzumab-POEGMA-3.
Fig. 9 shows gel permeation chromatography (GPC) detection curve of Trastuzumab-PMPC-1, Trastuzumab-PMPC-2, Trastuzumab-PMPC-3.
Figure 10 shows Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Trastuzumab-POEGMA-3 detects.
Figure 11 shows Trastuzumab-PMPC-1, Trastuzumab-PMPC-2, the SDS-PAGE of Trastuzumab-PMPC-3 detects.
Figure 12 shows the hydrated diameter that dynamic light scattering (DLS) measures Trastuzumab and Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, Trastuzumab-POEGMA-3.
Figure 13 shows dynamic light scattering and surveys the hydrated diameter that (DLS) determines Trastuzumab and Trastuzumab-PMPC-1.
Figure 14 shows the proton nmr spectra of Trastuzumab and Trastuzumab-POEGMA-1.
Figure 15 shows the circular dichroism curve of Trastuzumab and Trastuzumab-POEGMA-1.
Figure 16 shows the fluorescence immunoassay curve of Trastuzumab and Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, Trastuzumab-POEGMA-3.
Figure 17 shows Trastuzumab and Trastuzumab-PMPC-1, Trastuzumab-PMPC-2, Trastuzumab-PMPC-3 curve.
Figure 18 shows the fluorescence immunoassay curve of Trastuzumab after 0 ~ 3 time repeatedly lyophilization.
Figure 19 shows the activity before and after 3 times repeatedly lyophilization of Trastuzumab and Trastuzumab-POEGMA-1.
Figure 20 shows the activity before and after 3 times repeatedly lyophilization of Trastuzumab and Trastuzumab-PMPC-1.
Figure 21 shows the activity before and after enzyme hydrolysis of Trastuzumab and Trastuzumab-POEGMA-1.
The SDS-PAGE before and after enzyme hydrolysis that Figure 22 shows Trastuzumab and Trastuzumab-POEGMA-1 detects.
Figure 23 shows the activity before and after enzyme hydrolysis of Trastuzumab and Trastuzumab-PMPC-1.
The SDS-PAGE before and after enzyme hydrolysis that Figure 24 shows Trastuzumab and Trastuzumab-PMPC-1 detects.
Figure 25 shows the activity before and after Trastuzumab and Trastuzumab-POEGMA-1 heating.
Figure 26 shows the SDS-PAGE detection before and after the dyeing of Trastuzumab and Trastuzumab-POEGMA-2.9R.
The absworption peak of gel permeation chromatography (GPC) at 280nm and 568nm place that Figure 27 shows Trastuzumab and Trastuzumab-POEGMA-2.9R detects.
Figure 28 shows the SDS-PAGE detection before and after the dyeing of Trastuzumab and Trastuzumab-PMPC-12.6R.
The absworption peak of gel permeation chromatography (GPC) at 280nm and 568nm place that Figure 29 shows Trastuzumab and Trastuzumab-PMPC-12.6R detects.
Figure 30 shows the fluorescence spectrum of Trastuzumab-POEGMA-2.9R.
Figure 31 shows the working curve that BCA method (607nm OD value) measures the Trastuzumab concentration of Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-PMPC-fluorescent derivative.
Figure 32 shows the working curve measuring the fluorescence molecule concentration of Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-PMPC-fluorescent derivative at 568nm place (fluorescence molecule maximum absorption wavelength).
Figure 33 shows the fluorescence immunoassay curve of Trastuzumab-PMPC-12.6R.
SDS-PAGE before and after the dyeing that Figure 34 shows Trastuzumab and Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R detects.
Figure 35 shows Trastuzumab and Trastuzumab-POEGMA-2.9R, the absworption peak of gel permeation chromatography (GPC) at 280nm place of Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R detects.
Figure 36 shows the schematic diagram preparing Trastuzumab-TAM.
Figure 37 shows the relative intensity of fluorescence of Trastuzumab-TAM, Trastuzumab-POEGMA-2.9R under same concentrations, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R.
Figure 38 shows the active fluoro immunity curve of Trastuzumab-TAM, Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R.
Figure 39 shows Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R, Trastuzumab-POEGMA-30R and the Trastuzumab-TAM fluorescence immunoassay detection curve to antigen HER2.
Figure 40 shows the fluorescence signal intensity of Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R detectable antigens HER2, exceeds the multiple of the fluorescence signal intensity of Trastuzumab-TAM detectable antigens HER2.
Figure 41 shows the immunofluorescence picture of Trastuzumab-TAM and Trastuzumab-POEGMA-13.6R.
Figure 42 shows the flow cytometer curve of Trastuzumab-TAM and Trastuzumab-POEGMA-13.6R.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Described method is conventional method if no special instructions.Described reactant all can obtain from open commercial sources if no special instructions.
The invention provides the preparation method of antibody-macromolecule coalition and fluorescent derivative thereof, described antibody comprises at least one disulfide bond, and described macromolecule is attached on described antibody by the initiator of the disulfide bond being connected to the reduction of described antibody.
Particularly, described preparation method can comprise two kinds: the first is situ aggregation method, and described method comprises: a1) coalition of Dispersal risk-initiator, wherein said initiator be attached to described antibody disulfide bond or freely on sulfydryl; B1) described antibody-initiator coalition mixes with high polymer monomer or with high polymer monomer and fluorescent monomer in buffer, causes the polymerization of described high polymer monomer and prepare the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition under catalyst action.The second is Graft Method, Graft Method is previously prepared macromolecule, again with specific site coupling Dispersal risk-macromolecule coalition and the fluorescent derivative thereof of antibody, described method comprises: a2) initiator causes high polymer monomer or high polymer monomer and fluorescent monomer polymerization under catalyst action, generates described macromolecule or high molecular fluorescent derivative; B2) described macromolecule is by described initiator and the disulfide bond of described antibody or sulfydryl coupling freely, prepares the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition.
Polymerization will be carried out under hypoxia or atmosphere of inert gases, and the response time can be 5 minutes to 48 hours, such as 5 minutes, 15 minutes, 60 minutes, 12 hours, 24 hours or 48 hours.Reaction temperature can be 20 DEG C to 100 DEG C, such as 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C or 100 DEG C.
Macromolecule in described antibody-macromolecule coalition and fluorescent derivative thereof is selected from homopolymer, many heteropolymers, block polymer, copolymer, terpolymer.This macromolecule includes but not limited to functionalized macromolecular (such as 5-vinyl tetrazolium polymer), and its molecular weight distribution is less than 2.0.This macromolecule can comprise one or more star block copolymer, linear polymer, graft polymers, brush polymer, bottle brush copolymer, crosslinked shape copolymer (such as embedding the block copolymer of 5-vinyl tetrazolium monomer).
Macromolecule in described antibody-macromolecule coalition and fluorescent derivative thereof includes but not limited to polyesters macromolecule, PMAm, polymethacrylates, polyethers, polystyrene, polynorbornene etc.Such as polyolefin polymers includes but not limited to polyethylene, polypropylene, polychloroprene, polyvinyl ester, poly-(vinylacetate), polyvinyl halides, poly-(vinyl chloride), polysiloxanes, polystyrene, polyurethane, polyacrylate, poly-((methyl) acrylic acid methyl ester .), poly-((methyl) ethyl acrylate), poly-((methyl) n-butyl acrylate), poly-((methyl) Isobutyl 2-propenoate), poly-((methyl) tert-butyl acrylate), poly-((methyl) Hexyl 2-propenoate), poly-((methyl) isodecyl acrylate), poly-((methyl) lauryl acrylate), poly-((methyl) phenyl acrylate), poly-(acrylic acid methyl ester .), poly-(isopropyl acrylate), poly-(Isobutyl 2-propenoate), poly-(octadecyl acrylate), poly-(acrylate), poly-(Methacrylamide), poly-(ethyl acrylamide), poly-(ethyl methacrylamide), NIPA, (just gather, different, N-tert-butyl acrylamide) etc.Comprise in addition these other functionalization derivants high molecular (as with can replace, additive reaction, alkylated reaction, olefination, hydroxylating, oxidation reaction and other chemical reaction functional group).In addition polymer side chain can be betaine side chain, carboxyl betaine side chain, sulfuryl betaine side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol etc.Such as, the present invention can utilize POEGMA (poly-(few PEG methyl ether methacrylate)) or PMPC (poly-2-methacryloxyethyl Phosphorylcholine) to prepare Trastuzumab-POEGMA (antibody-macromolecule) or Trastuzumab-PMPC (antibody-macromolecule) coalition.Macromolecule POEGMA due to oligomeric ethylene glycol side chain, therefore possess hydrophilic, water solublity, without features such as deposition, hypotoxicity and reduced immunogenicities; Macromolecule PMPC, with the anion and cation macromolecule of positive and negative charge, therefore possesses extraordinary water solublity, without features such as deposition, hypotoxicity and reduced immunogenicities.
High polymer monomer used in the present invention can be water solublity, and water miscible high polymer monomer can comprise the reactive group of easily leaving away, with nucleopilic reagent generation substitution reaction.Such as, can add epoxychloropropane monomer in the process that macromolecular chain increases, epoxychloropropane monomer can be embedded on high molecular skeleton, and chlorine atom can as leaving group and nucleopilic reagent generation substitution reaction simultaneously.The end of Polyethylene Glycol (PEG) is replaced by amido, as the chlorine atom generation nucleophilic substitution on nucleopilic reagent and high polymer main chain, thus can prepare the high polymer main chain of PEGization.Macromolecule involved in the present invention comprises the hydrophilic macromolecule of the several functions utilizing said method to prepare.
High polymer monomer can part the high molecular propagation process of participation, be connected on high molecular main chain by identical chemical reaction.Described high polymer monomer can comprise two reactive groups, and these two reactive groups react formation macromolecule each other.Such as, lactic acid carboxyl and hydroxyl two reactive groups.High polymer monomer used in the present invention may further include one or more other reactive group, can be embedded in the skeleton of polymer when polymerization reaction take place.
The step a1 of situ aggregation method) and b1) in operable buffer solution as follows:
Step a1) in buffer solution be Na 2hPO 4~ citric acid solution, K 2hPO 4~ KH 2pO 4buffer solution, TrisHCl buffer solution, Hanks ' buffer solution or PBS buffer solution, preferred TrisHCl buffer solution; PH value is 6.0 ~ 8.0, preferably 7.4; The concentration of antibody is 10 ~ 200 μm, preferably 10 μm; Reducing agent is three (2-carboxyethyl) phosphine (TCEP), dithiothreitol, DTT (DTT), sodium borohydride, sodium cyanoborohydride etc., preferred TCEP; The ratio of reducing agent and antibody is 1: 1 ~ 200: 1, preferably 5: 1; Reaction temperature is 4 ~ 40 DEG C, preferably 37 DEG C; Response time is 0.5 ~ 24 hour, preferably 3 hours, prepares reduced form antibody.The concentration of reduced form antibody is 10 ~ 200 μMs, preferably 10 μMs; The ratio of initiator and antibody is 1: 1 ~ 200: 1, preferably 50: 1; Reaction temperature is 4 ~ 40 DEG C, preferably 37 DEG C; Response time is 0.5 ~ 24 hour, preferably 14 hours.
Described step b1) Dispersal risk-macromolecule coalition process in, buffer solution is Na 2hPO 4~ citric acid solution, K 2hPO 4~ KH 2pO 4buffer solution, TrisHCl buffer solution, Hanks ' buffer solution or PBS buffer solution, preferred TrisHCl buffer solution; PH value is 6.0 ~ 8.0, preferably 7.4; The concentration of antibody-initiator coalition is 10 ~ 200 μMs, preferably 20 μm; The ratio of high polymer monomer and antibody-initiator coalition is 200: 1 ~ 20000: 1, preferably 1000: 1,2000: 1 and 5000: 1; Catalyst is CuCl or CuBr, preferred CuCl; Part is TMEDA, bpy, Me 6-TREN, PPh 3, PMDETA, HMTETA, preferred HMTETA; The ratio of catalyst and part is 2: 1 ~ 10: 1, preferably 10: 1; Reaction temperature is 4 ~ 40 DEG C, preferably 25 DEG C; Response time is 0.5 ~ 24 hour, preferably 3 hours.
Described step b1) Dispersal risk-macromolecule coalition and fluorescent derivative thereof process in, buffer solution is Na 2hPO 4~ citric acid solution, K 2hPO 4~ KH 2pO 4buffer solution, TrisHCl buffer solution, Hanks ' buffer solution or PBS buffer solution, preferred TrisHCl buffer solution; PH value is 6.0 ~ 8.0, preferably 7.4; The concentration of antibody-initiator coalition is 10 ~ 200 μMs, preferably 20 μMs; The ratio of high polymer monomer and antibody-initiator coalition is 200: 1 ~ 20000: 1, preferably 1000: 1; The ratio of high polymer monomer and fluorescent monomer is 1000: 1 ~ 10: 1, preferably 10: 1,20: 1 and 50: 1; Catalyst is CuCl or CuBr, preferred CuCl; Part is TMEDA, bpy, Me 6-TREN, PPh 3, PMDETA, HMTETA, preferred HMTETA; The ratio of catalyst and part is 2: 1 ~ 10: 1, preferably 10: 1; Reaction temperature is 4 ~ 40 DEG C, preferably 25 DEG C; Response time is 0.5 ~ 24 hour, preferably 3 hours.
Fig. 1 shows the detailed description of the invention that the present invention adopts Graft Method (branch above) and situ aggregation method (branch below) Dispersal risk-macromolecule coalition and fluorescent derivative thereof respectively.Wherein, Trastuzumab-POEGMA and fluorescent derivative thereof or Trastuzumab-PMPC and fluorescent derivative thereof is prepared at interchain disulfide bond site growth in situ polyphosphazene polymer (few (ethylene glycol) methyl ether methacrylate) (POEGMA) of monoclonal antibody Herceptin or poly-2-methacryloxyethyl Phosphorylcholine (PMPC).Wherein, the concrete steps of situ aggregation method are: 1) interchain disulfide bond of antibody is reduced agent and opens (reduction) and generate sulfydryl (Trastuzumab-SH) freely, 2) nucleophilic substitution is passed through, the ATRP initiator molecule (DBEB) that two bromo maleimides are modified is inserted in the disulfide bond of interchain, antibody-initiator the coalition (Trastuzumab-Br) 3 formed) Trastuzumab-Br direct initiated polymerization thing monomer OEGMA (or PMPC) is by atom transition free radical polymerization reaction (ATRP) and initiated polymerization thing monomer OEGMA and fluorescent monomer (or PMPC and fluorescent monomer) copolymerization, growth in situ goes out macromolecule POEGMA (or PMPC) and fluorescent derivative thereof, prepare Trastuzumab-POEGMA (or Trastuzumab-PMPC) and fluorescent derivative thereof.Wherein, the concrete steps of Graft Method Dispersal risk-macromolecule coalition and fluorescent derivative thereof are: 1) interchain disulfide bond of antibody is reduced agent and opens (reduction) and generate sulfydryl (Trastuzumab-SH) freely; 2) atom transition free radical polymerization reaction (ATRP) of ATRP initiator molecule (DBEB) initiated polymerization thing monomer OEGMA (or MPC) modified of two bromo maleimides and initiated polymerization thing monomer OEGMA and fluorescent monomer (or PMPC and fluorescent monomer) copolymerization, prepares macromolecule POEGMA (or PMPC) and fluorescent derivative thereof; 3) by step 2) obtain macromolecule and be coupled to step 1) on the Trastuzumab-SH that obtains, prepare Trastuzumab-POEGMA (or Trastuzumab-PMPC) and fluorescent derivative thereof.
Embodiment 1 and 2 specifically illustrates embodiments of the present invention.
Concrete detection method in the present invention is as follows:
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): the sample 80 μ L of preparation 1mg/ml concentration, adds 20 μ L sample loading buffers (Loading Buffer) (× 5, not containing β mercaptoethanol).Above-mentioned 10 μ L samples are loaded in polyacrylamide gel, electrophoresis 2 ~ 4 hours (electrophoresis liquid is: 25mM Tris, 250mM Glycine, 0.1%SDS) under the condition of 80 ~ 120V, 40mA.
Gel permeation chromatography (GPC): gpc analysis uses high performance liquid chromatography (HPLC) analytical system of Waters company.Use UV-detector (Waters 2489) at 280nm and 568nm.Chromatographic column is GS-520HQ and GS-320HQ (being connected with guard column), and mobile phase is 50mM TrisHCl (150mM NaCl, pH=7.4), and temperature is 25 DEG C, and flow velocity is 0.5ml/min.
Dynamic light scattering (DLS): DLS test uses Malvem Zetasizer Nano-zs90.Sample before testing will by the filter membrane of 0.22 μm, and the concentration of sample is 15 μMs.Date processing uses software Zetasizer software6.32.
Circular dichroism measures: use circular dichroism instrument (Photophysics Ltd.Pistar π-180) at 25 DEG C, measure antibody-macromolecule coalition (Trastuzumab-polymer).Sample concentration is about 1.4 μMs, and uv absorption wavelength scanning range is 195 ~ 260nm.
BCA method measures Trastuzumab concentration: standard substance are added in the standard sample wells of 96 orifice plates by 0,1,2,4,8,12,16 and 20 μ L, add PBS and supply 20 μ L by configuration bovine serum albumin (BSA) standard substance 0.5mg/mL (PBS).Add proper volume (2 ~ 20 μ L) sample in the sample well of 96 orifice plates, add PBS and supply 20 μ L.Every hole adds 200 μ LBCA working solutions, places 30 minutes for 37 DEG C.Measure the OD value of 562nm, according to the protein concentration of standard curve calculation sample.
The active fluoro immune detection of Trastuzumab-macromolecule coalition and fluorescent derivative thereof: add antigen (HER2,1.3 μ g/mL) 100 μ L in 96 orifice plates, hold over night at 4 DEG C.Removing antigen (HER2) uses PBS (200 μ L) to wash 3 times afterwards, thereafter bovine serum albumin (BSA is added, 1%) 200 μ L, disturbance 3 hours under room temperature, 3 times are washed with PBS (200 μ L) after removing BSA, add the Trastuzumab of serial dilution and Trastuzumab-macromolecule coalition and fluorescent derivative (27 μ g/mL thereof, 9 μ g/mL, 3 μ g/mL, 1 μ g/mL, 0.333 μ g/mL, 0.111 μ g/mL, 0.037 μ g/mL, 0.012 μ g/mL) 100 μ L, disturbance 1 hour under room temperature, with PBS-T (0.1% tween 20, 200 μ L) wash 2 times, PBS (200 μ L) washs 1 time, add the anti-(Goat anti human's Trastuzumab-PE of fluorescence two, Santa Cruz biotechnology or anti-human igg (Fc the is special)-FITC antibody produced in goat, Sigma-Aldrich) 100 μ L, disturbance 1 hour under room temperature, at microplate reader (Molec μ Lar Devices after washing 2 times with PBS (200 μ L), SpectraMax M3) upper reading.Each sample in triplicate.
The fluorescence immunoassay of Trastuzumab-macromolecule coalition fluorescent derivative detectable antigens HER2 detects (direct method): in 96 orifice plates, add serial dilution antigen HER2 (32.5 μ g/mL, 6.5 μ g/mL, 1.3 μ g/mL, 0.26 μ g/mL, 0.052 μ g/mL, 0.0104 μ g/mL) 100 μ L, hold over night at 4 DEG C.Removing antigen (HER2) uses PBS (200 μ L) to wash 3 times afterwards, thereafter bovine serum albumin (BSA is added, 1%) 200 μ L, disturbance 3 hours under room temperature, 3 times are washed with PBS (200 μ L) after removing BSA, add Trastuzumab-macromolecule coalition fluorescent derivative (10 μ g/mL) 100 μ L, disturbance 1 hour under room temperature, with PBS-T (0.1% tween 20,200 μ L) wash 3 times, at the upper reading of microplate reader (Molec μ Lar Devices, SpectraMax M3) after PBS (200 μ L) washs 1 time.Each sample in triplicate.
Fluorescence spectrometry: use fluorescence spectrophotometer to measure antibody-macromolecule coalition fluorescent marker (Trastuzumab-POEGMA-dyestuff or Trastuzumab-PMPC-dyestuff) at 25 DEG C.Sample concentration is about 0.1 μM, and excitation wavelength is 540nm, and emission wavelength sweep limits is 560 ~ 700nm; Emission wavelength is 615nm, and excitation wavelength sweep limits is 500 ~ 605nm.
Immunofluorescence assay: by cell to be measured (about 1, 000, 000/mL) rinse with PBS the paraformaldehyde (PBS solution) that (each 5 minutes) after twice add 4% and at room temperature fix 15 minutes, after PBS washs three times, (each 5 minutes) add 0.3%Triton-X100 rupture of membranes (10min), PBS washs three times (each 5 minutes), add 10% lowlenthal serum, antibody (9 μ g/mL are added after closing 30min under room temperature, PBS solution) 37 DEG C hatch 60 minutes, PBS washs three times (each 5 minutes, note lucifuge), distillation washing once after (2min) at fluorescence microscopy Microscopic observation and Taking Pictures recording experimental result.The microscope used is Nikon Ti, and imaging and analysis software are NIS-Elements BR 3.2.
Flow cytometry analysis is tested: by cell to be measured (about 200,000/mL, 100 μ L, DMEM culture medium, 10%FBS) at room temperature hatch 1 hour with antibody (10 μ g/mL, 100 μ L, PBS solution), PBS washes twice (each 5 minutes), analyzes with flow cytometer.The FACSAria II that the flow cytometer used is U.S. company BD, analysis software is FCS Express 4Research Edition.
Use the nuclear magnetic resonance analyser of Japanese JEOL company working sample at 25 DEG C 1h NMR.
Embodiment 1:
1, ATRP initiator DBEB (Formula I-2) is prepared.
Preparation 3,4-bis-bromo-1-((2-(2-(2-hydroxy ethoxy) ethyoxyl) ethyoxyl) methyl)-1H-pyrroles-2,5-diketone (Formula I-1): the oxolane two bromo maleimides (1.02g, 4mmol) being dissolved in 20ml drying, slowly add triphenylphosphine (1.05g, 4mmol), diisopropyl azodiformate (DIAD, 790 μ L, 4mmol) and four Polyethylene Glycol (1.4ml, 8mmol) under room temperature and stir 20 hours.With Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound described in Formula I-1 provided by the invention (productive rate is 23% for yellow oil, 398mg), has reclaimed the two bromo maleimides of 50% simultaneously.
Formula I-1 compound is yellow oil. 1H?NMR(CDCl 3，400MHz)δ3.59-3.71(m，14H)，3.81-3.84(m，2H). 13C?NMR(CDCl 3，100MHz)δ39.0，61.8，67.7，70.1，70.4，70.6，70.8，72.6，129.5，164.0.MS(IES)m/z：452[M+Na] +，454[M+Na] +，456[M+Na] +。
As from the foregoing, above-claimed cpd structure is correct, is compound shown in Formula I-1.
The synthesis of 2-(2-(2-(3,4 two bromo maleimide-N-ethyoxyl) ethyoxyl) ethyoxyl) ethyl 2-bromo-2 Methylpropionic acid ester (DBEB) (Formula I-2)
Compound shown in Formula I-1 (215mg, 0.5mmol) is dissolved in 3ml dichloromethane, at 0 DEG C, adds 1ml NEt 3and stir 0.5 hour.DMF (0.3ml) solution of 2-bromo-2-methyl-prop acylbromide (138mg, 0.6mmol) is slowly dripped at 0 DEG C.Stir and slowly rise to room temperature after 3 hours, be extracted with ethyl acetate (3 × 25mL) after adding 10ml water, anhydrous magnesium sulfate drying is used after merging organic facies, cross and filter magnesium sulfate, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (yellow oil described in Formula I-2 provided by the invention, 145mg, productive rate is 50%).
Formula I-2 compound is yellow oil. 1H?NMR(CDCl 3，400MHz)δ1.94(s，6H)，3.61-3.81(m，14H)，4.33(t，2H，J＝4.8). 13C?NMR(CDCl 3，100MHz)δ30.8，39.0，55.9，65.2，67.6，68.8，70.1，70.6，70.71，70.74，129.5，163.8，171.6，.MS(IES)m/z：602[M+Na] +，604[M+Na] +。
As from the foregoing, above-claimed cpd structure is correct, is compound shown in Formula I-2.
2, ATRP initiator DBEB is inserted into the disulfide bond between antibody chain.
Monoclonal antibody Herceptin (7.5mg, 0.05 μm of ol) is dissolved in 1ml TrisHCl (50mM, 150mM NaCl, pH=7.4) in, reducing agent three (2-carboxyethyl) phosphine (TCEP, 50 μ l are added at 37 DEG C, aqueous solution 5mM), hatches 3 hours.Use desalting column (AKTA, GE Healthcare, HiTrapTM Desalting Column5ml, mobile phase is 50mM TrisHCl, 150mM NaCl, pH=7.4) micromolecule removing is obtained the reduced form Trastuzumab (Trastuzumab-SH) after purification.The DFM solution of ATRP initiator DBEB (5 μ L, 50mM) is joined in the TrisHCl buffer of above-mentioned Trastuzumab-SH (solution becomes faint yellow, see Fig. 2), hatch 14 hours at 37 DEG C, remove unnecessary ATRP initiator through desalting column and obtain antibody-initiator coalition (Trastuzumab-Br).Through above-mentioned two-step reaction, successfully ATRP initiator is inserted on the interchain disulfide bond of antibody.Fig. 2 shows Trastuzumab antibody, be reduced after antibody Herceptin-SH and the photo of antibody-initiator coalition Trastuzumab-Br, in Fig. 2, left side, centre and right side be respectively Trastuzumab antibody, be reduced after antibody Herceptin-SH and antibody-initiator coalition Trastuzumab-Br.
3, verify that ATRP reaction initiator molecule is inserted on the interchain disulfide bond of antibody Herceptin
Trypsin is used to be hydrolyzed by the antibody Herceptin-Br with initiator, by efficient liquid phase-mass spectrum (LC-MS/MS) test, detect the polypeptide fragment (as shown in Figure 3) that initiator molecule is inserted into the disulfide bond (SC223DK ~ GEC214) between light chain and heavy chain, detect initiator molecule simultaneously and be only linked to polypeptide fragment (Fig. 4) on this position heavy chain, Fig. 5 is the cartoon figure of this polypeptide fragment place antibody location.In addition, by efficient liquid phase-mass spectrum (LC-MS/MS) test, detect the polypeptide fragment that initiator molecule is linked to (THTC229PPC232PAPELLGGPSVFLFPPKPK) between inner two cysteine residues of heavy chain, as shown in Figure 6.Fig. 7 is the cartoon figure of this polypeptide fragment place antibody location.In efficient liquid phase-mass spectrum (LC-MS/MS) test process, do not find that other site is connected with the polypeptide fragment of initiator molecule.
Fig. 3 to Fig. 7 illustrates, ATRP reaction initiator molecule is inserted on the interchain disulfide bond of antibody (Trastuzumab) really.
4, the disulfide bond position original position between antibody chain causes ATRP polymerization reaction
CuCl (0.4mg) and hexamethyl trien (4mg) are dissolved in 1ml deionized water.By 50 ~ 250mg OEGMA (or MPC, 60 ~ 300mg) be dissolved in TrisHCl (50mM, 150mM NaCl, pH=7.4) buffer solution of Trastuzumab-Br (10 μMs, 5ml), Trastuzumab-Br: OEGMA is 1: 1000,1: 2000 or 1: 5000; Trastuzumab-Br: MPC is 1: 2000,1: 5000 or 1: 10000.Argon 30 minutes (deoxygenation) is blasted respectively in above-mentioned two solution, import in second solution (passing through bidirectional needle) by first solution under room temperature, reaction blasts air (termination polyreaction) for 3 hours in backward reactant liquor under an argon atmosphere.
5, antibody-macromolecule coalition (Trastuzumab-POEGMA and Trastuzumab-PMPC) is detected
There is the reaction of original position ATRP polymerization in antibody-initiator coalition trigger monomer OEGMA (or MPC), course of reaction carries out tracing detection by gel permeation chromatography (GPC) and polyacrylamide gel electrophoresis (SDS-PAGE).Fig. 8 shows the GPC curve (280nm) of the Trastuzumab-POEGMA of different molecular weight.Left several first, second, and third curve represents Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3 respectively, and Article 4 curve represents Trastuzumab-Br; Article 5 curve represents Trastuzumab.The weight average molecular weight (Mw) of the Trastuzumab-POEGMA of the different molecular weight measured is respectively 181.7kDa, 334.8kDa and 508.0kDa, and the molecular weight distribution (PDI) of its correspondence is respectively 1.4,1.4 and 1.3.
Fig. 9 shows the GPC curve (280nm) of the Trastuzumab-PMPC of different molecular weight.Left several first, second, and third curve represents Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3 respectively, and Article 4 curve represents Trastuzumab-Br; Article 5 curve represents Trastuzumab.The weight average molecular weight (Mw) of the Trastuzumab-PMPC of the different molecular weight measured is respectively 184.2kDa, 253.5kDa and 556.2kDa, and the molecular weight distribution (PDI) of its correspondence is respectively 1.3,1.3 and 2.1.
By the detection of the characteristic absorption peak 280nm at protein, the insertion of initiator and the productive rate (ratio of antibody-macromolecule coalition and unreacted antibody) of home position polymerization reaction can be judged.After GPC (uv absorption of 280nm) detects and finds ATRP polymerization reaction, the retention time of reactant mixture is all less than the retention time of unreacted antibody Herceptin (18.3 minutes).The retention time shortening of characteristic absorption peak shows to generate molecular weight larger antibody-macromolecule coalition through the reaction of original position ATRP polymerization.By carrying out integration to characteristic absorption peak (280nm), after can calculating polyreaction, the ratio shared by antibody-macromolecule coalition (Trastuzumab-POEGMA and Trastuzumab-PMPC) is more than 99%.Can infer that the insertion reaction of initiator and the efficiency of original position ATRP polymerization reaction are all greater than 99% thus.
The SDS-PAGE (Non) that Figure 10 shows the Trastuzumab-POEGMA of different molecular weight detects.Swimming lane 1 is Trastuzumab-Br, and swimming lane 2 is protein tag, and swimming lane 3 is Trastuzumab, and swimming lane 4 ~ 6 is respectively Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3.The SDS-PAGE that Figure 11 shows the Trastuzumab-PMPC of different molecular weight detects.Swimming lane 1 is protein tag, and swimming lane 2 is Trastuzumab, and swimming lane 3 ~ 5 is respectively Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3.SDS-PAGE analyzes and shows equally, and the molecular weight of antibody-macromolecule coalition is greater than antibody itself, and antibody is almost all converted into antibody-macromolecule coalition after the insertion reaction and the reaction of original position ATRP polymerization of initiator.
Further sign has been done to the Trastuzumab-POEGMA after purification and Trastuzumab-PMPC.Figure 12 shows the hydration radius that dynamic light scattering (DLS) measures the Trastuzumab-POEGMA of Trastuzumab and different molecular weight, Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 after Figure 12 shows purification and Trastuzumab-POEGMA-3 all only have (left several second ~ four peak, a level and smooth nano-particle distribution characteristics peak, hydrated diameter is respectively 15.2nm, 21.9nm and 34.3nm, PDI is respectively 0.19,0.23 and 0.22), the not nano-particle distribution characteristics peak of Trastuzumab (left several first peak) itself.Trastuzumab-PMPC-1 after Figure 13 shows purification only has a level and smooth nano-particle distribution characteristics peak (hydrated diameter is 22.0nm, and its PDI is 0.3), not the nano-particle distribution characteristics peak of antibody itself.Figure 14 shows the proton nmr spectra of Trastuzumab and Trastuzumab-POEGMA-1, and Figure 14 shows the feature hydrogen signal that Trastuzumab-POEGMA-1 has macromolecule POEGMA, shows that antibody (Trastuzumab) is connected with macromolecule (POEGMA) really.Figure 15 shows the circular dichroism curve of Trastuzumab (solid line) and Trastuzumab-POEGMA-1 (dotted line), Figure 15 shows antibody before and after home position polymerization reaction (Trastuzumab) circular dichroism curve and there is no significant change, after showing to combine with macromolecule (POEGMA), the secondary structure of antibody (Trastuzumab) is without significant change.
Trastuzumab is the common monoclonal antibody medicine of a class, is used for the treatment of the Cancerous disease such as breast carcinoma and gastric cancer of human epidermal growth factor receptor 2 (HER2) process LAN.Trastuzumab can combination specific with HER2, and the size of binding ability can be used for characterizing the height of Trastuzumab activity.Fluorescence immunoassay detects and shows, compared with Trastuzumab, Trastuzumab-POEGMA and Trastuzumab-PMPC still maintains good activity.Figure 16 shows the quasi integration concentration (EC that Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3 are combined with antigen HER2 50) be respectively 3.4 μ g/ml, 4.1 μ g/ml and 6.8 μ g/ml, the EC that Trastuzumab itself is combined with antigen HER2 50be 1.3 μ g/ml, wherein upper number Article 1 curve is the fluorescence immunoassay curve of Trastuzumab, and upper several second ~ Article 4 curve is respectively the fluorescence immunoassay curve of Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3.Figure 17 shows the quasi integration concentration (EC that Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3 are combined with antigen HER2 50) be respectively 2.2 μ g/ml, 3.5 μ g/ml and 16.3 μ g/ml, the EC that Trastuzumab itself is combined with antigen HER2 50be 1.1 μ g/ml, wherein upper number Article 1 curve is the fluorescence immunoassay curve of Trastuzumab, and upper several second ~ Article 4 curve is respectively the fluorescence immunoassay curve of Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3.Figure 16 and Figure 17 shows jointly, compared with Trastuzumab, adopts specific site to modify and the method for in-situ polymerization is prepared Trastuzumab-POEGMA or Trastuzumab-PMPC and all can be kept good activity, its activity along with the increase of high molecular molecular weight on a declining curve.
Trastuzumab is the important monoclonal antibody medicine (pharmaceutical grade protein) of a class, pharmaceutical grade protein due to himself less stable, easy inactivation (losing curative effect) in storage with transportation.Utilize macromolecule modified protein effectively can improve the stability of protein.Compared with Trastuzumab, Trastuzumab-POEGMA-1 and Trastuzumab-PMPC-1 substantially increases the stability of Trastuzumab.
Lyophilization is the preserving type that monoclonal antibody medicine (pharmaceutical grade protein) is conventional.Trastuzumab is after experience three times repeatedly lyophilization, activity almost completely loses, as shown in figure 18, uppermost circular node curve is fluorescence immunoassay curve before Trastuzumab lyophilization, the curve of square nodes is the fluorescence immunoassay curve of Trastuzumab after 1 lyophilization, the curve of positive three corner nodes is the fluorescence immunoassay curve of Trastuzumab after 2 lyophilizations, the curve of del node is the fluorescence immunoassay curve of Trastuzumab after 3 lyophilizations.Figure 19 shows under specific concentration (9 μ g/mL), after 3 times repeatedly lyophilization, Trastuzumab-POEGMA-1 maintains the activity of 79%, Trastuzumab then only maintains the activity of 15%, compared with Trastuzumab, Trastuzumab-POEGMA-1 through 3 times repeatedly lyophilization rear stability improve 4.3 times.Figure 20 shows under specific concentration (9 μ g/mL), and after 3 times repeatedly lyophilization, Trastuzumab-PMPC-1 maintains the activity of 62%, compared with Trastuzumab, Trastuzumab-PMPC through 3 times repeatedly lyophilization rear stability improve 3.1 times.
Papain (papain) is commonly used to be hydrolyzed antibody, Dispersal risk Fab fragment and Fc fragment.After Figure 21 shows and is hydrolyzed to Trastuzumab and Trastuzumab-POEGMA-1 (0.5mg/ml) under identical condition (papain of 0.08% is hydrolyzed 16 hours at 37 DEG C), under specific concentration (9 μ g/mL), Trastuzumab-POEGMA-1 maintains the activity of 60%, Trastuzumab then maintains the activity of 11%, compared with Trastuzumab, the enzyme stability of Trastuzumab-POEGMA-1 improves 4.5 times.
Figure 22 is that the SDS-PAGE (reduced form) before and after Trastuzumab and Trastuzumab-POEGMA-1 Papain enzymolysis detects, swimming lane 1 is protein tag, swimming lane 2 is Trastuzumab (before enzymolysis), swimming lane 3 is Trastuzumab (after enzymolysis), swimming lane 4 is Trastuzumab-POEGMA-1 (before enzymolysis), and swimming lane 5 is Trastuzumab-POEGMA-1 (after enzymolysis).The analysis of Figure 22 shows equally, the most heavy chain of Trastuzumab is that two peptide sections are (compared with swimming lane 2 by enzymolysis, there are two new bands in swimming lane 3), and Trastuzumab-POEGMA-1 most heavy chain is not two peptide sections (compared with swimming lane 4, very shallow two new bands appear in swimming lane 5) by enzymolysis.Can absolutely prove in conjunction with Figure 21 and Figure 22, compared with Trastuzumab, Trastuzumab-POEGMA-1 has better enzyme stability.
Figure 23 shows under identical enzymatic hydrolysis condition and specific concentration (9 μ g/mL), and Trastuzumab-PMPC-1 maintains the activity of 34%, and the enzyme stability of Trastuzumab-PMPC-1 improves 2.4 times compared with Trastuzumab.
Figure 24 is that the SDS-PAGE (reduced form) before and after Trastuzumab and Trastuzumab-PMPC Papain enzymolysis detects, swimming lane 1 is protein tag, swimming lane 2 is Trastuzumab (before enzymolysis), swimming lane 3 is Trastuzumab (after enzymolysis), swimming lane 4 is Trastuzumab-PMPC-1 (before enzymolysis), and swimming lane 4 is Trastuzumab-PMPC-1 (after enzymolysis).Can absolutely prove in conjunction with Figure 23 and Figure 24, Trastuzumab-PMPC-1 has better enzyme stability compared with Trastuzumab.
After Figure 25 shows and hatch 3 days in 37 DEG C of waters, under specific concentration, under (9 μ g/mL), Trastuzumab-POEGMA-1 maintains the activity of 63%, and Trastuzumab then maintains the activity of 61%.Compared with Trastuzumab, the heat stability of Trastuzumab-POEGMA-1 slightly improves.
Embodiment 2:
1, rhodamine B derivative fluorescent monomer (formula I-6) is prepared
1) synthesis of methacrylic acid-3 bromo propyl ester (Formula I-3).
Methacrylic chloride (1.04g, 10mmol) and 3ml triethylamine are slowly added drop-wise in the dichloromethane (20ml) of 3-bromopropyl alcohol (1.39g, 10mmol) and the mixed solution of DFM (1ml).React under room temperature after 12 hours, the dilute hydrochloric acid of 0.1M is slowly added drop-wise in reactant liquor, until pH < 7.0, be extracted with ethyl acetate (3 × 25mL), organic facies anhydrous magnesium sulfate drying, concentrated after filtering, rapid column chromatography obtains compound (colorless oil described in Formula I-3 provided by the invention, 1.13g, 55%).
Formula I-3 compound is yellow oil. 1H?NMR(400MHz，CDCl 3)δ1.95(s，3H)，2.21-2.27(m，2H)，3.49(t，2H，J＝6.4)，4.29(t，2H，J＝6.4)，5.58(s，1H)，6.11(s，1H).MS(ESI)m/z：229[M+Na] +。
As from the foregoing, above-claimed cpd structure is correct, is compound shown in Formula I-3.
2) synthesis of rhodamine B alkali (Rhodamine B base) (Formula I-4).
Rhodamine B (6.6g, 13.8mmol) is dissolved in 150ml ethyl acetate, adds sodium hydrate aqueous solution (1M, 150ml), fully stir.After 3 hours, separate organic facies, aqueous phase ethyl acetate (50ml) extracting twice.Merge organic facies, use sodium hydrate aqueous solution and aqueous NaCl wash organic facies respectively, anhydrous magnesium sulfate drying, filter, concentrate and obtain compound described in Formula I-4 provided by the invention (red foaming material, 5.13g, 84%).
Compound shown in Formula I-4 is red foaming material. 1H?NMR(400MHz，CD 3OD)δ1.27(t，12H，J＝6.8)，3.62(q，8H，J＝6.8)，6.88(d，2H，J＝2.4)，6.96(dd，2H，J＝2.7，9.6)，7.21-7.27(m，3H)，7.57-7.65(m，2H)，8.07-8.09(m，1H).MS(ESI)m/z：443[M+H] +。
As from the foregoing, above-claimed cpd structure is correct, is compound shown in formula I-4.
3) synthesis of rhodamine B piperazine amide (Rhodamine B piperazine amide) (Formula I-5).
The hexane solution (9mL, 9mmol) of the trimethyl aluminium of 1M is slowly added drop-wise in dichloromethane (10ml) solution of piperazine (1.55g, 18mmol).Stirred at ambient temperature slowly drips dichloromethane (4ml) solution of I-4 compound (2g, 4.5mmol) after 1 hour, be heated to boiling, slowly drips the dilute hydrochloric acid of 0.1M after 24 hours.By above-mentioned reacting liquid filtering, wash with the mixed solution of dichloromethane and dichloromethane and methanol (4: 1).After concentrated filtrate, residue is dissolved in dichloromethane, cross and filter insoluble matter, be used for by residue in the mixed solution of dilute aqueous solution of sodium bicarbonate and ethyl acetate, after layering, aqueous phase is extracted with ethyl acetate three times, removes responseless raw material.Remaining aqueous phase sodium chloride is saturated, and adding 1M salt acid for adjusting pH is faintly acid, extracts three times with the mixed solution of isopropyl alcohol/dichloromethane 2: 1, until the redness in aqueous phase disappears.Merge organic facies, anhydrous magnesium sulfate drying, concentrated after filtering, dry method loading, rapid column chromatography obtains compound described in formula I-5 provided by the invention (colorless oil, 1.42g, 62%).
Formula I-5 compound is atropurpureus solid. 1H?NMR(400MHz，CDCl 3)δ1.34(t，12H，J＝7.2)，2.79(br?s，4H)，3.51(br?s，4H)，3.59-3.74(m，8H)，4.18(br?s，1H)，6.76(d，2H，J＝2.4)，6.98(dd，2H，J＝2.4，9.6)，7.22(d，2H，J＝9.6)，7.34-7.36(m，1H)，7.54-7.56(m，1H)，7.65-7.70(m，2H).MS(ESI)m/z：511[M+H] +。
As from the foregoing, above-claimed cpd structure is correct, is compound shown in formula I-5.
4) synthesis of rhodamine B 4-(3-methacryloxypropyl) piperazine amide (RMPA) (Formula I-6).
By 3-bromopropionyl bromide (412mg, 2mmol) and N, N-diisopropyl ethyl amine (0.7ml) is slowly added drop-wise in DFM (10ml) solution of rhodamine B piperazine amide (I-5), stir at 40 DEG C after 24 hours and be chilled to room temperature, above-mentioned solution is poured in the two-phase system of dichloromethane (25ml) and water (25ml), after layering, extracting three times with dichloromethane (25ml).Collect organic facies, anhydrous sodium sulfate drying, concentrated after filtering, dry method loading, rapid column chromatography obtains compound described in Formula I-6 provided by the invention (atropurpureus solid, 92mg, 72%).
Formula I-6 compound is atropurpureus solid. 1H?NMR(400MHz，CD 3OD)δ1.29(t，12H，J＝7.2)，1.75-1.81(m，2H)，1.89(s，3H)，2.19(br?s，4H)，2.33(t，2H，J＝7.2)，3.37(br?s，4H)，3.68(q，8H，J＝7.2)，4.13(t，2H，J＝6.4)，5.59(s，1H)，6.04(s，1H)，6.96(d，2H，J＝2.0)，7.06(dd，2H，J＝2.0，9.6)，7.26(d，2H，J＝9.6)，7.48-7.51(m，1H)，7.62-7.66(m，1H)，7.72-7.77(m，2H). 13C?NMR(100MHz，CD 3OD)δ11.6，17.1，25.4，45.6，48.0，54.5，62.7，96.0，113.5，114.1，124.8，127.5，129.8，130.0，130.3，130.7，132.0，135.5，136.4，155.7，155.9，157.9，167.4，167.9.MS(ESI)m/z：637[M+H] +。
As from the foregoing, above-claimed cpd structure is correct, is compound shown in Formula I-6.
2, Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-PMPC-fluorescent derivative is prepared
CuCl (0.4mg) and hexamethyl trien (4mg) are dissolved in 1ml deionized water.By 50mgOEGMA (or 30mg PMPC) and fluorescent monomer (formula I-6,2.5mg, 5%) be dissolved in TrisHCl (50mM, 150mM NaCl, pH=7.4) buffer solution of Trastuzumab-Br (10 μMs, 5ml).Argon 30 minutes (deoxygenation) is blasted respectively in above-mentioned two solution, being dissolved in first under room temperature imports in second solution (passing through bidirectional needle), and reaction blasts air (termination polyreaction) for 3 hours in backward reactant liquor under an argon atmosphere.
Antibody-initiator coalition trigger monomer OEGMA (or MPC) and fluorescent monomer generation original position ATRP copolyreaction, course of reaction carries out tracing detection by polyacrylamide gel electrophoresis (SDS-PAGE) and gel permeation chromatography (GPC).
The SDS-PAGE that Figure 26 shows Trastuzumab-POEGMA-fluorescent derivative detects, before coomassie brilliant blue staining (left side), after coomassie brilliant blue staining (right side).Swimming lane 1 is protein tag; Swimming lane 2 is Trastuzumab; Swimming lane 3 is Trastuzumab-POEGMA-fluorescent derivative.Figure 26 Trastuzumab-POEGMA-fluorescent derivatization object location (swimming lane 3) is red stripes, shows that red fluorescent monomer has been copolymerized on high molecular main chain; SDS-PAGE picture after coomassie brilliant blue staining can see there is obvious blue bands at antibody-macromolecule coalition fluorescent derivatization object location (swimming lane 3), and the molecular weight of its correspondence is greater than the molecular weight of antibody (swimming lane 2) itself.Relatively before and after coomassie brilliant blue staining SDS-PAGE picture we think that antibody-initiator coalition (Trastuzumab-Br) is almost all converted into Trastuzumab-POEGMA-fluorescent derivative after original position ATRP copolyreaction.
Figure 27 shows the gel permeation chromatography figure (GPC) of Trastuzumab-POEGMA-fluorescent derivative at 280nn and 568nm place uv absorption (absorption maximum of fluorescence molecule is at 568nm place).Right side graph is Trastuzumab; Left side solid line is Trastuzumab-POEGMA-fluorescent derivative (280nm); Left-hand broken line is Trastuzumab-POEGMA-fluorescent derivative (568nm).By the detection of the characteristic absorption peak 280nm at protein, the insertion of initiator and the productive rate (ratio of antibody-macromolecule coalition and unreacted antibody) of home position polymerization reaction can be judged.After GPC (uv absorption of 280nm) detects and finds ATRP polymerization reaction, the retention time of reactant mixture is less than the retention time of unreacted antibody.The retention time shortening of characteristic absorption peak shows to generate molecular weight larger antibody-macromolecule coalition through the reaction of original position ATRP polymerization.By carrying out integration to characteristic absorption peak (280nm), the ratio after polyreaction shared by Trastuzumab-POEGMA-fluorescent derivative of can calculating is more than 99%.Can infer that the insertion reaction of initiator and the efficiency of original position ATRP polymerization reaction are all greater than 99% thus.After the GPC of absworption peak at 568nm (maximal ultraviolet absorption of fluorescent monomer) place detects and finds ATRP polymerization reaction, identical at 280nm of the retention time of reactant mixture and absworption peak, illustrate that fluorescent monomer has been copolymerized on the high polymer main chain of antibody-macromolecule coalition really, successfully prepared Trastuzumab-POEGMA-fluorescent derivative.
The SDS-PAGE that Figure 28 shows Trastuzumab-PMPC-fluorescent derivative detects, before coomassie brilliant blue staining (left side), after coomassie brilliant blue staining (right side).Swimming lane 1 is protein tag; Swimming lane 2 is Trastuzumab; Swimming lane 3 is Trastuzumab-PMPC-fluorescent derivative.Figure 28 directly can see that at Trastuzumab-PMPC-fluorescent derivatization object location (swimming lane 3) be red stripes, shows that red fluorescent monomer has been copolymerized on high molecular main chain; SDS-PAGE picture after coomassie brilliant blue staining can see that Trastuzumab-PMPC-fluorescent derivatization object location (swimming lane 3) has obvious blue bands, and the molecular weight of its correspondence is greater than the molecular weight of Trastuzumab (swimming lane 2) itself.Relatively before and after coomassie brilliant blue staining SDS-PAGE picture we think that Trastuzumab-Br is almost all converted into Trastuzumab-PMPC-fluorescent derivative after original position ATRP copolyreaction.
Figure 29 shows the gel permeation chromatography figure (GPC) of Trastuzumab-PMPC-fluorescent derivative in 280nm and 568nm place uv absorption.Right side graph is Trastuzumab; Left side solid line is Trastuzumab-PMPC-fluorescent derivative (280nm absorption); Left-hand broken line is Trastuzumab-PMPC-fluorescent derivative (568nm absorption).After GPC (uv absorption of 280nm) detects and finds ATRP polymerization reaction, the retention time of reactant mixture is less than the retention time of unreacted antibody.The retention time shortening of characteristic absorption peak shows to generate molecular weight larger antibody-macromolecule coalition through the reaction of original position ATRP polymerization.By carrying out integration to characteristic absorption peak (280nm), the ratio after polyreaction shared by Trastuzumab-PMPC-fluorescent derivative of can calculating is more than 99%.After the GPC of absworption peak at 568nm (maximal ultraviolet absorption of fluorescent monomer) place detects and finds ATRP polymerization reaction, identical at 280nm of the retention time of reactant mixture and absworption peak, illustrate that fluorescent monomer has been copolymerized on the high polymer main chain of antibody-macromolecule coalition really, successfully prepared Trastuzumab-PMPC-fluorescent derivative.
After purified (desalting column purification), further sign is done to Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-PMPC-fluorescent derivative.Trastuzumab-POEGMA-dyestuff is mixed with the aqueous solution being approximately 10nM, at 25 DEG C, measures its fluorescence excitation and emission spectra (is selected 540nm as excitation wavelength, carried out emission spectra scanning at 560nm ~ 700nm; Select 615nm as emission wavelength, carry out excitation spectrum scanning at 500nm ~ 605nm, Figure 30).Fluorescence spectrum shows, the maximum fluorescence generation wavelength of Trastuzumab-POEGMA-fluorescent derivative is 589nm, and Trastuzumab-PMPC-fluorescent derivative is identical with the fluorescence spectrum of Trastuzumab-POEGMA-fluorescent derivative.
The number of the fluorescence molecule that Trastuzumab-POEGMA-fluorescent derivative is connected with each antibody in Trastuzumab-PMPC-fluorescent derivative is adopted and is determined with the following method.
First the concentration of antibody is measured by BCA method, secondly using the antibody of known gradient concentration as standard substance, the antibody concentration of BCA method (the OD value at 607nm place bioassay standard product and unknown sample) mensuration Trastuzumab-POEGMA-fluorescent derivative (or Trastuzumab-PMPC-fluorescent derivative) is adopted.As shown in figure 31, the working curve of standard substance has good linear relationship.Then the OD value of known gradient concentration fluorescence molecule standard substance is measured at uv-absorption maximum wavelength (568nm) place of fluorescence molecule, with OD value for vertical coordinate, the concentration of fluorescence molecule is abscissa drawing curve, is measured the fluorescence molecule concentration of Trastuzumab-POEGMA-fluorescent derivative (or Trastuzumab-PMPC-fluorescent derivative) by this working curve.As shown in figure 32, the working curve of fluorescence molecule standard substance has good linear relationship.Fluorescence molecule concentration and the mol ratio of antibody concentration of Trastuzumab-POEGMA-fluorescent derivative (or Trastuzumab-PMPC-fluorescent derivative) are then the numbers of the fluorescence molecule that in Trastuzumab-POEGMA-fluorescent derivative (or Trastuzumab-PMPC-fluorescent derivative), each antibody is connected.
Table 1 shows the parameter of Trastuzumab-POEGMA-30R and Trastuzumab-PMPC-12.6R.As shown in table 1, average each Trastuzumab-POEGMA-fluorescent derivative is connected with 30.0 fluorescence molecules, and average each Trastuzumab-PMPC-fluorescent derivative is connected with 12.6 fluorescence molecules.
The parameter of table 1 Trastuzumab-POEGMA-30R and Trastuzumab-PMPC-12.6R
Sample ID Fluorescence molecule R concentration (μM) Trastuzumab Her concentration (μM) R/Her
Trastuzumab-POEGMA-30R 135.1 4.5 30
Trastuzumab-PMPC-12.6R 59.1 4.7 12.6
Fluorescence immunoassay experiment shows, Trastuzumab-PMPC-fluorescent derivative still maintains good activity compared with Trastuzumab.Figure 33 shows the EC of Trastuzumab-PMPC-fluorescent derivative and antigen HER2 binding ability 50be the EC that 1.9 μ g/ml, Herceptin itself and antigen HER2 are combined 50be 1.1 μ g/ml, curve is above the fluorescence immunoassay curve of Trastuzumab, and curve is below the fluorescence immunoassay curve of Trastuzumab-PMPC-fluorescent derivative.Figure 33 shows that the method adopting specific site to modify also in-situ polymerization is prepared Trastuzumab-PMPC-fluorescent derivative and can be kept good activity.
By changing the ratio of OEGMA, fluorescent monomer and Trastuzumab-Br, the Trastuzumab-POEGMA-fluorescent derivative containing different fluorescence molecule number can be prepared.
Figure 34 shows Trastuzumab-POEGMA-2.9R and (average each Trastuzumab is connected with 2.9 fluorescence molecules, below roughly the same), the SDS-PAGE of Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R detects, before coomassie brilliant blue staining (left side), after coomassie brilliant blue staining (right side).Swimming lane 1 is protein tag; Swimming lane 2 is Trastuzumab; Swimming lane 3 is Trastuzumab-POEGMA-2.9R; Swimming lane 4 is Trastuzumab-POEGMA-7.2R; Swimming lane 5 is Trastuzumab-POEGMA-13.6R; Swimming lane 6 is Trastuzumab-POEGMA-30R.Figure 34 shows original Trastuzumab-POEGMA-fluorescent derivatization object location (swimming lane 3 ~ 6) and is red stripes; SDS-PAGE picture after coomassie brilliant blue staining can see there is obvious blue bands at antibody-macromolecule coalition fluorescent derivatization object location (swimming lane 3 ~ 6), and the molecular weight of its correspondence is greater than the molecular weight of antibody (swimming lane 2) itself.Relatively before and after coomassie brilliant blue staining SDS-PAGE picture we think that antibody-initiator coalition (Trastuzumab-Br) is almost all converted into Trastuzumab-POEGMA-fluorescent derivative after original position ATRP copolyreaction.
Figure 35 shows the gel permeation chromatography (280nm) of different Trastuzumab-POEGMA-fluorescent derivative.Left several first, second, third represents Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R respectively with Article 4 curve, and Article 5 curve represents Trastuzumab.The weight average molecular weight (Mw) of Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R is respectively 209.8kDa, 194.1kDa, 182.9kDa and 177.6kDa, and the molecular weight distribution (PDI) of its correspondence is respectively 1.3,1.3,1.2 and 1.2.
Fluorescence molecule is coupled to Dispersal risk-fluorescent marker (or derivant) on antibody, has been widely used in laboratory and Clinical detection.
In order to Trastuzumab-POEGMA-fluorescent derivative the present invention prepared is compared with conventional method (directly fluorescence molecule being coupled to antibody interchain disulfide bond) Dispersal risk-fluorescent marker, our Trastuzumab-fluorescent marker that used conventional method to prepare.Preparation method is as follows:
First, reduced form Trastuzumab (Trastuzumab-SH) is prepared according to preceding method.Then, the DFM solution (20 times of equivalents) of fluorescent marker TAMRA-C6-maleimide is joined in the TrisHCl buffer of above-mentioned Trastuzumab-SH, 14 hours are hatched at 37 DEG C, remove unnecessary fluorescent marker TAMRA-C6-maleimide through desalting column and obtain antibody-fluorescent label (Trastuzumab-TAM), and determine each antibody and be on average connected, as Figure 36 with 2.9 fluorescence molecules.
Figure 37 shows different Trastuzumab-POEGMA-fluorescent derivative and the relative intensity of fluorescence of Trastuzumab-TAM under same concentrations (27ng/mL).As shown in the figure, the first to the five block diagram represents the relative intensity of fluorescence of Trastuzumab-TAM, Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30.0R respectively.Fluorescence intensity increases along with the increase of fluorescence molecule number in Trastuzumab-POEGMA-fluorescent derivative, when fluorescence molecule number is 13.6, fluorescence intensity reaches the highest (being about 19.8 times of Trastuzumab-TAM), and when fluorescence molecule reaches 30.0, fluorescence intensity declines (being about 17.4 times of Trastuzumab-TAM) to some extent.
Fluorescence immunoassay experiment shows, Trastuzumab-TAM and Trastuzumab-POEGMA-fluorescent derivative still maintain good activity.Upper number Article 1 curve is the fluorescence immunoassay curve of Trastuzumab as shown in figure 38, Article 2 curve is the fluorescence immunoassay curve of Trastuzumab-TAM, third and fourth curve is respectively the fluorescence immunoassay curve of Trastuzumab-POEGMA-2.9R and Trastuzumab-POEGMA-7.2R, and the 5th, six article of curve is respectively the fluorescence immunoassay curve of Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30.0R.As shown in Figure 38, along with the increase of the fluorescence molecule number of Trastuzumab-POEGMA-fluorescent derivative, its activity is on a declining curve.
The fluorescence signal intensity that Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-TAM detect antigen HER2 is compared by fluorescence immunoassay (direct method).
Figure 39 shows the fluorescence immunoassay curve that different Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-TAM detect antigen HER2.Upper several first, second, third and fourth bar curve represents Trastuzumab-POEGMA-30R, Trastuzumab-POEGMA-13.6R, Trastuzumab-POEGMA-7.2R and Trastuzumab-POEGMA-2.9R respectively, and Article 5 curve represents Trastuzumab-TAM.Figure 39 shows that the Trastuzumab-POEGMA-fluorescent derivative adopting method of the present invention to prepare is compared with conventional method, is significantly improved to the fluorescence signal intensity that antigen HER2 detects.
The consumption choosing antigen HER2 is 650ng/well, data mapping corresponding to Figure 40 is (with the number (n) of the fluorescence molecule connected in Trastuzumab-POEGMA-fluorescent derivative for abscissa, with the ratio of the fluorescence signal intensity of the fluorescence signal intensity of Trastuzumab-POEGMA-fluorescent derivative and Trastuzumab-TAM for vertical coordinate), as shown in figure 40, along with the increase of fluorescence molecule number, the ratio of the fluorescence signal intensity of Trastuzumab-POEGMA-fluorescent derivative and the fluorescence signal intensity of Trastuzumab-TAM increases gradually.When the fluorescence molecule number of Trastuzumab-POEGMA-fluorescent derivative reaches 30, fluorescence signal intensity is 13.1 times of Trastuzumab-TAM.
Figure 41 shows Trastuzumab-POEGMA-13.6R with Trastuzumab-TAM to the Immunofluorescence test photo of process LAN antigen HER2 cell.Two (A and the C) photos in left side are negative control group (the low express cell of A431, antigen HER2), and two, right side (B and D) photo is positive test group (SK-BR-3, antigen HER2 overexpressing cell).Two (A and B) photos are respectively Trastuzumab-TAM to two groups of cell dyeings above, below two (C and D) photos be respectively Trastuzumab-POEGMA-13.6R to two groups of cell dyeings.As shown in Figure 41, Trastuzumab-TAM can optionally dye to SK-BR-3, but fluorescence signal is lower; Trastuzumab-POEGMA-13.6R optionally to SK-BR-3 dyeing, can show very high fluorescence signal equally simultaneously.
Figure 42 shows Trastuzumab-POEGMA-13.6R with Trastuzumab-TAM to the flow cytomery of SK-BR-3 cell.Left several Article 1 curve is negative control, and Article 2 curve is the cell that Trastuzumab-TAM dyes, and Article 3 curve is the cell that Trastuzumab-POEGMA-13.6R dyes.Wherein door 1 (M1) inner cell is negative group, and door 2 (M2) inner cell is positive group.
Table 2 shows the parameter of Trastuzumab-POEGMA-13.6R and Trastuzumab-2.9TAM.As shown in table 2, only have the cell of 12.2% can be dyeed by Trastuzumab-2.9TAM, on average each fluorescence intensity being colored cell is 90.8; Have the cell of 48.0% can be dyeed by Trastuzumab-POEGMA-13.6R, on average each fluorescence intensity being colored cell is 305.1, and being added by the fluorescence intensity of cell that Trastuzumab-POEGMA-13.6R dyes and be worth is 13.2 times of Trastuzumab-2.9TAM.
The flow cytomery parameter of table 2 Trastuzumab-POEGMA-13.6R and Trastuzumab-2.9TAM
The method of Dispersal risk provided by the invention-macromolecule coalition and fluorescent derivative thereof can be connected to the antibody of major general 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and > 99% with macromolecule, and each antibody can connect one or more (1 ~ 10) macromolecule.Antibody and high molecular connection site can be disulfide bond in chain, the disulfide bond of interchain and the site such as sulfydryl (cysteine) or the disulfide bond (or free sulfhydryl group) introduced by chemical modification freely.
Antibody prepared by the present invention-macromolecule coalition and fluorescent derivative thereof can have multiple different purposes.Such as, when selecting the antibody of therapeutic efficiency, coalition can improve acceptable antibody and thus improve therapeutic effect at the assemble index of disease site.Compared with simple antibody, antibody-macromolecule coalition and fluorescent derivative thereof can improve the water solublity of antibody, stability, pharmacokinetics and bio distribution and reduce its immunogenicity.
Antibody is combined with macromolecule, and (antibody-macromolecule coalition) effectively can improve the half-life of antibody in vivo or in blood.The raising of half-life can reach 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, even 20,30,40,50 times etc.Antibody is combined with macromolecule, and (antibody-macromolecule coalition) effectively can improve antibody dissolubility in aqueous, deliquescent raising can reach 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, even more.Antibody is combined with macromolecule, and (antibody-macromolecule coalition) effectively can improve the pharmacokinetics of antibody.Such as, antibody-macromolecule coalition can slow down the metabolism of active substance or stimulates inert matter to accelerate metabolism formation metabolite thus reduce the metabolism of active substance.Antibody is combined with macromolecule (antibody-macromolecule coalition) can by reducing antibody immunne response in vivo thus effectively reducing the immunogenicity of antibody.Immunogenic reduction can reach 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, even more.
Without departing from the inventive concept of the premise, carry out any possible change or replace all belonging to protection scope of the present invention.

Claims (25)

1. the method for Dispersal risk-macromolecule coalition and fluorescent derivative thereof, wherein, described antibody comprises at least one disulfide bond or sulfydryl freely, and described macromolecule is by being connected to disulfide bond or the sulfydryl and being attached on described antibody freely of described antibody.
2. the method for Dispersal risk-macromolecule coalition as claimed in claim 1 and fluorescent derivative thereof, described method comprises:
A1) coalition of Dispersal risk-initiator, wherein said initiator be attached to described antibody disulfide bond or freely on sulfydryl;
B1) described antibody-initiator coalition mixes with high polymer monomer or with high polymer monomer and fluorescent monomer in buffer, under catalyst action, cause the polymerization of described high polymer monomer and prepare the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition
At preferred step a1) in, the disulfide bond of described antibody is reduced into sulfydryl freely, and then described initiator is connected on described sulfydryl.
3. the method for Dispersal risk-macromolecule coalition as claimed in claim 1 and fluorescent derivative thereof, described method comprises:
A2) initiator causes high polymer monomer or high polymer monomer and fluorescent monomer polymerization under catalyst action, generates described macromolecule or high molecular fluorescent derivative;
B2) described macromolecule is by described initiator and the disulfide bond of described antibody or sulfydryl coupling freely, prepares the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition,
At preferred step b2) in, the disulfide bond of described antibody is reduced into sulfydryl freely, then described macromolecule is connected on described sulfydryl by described initiator, prepares the fluorescent derivative of described antibody-macromolecule coalition or antibody-macromolecule coalition.
4. the method for the Dispersal risk as described in any one of claims 1 to 3-macromolecule coalition and fluorescent derivative thereof, wherein, described disulfide bond is selected from least one in the disulfide bond of disulfide bond in chain, interchain.
5. the method for the Dispersal risk as described in any one of claims 1 to 3-macromolecule coalition and fluorescent derivative thereof, wherein, the described disulfide bond that described antibody comprises by being selected from gene mutation, introducing alpha-non-natural amino acid, oxidoreduction, enzymatic chemistry or biological modification method are introduced or freely sulfydryl.
6. the method for Dispersal risk-macromolecule coalition as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described polymerization is selected from the one in atom transition free radical polymerization reaction, reversible addion-fragmentation chain transfer polyreaction, ring opening metathesis polyreaction, ring opening polyaddition.
7. the method for Dispersal risk-macromolecule coalition as claimed in claim 6 and fluorescent derivative thereof, wherein, the catalyst for causing described atom transition free radical polymerization reaction is selected from least one of copper ion, second bipyridine and derivative ligand thereof, π receptor derivative part, nitrogen-atoms cheland and fat polyamine class part.
8. the method for Dispersal risk-macromolecule coalition as claimed in claim 6 and fluorescent derivative thereof, wherein, catalyst for causing described reversible addion-fragmentation chain transfer polyreaction is selected from water miscible radical initiator, preferably, described water miscible radical initiator is selected from 4, 4 '-azo (4-cyanopentanoic acid), 2, 2 '-azo [2-(2-imidazoline-2-base) propane] dihydrochloride, 2, 2 '-azo [2-(2-imidazoline-2-base) propane]-anhydrous pyrosulfate, 2, the at least one of 2 '-azo (2-ethyl third amidine) dihydrochloride.
9. the method for Dispersal risk-macromolecule coalition as claimed in claim 6 and fluorescent derivative thereof, wherein, is selected from water miscible Grubbs catalyst for the catalyst causing the polymerization of described ring opening metathesis.
10. the method for as claimed in claim 2 or claim 3 Dispersal risk-macromolecule coalition and fluorescent derivative thereof, wherein, described in be aggregated in hypoxia or atmosphere of inert gases under carry out, the response time is 5 to 48 hours, and reaction temperature is 0 to 80 DEG C.
The method of 11. Dispersal risk-macromolecule coalitions as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described initiator be selected from the initiator for atom transition free radical polymerization reaction, the initiator for reversible addion-fragmentation chain transfer polymerization, for the initiator of ring opening metathesis polymerization and initiator for ring opening polyaddition.
The method of 12. Dispersal risk-macromolecule coalitions as claimed in claim 11 and fluorescent derivative thereof, wherein, the described initiator for atom transition free radical polymerization reaction is 2-(2-(2-(3,4-bis-bromo maleimide-N-ethyoxyl) ethyoxyl) ethyoxyl) ethyl 2-bromo-2 Methylpropionic acid ester.
The method of the 13. antibody-macromolecule coalitions prepared as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described initiator molecule is at least one in chemical formula 1, chemical formula 2 or chemical formula 3:
In chemical formula 1 ~ 3,
R 1for can Atom Transfer Radical Polymerization reaction, the functional group of reversible addion-fragmentation chain transfer polyreaction or ring opening metathesis polyreaction or its functional group derivant;
Wherein, can Atom Transfer Radical Polymerization reaction functional group be selected from N-(2-aminoethyl)-2-bromo-2-methyl propanamide, N-(2-aminoethyl)-2-chloro-2-methyl propanamide, 2-bromo-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide and 2-chloro-N-(2-(2-diazanyl acetylamino) ethyl)-2-methyl propanamide;
The functional group can causing reversible addion-fragmentation chain transfer polyreaction is selected from R ' C (=S) SR, and wherein R group is cysteine, hydrazine, azanol, and R ' group is phenyl, alkyl and phthalimidomethyl;
The functional group can causing ring opening metathesis polyreaction is selected from A-B type functional group, and A is selected from cysteine, hydrazine and azanol, and B is alkene;
R 2and R 3identical or different, R 2and R 3be selected from the derivant easily by the functional group of nucleophilic displacement of fluorine and described functional group independently of one another, preferably, be describedly easily selected from H, I, Br, Cl, C by the functional group of nucleophilic displacement of fluorine 6h 5s, CH 3c 6h 5s, at least one to toluene ring sulfonyl, R 2and R 3can not be H simultaneously;
X and Y is identical or different, X and Y is selected from NH, O, S, Se atom independently of one another,
Z comprises N or CH.
The method of 14. Dispersal risk-macromolecule coalitions as claimed in claim 1 and fluorescent derivative thereof, wherein, the macromolecule in described antibody-macromolecule coalition is selected from least one of homopolymer, many heteropolymers, block polymer, copolymer, terpolymer.
The method of 15. Dispersal risk-macromolecule coalitions as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described high polymer monomer is selected from least one in lactic acid, chloropropylene oxide, acrylate, methacrylate, acrylamide, Methacrylamide, norborene and oxanorbornene.
The method of 16. Dispersal risk-macromolecule coalitions as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described high polymer monomer has any one structure represented of chemical formula 4 to 7:
Wherein, the R group in chemical formula 4 ~ 7 be selected from alkyl, phenyl, benzyl, carboxylic Radix Betae base, sulphonic acid betaine base, oligomeric ethylene glycol, Polyethylene Glycol, preferably, described alkyl is selected from methyl, ethyl, propyl group, isopropyl, the tert-butyl group.
The method of 17. Dispersal risk-macromolecule coalitions as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described high polymer monomer comprises two reactive groups, and described two reactive groups react each other and form described macromolecule.
The method of 18. Dispersal risk-macromolecule coalitions as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described high polymer monomer comprises one or more reactive group be embedded into when polymerization reaction take place in high molecular skeleton further.
The method of 19. Dispersal risk-macromolecule coalitions as claimed in claim 2 or claim 3 and fluorescent derivative thereof, wherein, described high polymer monomer is water solublity or biodegradable.
The method of 20. Dispersal risk-macromolecule coalitions as claimed in claim 1 and fluorescent derivative thereof, wherein, described macromolecule has side chain, and described side chain is selected from least one of betaine side chain, carboxyl betaine side chain, sulfuryl betaine side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol.
The method of 21. Dispersal risk-macromolecule coalitions as claimed in claim 1 and fluorescent derivative thereof, wherein, described macromolecule is selected from least one of POEGMA and PMPC.
The method of 22. Dispersal risk-macromolecule coalitions as claimed in claim 1 and fluorescent derivative thereof, wherein, antibody described in each-macromolecule coalition and fluorescent derivative thereof have at least one macromolecular chain.
The method of 23. Dispersal risk-macromolecule coalitions as claimed in claim 1 and fluorescent derivative thereof, wherein, described antibody is selected from medicine, agricultural, scientific research and other industrial circle relevant monoclonal antibody, polyclonal antibody.
The method of 24. Dispersal risk-macromolecule coalitions as claimed in claim 1 and fluorescent derivative thereof, wherein, described antibody is selected from infliximab, Rituximab, bevacizumab, adalimumab, Cetuximab, palivizumab, Gemtuzumab ozogamicin, ibritumomab tiuxetan and Trastuzumab.
25. 1 kinds of antibody-macromolecule coalitions and fluorescent derivative thereof, described antibody-macromolecule coalition and fluorescent derivative thereof are prepared by the method for any one of claim 1 to 24.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017075866A1 (en) * 2015-11-04 2017-05-11 清华大学 Method for preparing interferon-polymer conjugate ifn-poegma
CN107226858A (en) * 2016-03-23 2017-10-03 清华大学 Interferon macromolecule combination IFN-PMPC preparation and its application
CN108369235A (en) * 2015-12-18 2018-08-03 丹麦科达有限公司 Chromogenic peroxidase substrate
CN109265512A (en) * 2018-09-25 2019-01-25 清华大学 The preparation method of protein conjugate based on pyridine dicarbaldehyde
CN113248466A (en) * 2021-04-16 2021-08-13 海昌隐形眼镜有限公司 Rhodamine B dye, preparation method thereof and application thereof in preparing RGB blind correction contact lenses
CN115260412A (en) * 2018-03-23 2022-11-01 清华大学 preparation and application of pH-responsive protein macromolecule amphiphile
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103214577A (en) * 2007-03-22 2013-07-24 拜奥根Idec马萨诸塞公司 Binding proteins, including antibodies, antibody derivatives and antibody fragments, that specifically bind CD154 and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103214577A (en) * 2007-03-22 2013-07-24 拜奥根Idec马萨诸塞公司 Binding proteins, including antibodies, antibody derivatives and antibody fragments, that specifically bind CD154 and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HEREDIA ET AL.: "In Situ Preparation of Protein-"Smart"Polymer Conjugates with Retention of Bioactivity", 《J. AM. CHEM. SOC.》 *
WEIPING GAO ET AL.: "In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation", 《PROC. NATL. ACAD. SCI.》 *

Cited By (12)

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CN108369235A (en) * 2015-12-18 2018-08-03 丹麦科达有限公司 Chromogenic peroxidase substrate
CN108369235B (en) * 2015-12-18 2021-11-26 丹麦科达有限公司 Chromogenic peroxidase substrates
US11466314B2 (en) 2015-12-18 2022-10-11 Agilent Technologies, Inc. Chromogenic peroxidase substrates
US11993810B2 (en) 2015-12-18 2024-05-28 Agilent Technologies, Inc. Chromogenic peroxidase substrates
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CN115286669A (en) * 2022-07-15 2022-11-04 北京大学 Preparation method and application of antibody-macromolecule-drug conjugate

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