CN104288777B - Antibody-macromolecule combination, its fluorescent derivative and their preparation method - Google Patents
Antibody-macromolecule combination, its fluorescent derivative and their preparation method Download PDFInfo
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- CN104288777B CN104288777B CN201410480011.4A CN201410480011A CN104288777B CN 104288777 B CN104288777 B CN 104288777B CN 201410480011 A CN201410480011 A CN 201410480011A CN 104288777 B CN104288777 B CN 104288777B
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- antibody
- trastuzumab
- macromolecule
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- C09B11/10—Amino derivatives of triarylmethanes
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- A61K49/001—Preparation for luminescence or biological staining
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- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
Antibody macromolecule combination, its fluorescent derivative and their preparation method.The antibody includes at least one disulfide bond or free sulfydryl, and the macromolecule is attached to by the disulfide bond or free sulfydryl for being connected to the antibody on the antibody.The method in the specific site of antibody by modify simultaneously growth in situ macromolecule and its fluorescent derivative；Or previously prepared macromolecule and its fluorescent derivative, then prepare antibody macromolecule combination and its fluorescent derivative with the specific site coupling of antibody.The method simple and effective of the present invention, and prepared product not only retains the original bioactivity of antibody but also improves water solubility, stability, pharmacokinetics, bio distribution and the therapeutic efficiency of antibody, and reduce its immunogenicity, in addition the present invention prepares antibody high molecular fluorescent derivative compared with antibody fluorescence label prepared by conventional method, has stronger fluorescence signal and detection signal strength.
Description
Technical field
The present invention relates to biomedicine field, more particularly to the system of antibody-macromolecule combination and its fluorescent derivative
Preparation Method.
Background technology
Antibody has been widely used for biological medicine development, targeting as most important a kind of immune protein in organism
The multiple fields such as treatment and clinical diagnosis.Be used alone antibody there are half-life short, dissolubility is poor the problems such as.By macromolecule with
Antibody, which is connected, prepares antibody-macromolecule combination, can effectively improve the dissolubility of antibody, stability, pharmacokinetics and
Therapeutic efficiency simultaneously reduces its immunogenicity.The synthetic method of traditional antibody-macromolecule combination is by height well prepared in advance
Molecule is connected with antibody, is not often known there is conjugation sites, efficiency is low, yield is poor, product is difficult to separate, quality control
Difference, activity are difficult to keep many problems such as (reaction site are close to antibody activity site).Therefore, it is a kind of general there is an urgent need to design
Method, effectively to solve above-mentioned problem.
The content of the invention
The object of the present invention is to provide the special antibody of efficient, high yield synthesis site-macromolecule combinations and its glimmering
The method of light derivative, to improve the dissolubility of antibody, stability, pharmacokinetics and therapeutic efficiency and reduce its immunogene
Property.
It is described the present invention provides antibody-macromolecule combination of site-specific and its preparation method of fluorescent derivative
Antibody includes at least one disulfide bond or free sulfydryl, and the macromolecule is by being connected to the disulfide bond or freedom of the antibody
Sulfydryl and be attached on the antibody.
The preparation method may include two kinds：The first is situ aggregation method, the described method includes：A1 antibody-draw) is prepared
The combination of agent is sent out, wherein the initiator is attached on the disulfide bond or free sulfydryl of the antibody；B1) antibody-
Initiator combination is mixed with high polymer monomer in buffer solution or with high polymer monomer and fluorescent monomer, is made in catalyst
It the high polymer monomer is triggered to polymerize and is prepared the antibody-macromolecule combination or antibody-macromolecule combination with lower
Fluorescent derivative.Preferably, in step a1) in, the disulfide bond of the antibody is reduced into free sulfydryl, then the initiation
Agent is connected on the sulfydryl.Situ aggregation method in the specific site of antibody by modify simultaneously growth in situ macromolecule, tool
There are efficient, high yield, high activity conservation rate feature.
Be for second Graft Method, the previously prepared macromolecule of Graft Method, then with the specific site coupling of antibody prepare antibody-
Macromolecule combination and its fluorescent derivative, the described method includes：A2) initiator triggers high polymer monomer under catalyst action
Or high polymer monomer and fluorescent monomer polymerize, and generate the macromolecule or high molecular fluorescent derivative；B2) the high score
Son is coupled by the disulfide bond or free sulfydryl of the initiator and the antibody, prepares the antibody-macromolecule combination
Or the fluorescent derivative of antibody-macromolecule combination.Preferably, in step b2) in, the disulfide bond of the antibody is reduced into certainly
By sulfydryl, then the macromolecule be connected to by the initiator on the sulfydryl, prepare the antibody-macromolecule knot
Fit or antibody-macromolecule combination fluorescent derivative.
The disulfide bond is selected from disulfide bond, at least one of the disulfide bond of interchain in chain.The disulfide bond or sulfydryl
It is introduced by being selected from gene mutation, introducing alpha-non-natural amino acid, redox, the chemistry of enzymatic or biological modification method.
The polymerization may 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 combination thereof.It can be poly- by adding in catalyst regulation and control
It closes.For example, for the catalyst that ATRP polymerization is reacted to be triggered to include but not limited to copper ion and its ligand, (ligand includes：Bigeminy
Pyridine and its derivatives ligand, π receptor derivatives ligand, nitrogen-atoms cheland and some fat polyamine class ligands)；Trigger
The catalyst of RAFT polymerisations includes but not limited to water-soluble radical initiator, and such as 4,4 '-azo (4- cyanopentanoic acids),
2,2 '-azo [2- (2- imidazoline -2- bases) propane] dihydrochloride, 2,2 '-azo [2- (2- imidazoline -2- bases) propane]-nothing
Water pyrosulfate, 2,2 '-azo (the third amidine of 2- ethyls) dihydrochloride etc..Triggering the catalyst of ROMP polymerisations includes but unlimited
In water-soluble Grubbs catalyst etc..
Described be aggregated under hypoxemia or atmosphere of inert gases carries out, and the reaction time is 5 to 48 hours, and reaction temperature is
0 to 80 DEG C.
The initiator may be selected from ATRP initiators, RAFT initiators, ROMP initiators or draw with ring opening polyaddition
Send out agent.The ATRP initiators can be 2- (2- (2- (3,4- bis- bromo maleimide-N- ethyoxyls) ethyoxyl) ethyoxyl)
Ethyl 2- bromos -2 Methylpropionic acid ester (DBEB).
The initiator molecule can be at least one of chemical formula 1, chemical formula 2 or chemical formula 3：
In the chemical formula 1, R1For the functional group of the polymerisations such as ATRP, RAFT or ROMP and its functional group can be triggered
Derivative.Such as the functional group that ATRP can be triggered to react includes but not limited to：N- (2- aminoethyls) -2- bromo -2- methyl-props
Amide, N- (2- aminoethyls) -2- chloro -2- methyl propanamides, 2- bromos-N- (2- (2- diazanyls acetylamino) ethyl) -2- first
Base propionamide, 2- chloros-N- (2- (2- diazanyls acetylamino) ethyl) -2- methyl propanamides etc..It can trigger what RAFT reacted
Functional group includes but not limited to：R ' C (=S) SR, wherein R group can be cysteine, hydrazine, azanol；R ' group can be benzene
Base, alkyl, phthalimidomethyl；And the functional group that ROMP can be triggered to react simultaneously includes but not limited to A-B types functional group,
A can be cysteine, hydrazine, azanol, and B can be alkene.R2And R3It can be but not limited to H, I, Br, Cl, C6H5S、
CH3C6H5S, Ts etc. is easily by the functional group of nucleophilic displacement of fluorine and its functional group derivant；R2And R3It can be the same or different；X
The atoms such as NH, O, S, Se can be but not limited to Y, X and Y can be the same or different, and Z includes but not limited to N or CH.
In the chemical formula 2, R1For the functional group of the polymerisations such as ATRP, RAFT or ROMP and its functional group can be triggered
Derivative.Such as the functional group that ATRP can be triggered to react includes but not limited to：N- (2- aminoethyls) -2- bromo -2- methyl-props
Amide, N- (2- aminoethyls) -2- chloro -2- methyl propanamides, 2- bromos-N- (2- (2- diazanyls acetylamino) ethyl) -2- first
Base propionamide, 2- chloros-N- (2- (2- diazanyls acetylamino) ethyl) -2- methyl propanamides etc..It can trigger what RAFT reacted
Functional group includes but not limited to：R ' C (=S) SR, wherein R group can be cysteine, hydrazine, azanol；R ' group can be benzene
Base, alkyl, phthalimidomethyl；And the functional group that ROMP can be triggered to react simultaneously includes but not limited to A-B types functional group,
A can be cysteine, hydrazine, azanol, and B can be alkene.R2And R3It can be but not limited to I, Br, Cl, C6H5S、CH3C6H5S、
To at least one of toluene ring sulfonyl, R2And R3Cannot be H simultaneously；X can be but not limited to the atoms such as NH, O, S, Se.
In the chemical formula 3, R1For the functional group of the polymerisations such as ATRP, RAFT or ROMP and its derivative can be triggered
Object.Such as the functional group that ATRP can be triggered to react includes but not limited to：N- (2- aminoethyls) -2- bromo -2- methyl propanamides,
N- (2- aminoethyls) -2- chloro -2- methyl propanamides, 2- bromos-N- (2- (2- diazanyls acetylamino) ethyl) -2- methyl propionyl
Amine, 2- chloros-N- (2- (2- diazanyls acetylamino) ethyl) -2- methyl propanamides etc..The functional group that RAFT can be triggered to react
Including but not limited to：R ' C (=S) SR, wherein R group can be cysteines, hydrazine, azanol；R ' group can be phenyl, alkane
Base, phthalimidomethyl；And the functional group that ROMP can be triggered to react simultaneously includes but not limited to A-B types functional group, A can be with
It is cysteine, hydrazine, azanol, B can be alkene.R2It can be but not limited to H, I, Br, Cl, C6H5S、CH3C6H5S, to toluene
At least one of ring sulfonyl；X can be but not limited to the atoms such as NH, O, S, Se.
Macromolecule in the antibody-macromolecule combination is selected from homopolymer, more heteropolymers, block polymer, copolymerization
At least one of object, terpolymer.
The high polymer monomer is selected from lactic acid, epichlorohydrin, acrylate, methacrylate, acrylamide, metering system
At least one of amide, norbornene and oxanorbornene.The high polymer monomer can be the monomer of PEG classes.
For any one table with chemical formula 4 to 7 of the high polymer monomer of the polymerisations such as ATRP, RAFT and ROMP
The structure shown：
Wherein, the R group in chemical formula 4~7 is selected from alkyl, phenyl, benzyl, carboxylic beet base, sulphonic acid betaine base, widow
Polyethylene glycol, polyethylene glycol, Preferably, the alkyl is selected from methyl, ethyl, propyl, isopropyl, tertiary butyl.
The high polymer monomer may include two reactive groups, and described two reactive groups react with each other to form the high score
Son.The high polymer monomer can further comprise that one or more is embedded into when polymerisation occurs in high molecular skeleton
Reactive group.The high polymer monomer can be water-soluble or biodegradable.
The macromolecule may be selected from at least one of POEGMA and PMPC.The macromolecule can have side chain, the side chain
Selected from betaine side chain, carboxyl betaine side chain, sulfuryl betaine side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol extremely
Few one kind.
Polymer fluorescent monomer used in the present invention can be the fluorescent derivative of above-mentioned polymer monomer, which spreads out
Bioluminescence structure can include the structures such as rhodamine structure, fluorescein structure, tonyred.
The species of the antibody includes but is not limited to medicine, agricultural, scientific research and the relevant Dan Ke of other industrial circles
The immune proteins such as grand antibody, polyclonal antibody, such as：Infliximab (infliximab), Rituximab
(rituximab), bevacizumab (bevacizumab), adalimumab (adalimumab), Cetuximab
(cetuximab), palivizumab (palivizumab), Gemtuzumab ozogamicin (Mylotarg), alemtuzumab (Alemtuzumab), replace
Emol monoclonal antibody (Zevalin), Trastuzumab (Herceptin) etc..
In addition, if needing, biodegradable material (polymer monomer, oligomer or polymer) can also introduce
Into the structure of antibody-macromolecule combination and its fluorescent derivative.
The preparation method of the present invention has the advantage that：First, the method for preparing antibody-macromolecule combination in the past is often selected
It the surface lysines of antibody is selected as conjugation sites, and then results in that reaction site is uncontrollable, the strong influence work of antibody
Property (antibody-antigen binding ability), the present invention specific site of antibody is modified, remain the work of antibody to greatest extent
Property；Secondly, preparation method of the invention can by reaction product, (antibody-macromolecule combination and its fluorescence spread out with simple and effective
Biology) it is separated with raw material (unreacted catalyst, polymer monomer and fluorescent monomer).In addition, the original position of the present invention
Polymerization step is easy.
Description of the drawings
Fig. 1 respectively illustrates situ aggregation method and grafting method prepares antibody-macromolecule combination and its fluorescent derivative
(Trastuzumab-POEGMA, Trastuzumab-PMPC, Trastuzumab-POEGMA- fluorescent derivatives and Trastuzumab-PMPC- fluorescent derivatives)
The step of.
Fig. 2 shows antibody Herceptin, be reduced after antibody Herceptin-SH and antibody-initiator combination Trastuzumab-
The photo of Br.
The peptide hydrolysis mass spectrum that Fig. 3,4 show antibody-initiator combination Trastuzumab-Br.
The cartoon figure of antibody location where Fig. 5 shows the peptide hydrolysis of antibody-initiator combination Trastuzumab-Br.
Fig. 6 shows the peptide hydrolysis mass spectrum of antibody-initiator combination Trastuzumab-Br.
The cartoon figure of antibody location where Fig. 7 shows the peptide hydrolysis of antibody-initiator combination Trastuzumab-Br.
Fig. 8 shows the gel infiltration color of Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, Trastuzumab-POEGMA-3
Compose (GPC) detection curve.
Fig. 9 shows the gel permeation chromatography (GPC) of Trastuzumab-PMPC-1, Trastuzumab-PMPC-2, Trastuzumab-PMPC-3
Detection curve.
Figure 10 shows the dodecyl sulphur of Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, Trastuzumab-POEGMA-3
Sour sodium-polyacrylamide gel electrophoresis (SDS-PAGE) detection.
Figure 11 shows the SDS-PAGE detections of Trastuzumab-PMPC-1, Trastuzumab-PMPC-2, Trastuzumab-PMPC-3.
Figure 12 show dynamic light scattering (DLS) measure Trastuzumab and Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2,
The hydrated diameter of Trastuzumab-POEGMA-3.
Figure 13 shows that dynamic light scattering surveys the hydrated diameter that (DLS) determines Trastuzumab and Trastuzumab-PMPC-1.
Figure 14 shows the nuclear magnetic resonance spectroscopy of Trastuzumab and Trastuzumab-POEGMA-1.
Figure 15 shows Trastuzumab and the circular dichroism curve of Trastuzumab-POEGMA-1.
Figure 16 show Trastuzumab and Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2, Trastuzumab-POEGMA-3 it is glimmering
Curve is immunized in light.
Figure 17 shows Trastuzumab and Trastuzumab-PMPC-1, Trastuzumab-PMPC-2, Trastuzumab-PMPC-3 curves.
Figure 18 shows fluorescence immunoassay curve of the Trastuzumab after 0~3 time is freeze-dried repeatedly.
Figure 19 shows that the process of Trastuzumab and Trastuzumab-POEGMA-1 are freeze-dried front and rear activity 3 times repeatedly.
Figure 20 shows that the process of Trastuzumab and Trastuzumab-PMPC-1 are freeze-dried front and rear activity 3 times repeatedly.
Figure 21 show Trastuzumab and Trastuzumab-POEGMA-1 by the activity before and after enzyme hydrolysis.
Figure 22 shows being detected by the SDS-PAGE before and after enzyme hydrolysis for Trastuzumab and Trastuzumab-POEGMA-1.
Figure 23 show Trastuzumab and Trastuzumab-PMPC-1 by the activity before and after enzyme hydrolysis.
Figure 24 shows being detected by the SDS-PAGE before and after enzyme hydrolysis for Trastuzumab and Trastuzumab-PMPC-1.
Figure 25 shows the activity before and after Trastuzumab and Trastuzumab-POEGMA-1 heating.
Figure 26 shows the SDS-PAGE detections before and after the dyeing of Trastuzumab and Trastuzumab-POEGMA-2.9R.
Figure 27 shows the gel permeation chromatography (GPC) of Trastuzumab and Trastuzumab-POEGMA-2.9R in 280nm and 568nm
The absorption blob detection at place.
Figure 28 shows the SDS-PAGE detections before and after the dyeing of Trastuzumab and Trastuzumab-PMPC-12.6R.
Figure 29 shows the gel permeation chromatography (GPC) of Trastuzumab and Trastuzumab-PMPC-12.6R in 280nm and 568nm
The absorption blob detection at place.
Figure 30 shows the fluorescence spectrum of Trastuzumab-POEGMA-2.9R.
Figure 31 shows that BCA methods (607nm OD values) measure Trastuzumab-POEGMA- fluorescent derivatives and Trastuzumab-PMPC-
The working curve of the Trastuzumab concentration of fluorescent derivative.
Figure 32 shows that at 568nm (fluorescence molecule maximum absorption wavelength) measures Trastuzumab-POEGMA- fluorescent derivatives
With the working curve of the fluorescence molecule concentration of Trastuzumab-PMPC- fluorescent derivatives.
Figure 33 shows the fluorescence immunoassay curve of Trastuzumab-PMPC-12.6R.
Figure 34 show Trastuzumab and Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-
SDS-PAGE detections before and after the dyeing of POEGMA-13.6R and Trastuzumab-POEGMA-30R.
Figure 35 show Trastuzumab and Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-
Absorption blob detection of the gel permeation chromatography of POEGMA-13.6R and Trastuzumab-POEGMA-30R (GPC) at 280nm.
Figure 36 shows the schematic diagram for preparing Trastuzumab-TAM.
Figure 37 show Trastuzumab-TAM under same concentrations, Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R,
The relative intensity of fluorescence of Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R.
Figure 38 show Trastuzumab-TAM, Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-
Curve is immunized in the active fluoro of POEGMA-13.6R and Trastuzumab-POEGMA-30R.
Figure 39 show Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R,
Trastuzumab-POEGMA-30R and Trastuzumab-TAM is to the fluorescence immunoassay detection curve of antigen HER2.
Figure 40 show Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and
The fluorescence signal intensity of Trastuzumab-POEGMA-30R detection antigens HER2 is higher by the fluorescence of Trastuzumab-TAM detection antigens HER2
The multiple of signal strength.
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.
Specific embodiment
With reference to specific embodiment, the present invention is further elaborated, but the present invention is not limited to following embodiments.Institute
It is conventional method unless otherwise instructed to state method.The reactant can obtain unless otherwise instructed from open commercial sources.
The present invention provides the preparation method of antibody-macromolecule combination and its fluorescent derivative, the antibody includes extremely
A few disulfide bond, the initiator for the disulfide bond that the macromolecule is reduced by being connected to the antibody are attached to the antibody
On.
Specifically, the preparation method may include two kinds：The first is situ aggregation method, the described method includes：A1) prepare
The combination of antibody-initiator, wherein the initiator is attached on the disulfide bond or free sulfydryl of the antibody；B1) institute
Antibody-initiator combination is stated to mix with high polymer monomer in buffer solution or with high polymer monomer and fluorescent monomer,
The high polymer monomer is triggered to polymerize and prepare the antibody-macromolecule combination or antibody-macromolecule under catalyst action
The fluorescent derivative of combination.It is for second Graft Method, the previously prepared macromolecule of Graft Method, then it is even with the specific site of antibody
Connection prepares antibody-macromolecule combination and its fluorescent derivative, the described method includes：A2) initiator draws under catalyst action
High polymer monomer or high polymer monomer and fluorescent monomer polymerization are sent out, generates the macromolecule or high molecular fluorescent derivatization
Object；B2) macromolecule is coupled by the disulfide bond or free sulfydryl of the initiator and the antibody, is prepared described anti-
The fluorescent derivative of body-macromolecule combination or antibody-macromolecule combination.
Polymerization will carry out under hypoxemia or atmosphere of inert gases, and the reaction time can be 5 minutes to 48 hours, such as
5 minutes, 15 minutes, 60 minutes, 12 it is small when, 24 it is small when or 48 it is small when.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 the antibody-macromolecule combination and its fluorescent derivative is selected from homopolymer, more heteropolymers, embedding
Section polymer, copolymer, terpolymer.The macromolecule includes but not limited to functionalized macromolecular (such as 5- vinyl tetrazoliums
Polymer), molecular weight distribution is less than 2.0.The macromolecule can include one or more star block copolymers, linear polymerization
Object, graft polymers, brush polymer, bottle brush copolymer, crosslinking shape copolymer (such as embedded 5- vinyl tetrazolium monomers
Block copolymer).
Macromolecule in the antibody-macromolecule combination and its fluorescent derivative includes but not limited to polyesters high score
Son, polymethacrylamide, polymethacrylates, polyethers, polystyrene, polynorbornene etc..Such as polyolefins polymerization
Object includes but not limited to polyethylene, polypropylene, polychloroprene, polyvinyl ester, poly- (vinylacetate), polyvinyl halides, poly-
(vinyl chloride), polysiloxanes, polystyrene, polyurethane, polyacrylate, poly- ((methyl) methyl acrylate), poly- ((methyl) third
Olefin(e) acid ethyl ester), poly- ((methyl) n-butyl acrylate), poly- ((methyl) isobutyl acrylate), poly- (the tertiary fourth of (methyl) acrylic acid
Ester), poly- ((methyl) Hexyl 2-propenoate), poly- ((methyl) isodecyl acrylate), poly- ((methyl) lauryl acrylate), poly- ((first
Base) phenyl acrylate), poly- (methyl acrylate), poly- (isopropyl acrylate), poly- (isobutyl acrylate), poly- (acrylic acid 18
Ester), poly- (acrylate), poly- (Methacrylamide), poly- (ethyl acrylamide), poly- (ethyl methacrylamide), poly- (N-
N-isopropylacrylamide), poly- (just, different, N-tert-butyl acrylamide) etc..Additionally spread out comprising these high molecular other functionalization
Biology (as with can occur substitution, addition reaction, alkylated reaction, olefination, hydroxylating, oxidation reaction with
And the functional group of other chemical reactions).In addition polymer side chain can be betaine side chain, carboxyl betaine side chain, in sulfuryl
Ammonium salt side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol etc..For example, the present invention can utilize POEGMA (poly- (oligomerization (second two
Alcohol) methyl ether methacrylate)) or PMPC (poly- 2- methacryloxyethyls Phosphorylcholine) prepare Trastuzumab-
POEGMA (antibody-macromolecule) or Trastuzumab-PMPC (antibody-macromolecule) combination.Macromolecule POEGMA is due to carrying widow
Side-chain of polyelycol, thus possess hydrophily, water solubility, without deposition, hypotoxicity and low immunogenicity the features such as；Macromolecule
PMPC carries the anion and cation macromolecule of positive and negative charge, thus possess it is extraordinary it is water-soluble, without deposition, hypotoxicity and
The features such as low immunogenicity.
High polymer monomer used in the present invention can be it is water-soluble, water-soluble high polymer monomer can include easily from
With nucleopilic reagent substitution reaction occurs for the reactive group gone.For example, epoxy chlorine can be added in during macromolecular chain increases
Oxide monomer, epoxychloropropane monomer can be embedded on high molecular skeleton, at the same chlorine atom can be used as leaving group with
Substitution reaction occurs for nucleopilic reagent.The end of polyethylene glycol (PEG) is substituted by amido, can be used as nucleopilic reagent and macromolecule master
Nucleophilic substitution occurs for the chlorine atom on chain, so as to prepare PEGylated high polymer main chain.High score attached bag according to the present invention
Include the hydrophilic macromolecule of the multiple functions prepared using the above method.
High polymer monomer can part the high molecular propagation process of participation, height is connected to by identical chemical reaction
On the main chain of molecule.The high polymer monomer can include two reactive groups, the two reactive groups react with each other to form height
Molecule.For example, two reactive groups of lactic acid carboxyl and hydroxyl.High polymer monomer used in the present invention may further include one
A or multiple other reactive groups can be embedded into the skeleton of polymer when polymerisation occurs.
The step a1 of situ aggregation method) and b1) in the buffer solution that can use it is as follows：
Step a1) in buffer solution be Na2HPO4~citric acid solution, K2HPO4~KH2PO4Buffer solution,
TrisHCl buffer solutions, Hanks ' buffer solutions or PBS buffer solutions, preferably TrisHCl buffer solutions；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- carboxyethyls) phosphines (TCEP), two
Sulphur threitol (DTT), sodium borohydride, sodium cyanoborohydride etc., preferably 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；Reaction time for 0.5~24 it is small when, preferably 3 it is small when, prepare reduction
Type antibody.The concentration of reduced form antibody is 10~200 μM, preferably 10 μM；The ratio of initiator and antibody is 1: 1~200: 1, excellent
Select 50: 1；Reaction temperature is 4~40 DEG C, preferably 37 DEG C；Reaction time for 0.5~24 it is small when, preferably 14 it is small when.
The step b1) prepare antibody-macromolecule combination during, buffer solution Na2HPO4~lemon acid buffering
Solution, K2HPO4~KH2PO4Buffer solution, TrisHCl buffer solutions, Hanks ' buffer solutions or PBS buffer solutions, preferably
TrisHCl buffer solutions；PH value is 6.0~8.0, preferably 7.4；The concentration of antibody-initiator combination is 10~200 μM,
It is preferred that 20 μm；The ratio of high polymer monomer and antibody-initiator combination is 200: 1~20000: 1, preferably 1000: 1,2000:
1 and 5000: 1；Catalyst is CuCl or CuBr, preferably CuCl；Ligand is TMEDA, bpy, Me6-TREN、PPh3、
PMDETA, HMTETA, preferably HMTETA；The ratio of catalyst and ligand is 2: 1~10: 1, preferably 10: 1；Reaction temperature for 4~
40 DEG C, preferably 25 DEG C；Reaction time for 0.5~24 it is small when, preferably 3 it is small when.
The step b1) prepare antibody-macromolecule combination and its fluorescent derivative during, buffer solution is
Na2HPO4~citric acid solution, K2HPO4~KH2PO4Buffer solution, TrisHCl buffer solutions, Hanks ' buffer solutions
Or PBS buffer solutions, preferred TrisHCl buffer solutions；PH value is 6.0~8.0, preferably 7.4；Antibody-initiator combination
Concentration be 10~200 μM, preferably 20 μM；The ratio of high polymer monomer and antibody-initiator combination 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, preferably CuCl；Ligand is TMEDA, bpy, Me6-TREN、PPh3, PMDETA, HMTETA, preferably
HMTETA；The ratio of catalyst and ligand is 2: 1~10: 1, preferably 10: 1；Reaction temperature is 4~40 DEG C, preferably 25 DEG C；Reaction
Time for 0.5~24 it is small when, preferably 3 it is small when.
Fig. 1 shows that Graft Method (branch above) is respectively adopted in the present invention and prepared by situ aggregation method (following branch)
The specific embodiment of antibody-macromolecule combination and its fluorescent derivative.Wherein, in the interchain two of monoclonal antibody Herceptin
Sulfide linkage site growth in situ polyphosphazene polymer (few (ethylene glycol) methyl ether methacrylate) (POEGMA) or poly- 2- methyl-props
Alkene trimethylammonium Phosphorylcholine (PMPC) and prepare Trastuzumab-POEGMA and its fluorescent derivative or Trastuzumab-PMPC and
Its fluorescent derivative.Wherein, situ aggregation method concretely comprises the following steps：1) interchain disulfide bond of antibody is reduced agent and opens (reduction)
Generate free sulfydryl (Trastuzumab-SH)；2) nucleophilic substitution, the ATRP initiators of two bromo maleimides modification are passed through
Molecule (DBEB) is inserted into the disulfide bond of interchain, antibody-initiator combination (Trastuzumab-Br) 3 of formation) Trastuzumab-Br
Directly trigger polymer monomer OEGMA (or PMPC) by atom transition free radical polymerization reaction (ATRP) and trigger polymerization
Object monomer OEGMA and fluorescent monomer (either PMPC and fluorescent monomer) copolymerization growth in situ go out macromolecule POEGMA (or
PMPC) and its fluorescent derivative, Trastuzumab-POEGMA (or Trastuzumab-PMPC) and its fluorescent derivative are prepared.Wherein, connect
Branch method prepares concretely comprising the following steps for antibody-macromolecule combination and its fluorescent derivative：1) interchain disulfide bond of antibody is reduced
Agent opens (reduction) and generates free sulfydryl (Trastuzumab-SH)；2) the ATRP initiator molecules of two bromo maleimides modification
(DBEB) trigger the atom transition free radical polymerization reaction (ATRP) of polymer monomer OEGMA (or MPC) and trigger polymerization
The copolymerization of object monomer OEGMA and fluorescent monomer (either PMPC and fluorescent monomer) prepare macromolecule POEGMA (or PMPC) and its
Fluorescent derivative；3) macromolecule obtained by step 2) is coupled on the obtained Trastuzumab-SH of step 1), prepare Trastuzumab-
POEGMA (or Trastuzumab-PMPC) and its fluorescent derivative.
Examples 1 and 2 specifically illustrate embodiments of the present invention.
Specific detection method in the present invention is as follows：
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)：The 80 μ L of sample of 1mg/ml concentration are prepared,
Add in 20 μ L sample loading buffers (Loading Buffer) (× 5, without β mercaptoethanols).Above-mentioned 10 μ L samples are loaded into poly-
On acrylamide gel, when electrophoresis 2~4 is small under conditions of 80~120V, 40mA, (electrophoresis liquid is：25mM Tris、250mM
Glycine, 0.1%SDS).
Gel permeation chromatography (GPC)：Gpc analysis uses the high performance liquid chromatography (HPLC) of Waters companies to analyze system
System.Using UV detector (Waters 2489) in 280nm and 568nm.Chromatographic column (is connected with for GS-520HQ and GS-320HQ
Guard column), mobile phase is 50mM TrisHCl (150mM NaCl, pH=7.4), and temperature is 25 DEG C, flow velocity 0.5ml/
min。
Dynamic light scattering (DLS)：DLS tests use Malvem Zetasizer Nano-zs90.Sample is being tested
Before will be by 0.22 μm of filter membrane, the concentration of sample is 15 μM.Data processing uses software Zetasizer software6.32.
Circular dichroism measures：Using circular dichroism instrument (Photophysics Ltd.Pistar π -180) at 25 DEG C
Measure antibody-macromolecule combination (Trastuzumab-polymer).Sample concentration is about 1.4 μM, and UV absorption wavelength scanning range is
195~260nm.
BCA methods measure Trastuzumab concentration：Bovine serum albumin(BSA) (BSA) standard items 0.5mg/mL (PBS) is configured, by standard items
It is added to by 0,1,2,4,8,12,16 and 20 μ L in the standard sample wells of 96 orifice plates, PBS is added to supply to 20 μ L.Add proper volume (2~
20 μ L) sample into the sample well of 96 orifice plates, adds PBS to supply to 20 μ L.Add 200 μ LBCA working solutions per hole, 37 DEG C are placed 30 points
Clock.The OD values of 562nm are measured, the protein concentration of sample is calculated according to standard curve.
The active fluoro immune detection of Trastuzumab-macromolecule combination and its fluorescent derivative：It is added in 96 orifice plates anti-
Former (HER2,1.3 μ g/mL) 100 μ L are stood overnight at 4 DEG C.It removes antigen (HER2) and washs 3 times with PBS (200 μ L) afterwards, thereafter
Bovine serum albumin(BSA) (BSA, 1%) 200 μ L are added in, when disturbance 3 is small at room temperature, are washed 3 times with PBS (200 μ L) after removing BSA,
The Trastuzumab and Trastuzumab-macromolecule combination and its fluorescent derivative (27 μ g/mL, 9 μ g/mL, 3 μ g/ of addition serial dilution
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, when disturbance 1 is small at room temperature,
It is washed 2 times with PBS-T (0.1% Tween-20,200 μ L), PBS (200 μ L) is washed 1 time, and adding in fluorescence secondary antibody, (Goat anti-Human is conspicuous
Sai Ting-PE, Santa Cruz biotechnology or anti-human igg (Fc the is special)-FITC antibody generated in goat,
Sigma-Aldrich) 100 μ L, when disturbance 1 is small at room temperature, in microplate reader (Molec μ Lar after washing 2 times with PBS (200 μ L)
Devices, SpectraMax M3) on reading.Each sample is in triplicate.
The fluorescence immunoassay detection (direct method) of Trastuzumab-macromolecule combination fluorescent derivatization analyte detection antigen HER2：96
Antigen HER2 (32.5 μ g/mL, 6.5 μ g/mL, 1.3 μ g/mL, 0.26 μ g/mL, the 0.052 μ g/ of serial dilution are added in orifice plate
ML, 0.0104 μ g/mL) 100 μ L, it stands overnight at 4 DEG C.It removes antigen (HER2) to be washed 3 times with PBS (200 μ L) afterwards, add thereafter
Enter 200 μ L of bovine serum albumin(BSA) (BSA, 1%), when disturbance 3 is small at room temperature, is washed 3 times, added with PBS (200 μ L) after removing BSA
Enter Trastuzumab-macromolecule combination fluorescent derivative (10 μ g/mL) 100 μ L, when disturbance 1 is small at room temperature, (0.1% spits with PBS-T
- 20,200 μ L of temperature) washing 3 times, PBS (200 μ L) wash 1 time after in microplate reader (Molec μ Lar Devices, SpectraMax
M3 reading on).Each sample is in triplicate.
Fluorescence spectrometry：It is (conspicuous that antibody-macromolecule combination fluorescent marker is measured at 25 DEG C using Fluorescence Spectrometer
Sai Ting-POEGMA- dyestuffs or Trastuzumab-PMPC- dyestuffs).Sample concentration is about 0.1 μM, a length of 540nm of excitation light wave, hair
It is 560~700nm to penetrate wavelength scanning range；Launch wavelength is 615nm, and excitation wavelength scanning range is 500~605nm.
Immunofluorescence assay：(every time 5 minutes) add after cell to be measured (about 1,000,000/mL) is rinsed twice with PBS
4% paraformaldehyde (PBS solution) fixes 15 minutes at room temperature, after PBS washings three times (every time 5 minutes) plus 0.3%
Triton-X100 ruptures of membranes (10min), PBS are washed three times (every time 5 minutes), are added 10% lowlenthal serum, close 30min at room temperature
Afterwards plus 37 DEG C of antibody (9 μ g/mL, PBS solution) is incubated 60 minutes, and PBS is washed three times (5 minutes every time, pay attention to being protected from light), distilled water
It washes once after (2min) in fluorescence microscopy Microscopic observation and photographs to record experimental result.Used microscope is Nikon Ti,
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 mediums, 10%FBS)
With antibody (10 μ g/mL, 100 μ L, PBS solution) be incubated at room temperature 1 it is small when, PBS washes twice (every time 5 minutes), uses streaming
Cell instrument is analyzed.Used flow cytometer is the FACSAria II, analysis software FCS of U.S. company BD
Express 4Research Edition。
Using the Nuclear Magnetic Resonance of Japanese JEOL companies at 25 DEG C determination sample1H NMR。
Embodiment 1：
1st, ATRP initiators DBEB (Formula I -2) is prepared.
Prepare 3,4- bis- bromo -1- ((2- (2- (2- hydroxy ethoxies) ethyoxyl) ethyoxyl) methyl) -1H- pyrroles -2,5-
Diketone (Formula I -1)：Two bromo maleimides (1.02g, 4mmol) are dissolved in the tetrahydrofuran of 20ml dryings, room temperature
Under be slowly added to triphenylphosphine (1.05g, 4mmol), diisopropyl azodiformate (DIAD, 790 μ L, 4mmol) and four poly- second
When glycol (1.4ml, 8mmol) and small stirring 20.Solvent, dry method loading are removed with Rotary Evaporators, rapid column chromatography obtains this
The compound of Formula I -1 (yellow oil, 398mg, yield 23%) provided is provided, while has recycled the two of 50%
Bromo maleimide.
- 1 compound of Formula I is yellow oil.1H NMR(CDCl3, 400MHz) and δ 3.59-3.71 (m, 14H),
(3.81-3.84 m, 2H)13C NMR(CDCl3, 100MHz) and δ 39.0,61.8,67.7,70.1,70.4,70.6,70.8,72.6,
129.5,164.0.MS (IES) m/z：452[M+Na]+, 454 [M+Na]+, 456 [M+Na]+。
From the foregoing, it will be observed that above-claimed cpd structure is correct, it is compound shown in Formula I -1.
2- (2- (2- (3,4 two bromo maleimide-N- ethyoxyls) ethyoxyl) ethyoxyl) ethyl 2- bromo -2- methyl
The synthesis of propionic ester (DBEB) (Formula I -2)
Compound shown in Formula I -1 (215mg, 0.5mmol) is dissolved in 3ml dichloromethane, 1ml is added at 0 DEG C
NEt3And stir 0.5 it is small when.The DMF (0.3ml) that the bromo- 2- methyl propionyl bromides (138mg, 0.6mmol) of 2- are slowly added dropwise at 0 DEG C is molten
Liquid.Stir 3 it is small when after be slowly increased to room temperature, be extracted with ethyl acetate (3 × 25mL) after adding in 10ml water, used after merging organic phase
Anhydrous magnesium sulfate is dried, and is filtered to remove magnesium sulfate, removes solvent, dry method loading with Rotary Evaporators, rapid column chromatography obtains this
The compound of Formula I -2 (yellow oil, 145mg, yield 50%) provided is provided.
- 2 compound of Formula I is yellow oil.1H NMR(CDCl3, 400MHz) and δ 1.94 (s, 6H), 3.61-3.81
(m, 14H), 4.33 (t, 2H, J=4.8)13C NMR(CDCl3, 100MHz) and δ 30.8,39.0,55.9,65.2,67.6,68.8,
70.1,70.6,70.71,70.74,129.5,163.8,171.6 .MS (IES) m/z：602[M+Na]+, 604 [M+Na]+。
From the foregoing, it will be observed that above-claimed cpd structure is correct, it is compound shown in Formula I -2.
2nd, ATRP initiators DBEB is inserted into the disulfide bond of antibody interchain.
Monoclonal antibody Herceptin (7.5mg, 0.05 μm of ol) is dissolved in 1ml TrisHCl (50mM, 150mM
NaCl, pH=7.4) in, the aqueous solution of reducing agent three (2- carboxyethyls) phosphine (TCEP, 50 μ l, 5mM) is added at 37 DEG C, it is small to be incubated 3
When.Use desalting column (AKTA, GE Healthcare, HiTrapTM Desalting Column5ml, mobile phase 50mM
TrisHCl, 150mM NaCl, pH=7.4) small molecule removed to obtain reduced form Trastuzumab (Trastuzumab-SH) after purification.
The DFM solution of ATRP initiators DBEB (5 μ L, 50mM) is added to (molten in the TrisHCl buffer solutions of above-mentioned Trastuzumab-SH
Liquid becomes faint yellow, sees Fig. 2), be incubated at 37 DEG C 14 it is small when, remove extra ATRP initiators through desalting column and obtain antibody-initiation
Agent combination (Trastuzumab-Br).Through above-mentioned two-step reaction, ATRP initiators are successfully inserted into the interchain disulfide bond of antibody
On.Fig. 2 shows Trastuzumab antibody, be reduced after antibody Herceptin-SH and antibody-initiator combination Trastuzumab-Br
Photo, in Fig. 2, left side, centre and right side be respectively Trastuzumab antibody, be reduced after antibody Herceptin-SH and antibody-initiation
Agent combination Trastuzumab-Br.
3rd, verification ATRP reaction initiator molecules are inserted on the interchain disulfide bond of antibody Herceptin
Antibody Herceptin-the Br with initiator is hydrolyzed using trypsase, passes through efficient liquid phase-mass spectrum (LC-MS/
MS) test, detect the polypeptide piece for the disulfide bond (SC223DK~GEC214) that initiator molecule is inserted between light chain and heavy chain
Section (as shown in Figure 3), has been detected simultaneously by the polypeptide fragment (Fig. 4) that initiator molecule has been only linked on the position heavy chain, Fig. 5
The cartoon figure of antibody location where the polypeptide fragment.In addition, being tested by efficient liquid phase-mass spectrum (LC-MS/MS), detect
Initiator molecule is linked to inside heavy chain between two cysteine residues
(THTC229PPC232PAPELLGGPSVFLFPPKPK) polypeptide fragment, as shown in Figure 6.Fig. 7 is anti-where the polypeptide fragment
The cartoon figure of body position.Other sites are not found in efficient liquid phase-mass spectrum (LC-MS/MS) test process is connected with initiation
The polypeptide fragment of agent molecule.
Fig. 3 to Fig. 7 illustrates that ATRP reaction initiators molecule is inserted into the interchain disulfide bond of antibody (Trastuzumab) really
On.
4th, ATRP polymerization reaction is triggered in situ in the disulfide bond position of antibody interchain
CuCl (0.4mg) and hexamethyl trien (4mg) are dissolved in 1ml deionized waters.By 50~250mg
OEGMA (or MPC, 60~300mg) is dissolved in TrisHCl (50mM, 150mM NaCl, the pH of Trastuzumab-Br (10 μM, 5ml)
=7.4) in buffer solution, Trastuzumab-Br: OEGMA 1: 1000,1: 2000 or 1: 5000；Trastuzumab-Br: MPC 1:
2000th, 1: 5000 or 1: 10000.Argon gas 30 minutes (deoxygenation) is blasted into above-mentioned two solution respectively, at room temperature by first
A solution, which is imported into second solution, (passes through bidirectional needle), is blasted when reaction 3 is small under an argon atmosphere in backward reaction solution
Air (termination polymerisation).
5th, antibody-macromolecule combination (Trastuzumab-POEGMA and Trastuzumab-PMPC) is detected
Antibody-initiator combination triggers monomer OEGMA (or MPC) that ATRP polymerization reaction in situ, reaction process occurs
Tracing detection is carried out by gel permeation chromatography (GPC) and polyacrylamide gel electrophoresis (SDS-PAGE).Fig. 8 shows difference
The GPC curves (280nm) of the Trastuzumab-POEGMA of molecular weight.Left first, second, and third curve of number represents He Sai respectively
Spit of fland-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3, Article 4 curve represent Trastuzumab-Br；Article 5 is bent
Line represents Trastuzumab.The weight average molecular weight (Mw) of the Trastuzumab-POEGMA of the different molecular weight measured be respectively 181.7kDa,
334.8kDa and 508.0kDa, corresponding molecular weight distribution (PDI) is respectively 1.4,1.4 and 1.3.
Fig. 9 shows the GPC curves (280nm) of the Trastuzumab-PMPC of different molecular weight.Left number first, second, and third
Curve represents Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3 respectively, and Article 4 curve represents He Sai
Spit of fland-Br；Article 5 curve represents Trastuzumab.The weight average molecular weight (Mw) of the Trastuzumab-PMPC of the different molecular weight measured point
Not Wei 184.2kDa, 253.5kDa and 556.2kDa, corresponding molecular weight distribution (PDI) is respectively 1.3,1.3 and 2.1.
Pass through the detection of the characteristic absorption peak 280nm in protein, it can be determined that the insertion of initiator and in-situ polymerization
The yield (ratio of antibody-macromolecule combination and unreacted antibody) of reaction.GPC (UV absorption of 280nm) detections are found
After ATRP polymerization reaction, the retention time of reaction mixture is both less than (18.3 points of the retention time of unreacted antibody Herceptin
Clock).The retention time shortening of characteristic absorption peak shows to generate antibody-high score of molecular weight bigger through ATRP polymerization in situ reaction
Sub- combination.By being integrated to characteristic absorption peak (280nm), after can calculating polymerisation, antibody-macromolecule combines
Ratio shared by body (Trastuzumab-POEGMA and Trastuzumab-PMPC) is more than 99%.It is possible thereby to it is inferred to the insertion of initiator
The efficiency of reaction and ATRP polymerization reaction in situ is all higher than 99%.
Figure 10 shows SDS-PAGE (non-reduced) detections of the Trastuzumab-POEGMA of different molecular weight.Swimming lane 1 is conspicuous
Sai Ting-Br, swimming lane 2 be protein tag, swimming lane 3 be Trastuzumab, swimming lane 4~6 be respectively Trastuzumab-POEGMA-1, Trastuzumab-
POEGMA-2 and Trastuzumab-POEGMA-3.Figure 11 shows the SDS-PAGE detections of the Trastuzumab-PMPC of different molecular weight.Swimming
Road 1 is protein tag, and swimming lane 2 is Trastuzumab, and swimming lane 3~5 is respectively Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and He Sai
Spit of fland-PMPC-3.SDS-PAGE analyses again show that the molecular weight of antibody-macromolecule combination is more than antibody in itself, and through drawing
Antibody almost all is converted into antibody-macromolecule combination after sending out the intercalation reaction of agent and original position ATRP polymerization reaction.
Further characterization has been done to Trastuzumab-POEGMA after purification and Trastuzumab-PMPC.Figure 12 shows dynamic
Light scattering (DLS) measures the hydration radius of the Trastuzumab-POEGMA of Trastuzumab and different molecular weight, and Figure 12 shows hertz after purification
Only there are one smooth nano particle distribution is special by Sai Ting-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3
Levy peak (left number second~four peak, hydrated diameter are respectively 15.2nm, 21.9nm and 34.3nm, PDI is respectively 0.19,
0.23 and 0.22), there is no Trastuzumab (left first peak of number) the nano particle distribution characteristics peaks of itself.Figure 13 is shown after purification
Trastuzumab-PMPC-1 only there are one smooth nano particle distribution characteristics peak (hydrated diameter 22.0nm, PDI 0.3),
There is no the nano particle distribution characteristics peak of antibody in itself.Figure 14 shows that Trastuzumab and the nuclear-magnetism of Trastuzumab-POEGMA-1 are total to
The hydrogen that shakes is composed, and Figure 14 shows that Trastuzumab-POEGMA-1 has the feature hydrogen signal of macromolecule POEGMA, shows antibody (Trastuzumab) really
It is connected with macromolecule (POEGMA).Figure 15 shows the circular dichroism of Trastuzumab (solid line) and Trastuzumab-POEGMA-1 (dotted line)
Curve, antibody (Trastuzumab) circular dichroism curve has no significant change before and after Figure 15 shows home position polymerization reaction, shows and high score
After sub (POEGMA) is combined, the secondary structure of antibody (Trastuzumab) is without significant change.
Trastuzumab is a kind of common monoclonal antibody medicine, for treating human epidermal growth factor receptor 2 (HER2) overexpression
The Cancerous diseases such as breast cancer and stomach cancer.Trastuzumab can and HER2 specificity combination, the size of binding ability can be used for table
Levy the height of Trastuzumab activity.Fluorescence immunoassay, which detects, to be shown compared with Trastuzumab, Trastuzumab-POEGMA and Trastuzumab-PMPC
Still maintain good activity.Figure 16 show Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-
Quasi integration concentration (the EC that POEGMA-3 is combined with antigen HER250) it is respectively 3.4 μ g/ml, 4.1 μ g/ml and 6.8 μ g/ml, He Sai
The EC that spit of fland is combined in itself with antigen HER250For 1.3 μ g/ml, wherein first curve of upper number is the fluorescence immunoassay curve of Trastuzumab,
Upper second~Article 4 of number curve is respectively Trastuzumab-POEGMA-1, Trastuzumab-POEGMA-2 and Trastuzumab-POEGMA-3
Fluorescence immunoassay curve.Figure 17 shows that Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3 are tied with antigen HER2
Quasi integration concentration (the EC of conjunction50) it is respectively 2.2 μ g/ml, 3.5 μ g/ml and 16.3 μ g/ml, Trastuzumab is tied in itself with antigen HER2
The EC of conjunction50For 1.1 μ g/ml, wherein first curve of upper number is the fluorescence immunoassay curve of Trastuzumab, upper second~Article 4 of number is bent
Line is respectively the fluorescence immunoassay curve of Trastuzumab-PMPC-1, Trastuzumab-PMPC-2 and Trastuzumab-PMPC-3.Figure 16 and Figure 17 are total to
It is same to show compared with Trastuzumab, Trastuzumab-POEGMA or He Sai is prepared using the method for specific site modification and in-situ polymerization
Spit of fland-PMPC can keep preferable activity, and activity is on a declining curve with the increase of high molecular molecular weight.
Trastuzumab is a kind of important monoclonal antibody medicine (pharmaceutical grade protein), pharmaceutical grade protein due to its own stability compared with
Difference is being stored with easily inactivating (losing curative effect) in transportational process.Egg can be effectively improved using macromolecule modified protein
The stability of white matter.Compared with Trastuzumab, Trastuzumab-POEGMA-1 and Trastuzumab-PMPC-1 substantially increase the steady of Trastuzumab
It is qualitative.
Freeze-drying is monoclonal antibody medicine (pharmaceutical grade protein) common preserving type.Trastuzumab freezes repeatedly three times in experience
After drying, activity is almost lost, and as shown in figure 18, uppermost circular node curve is glimmering before Trastuzumab freeze-drying
Curve is immunized in light, the curve of square nodes is fluorescence immunoassay curve, positive triangle node of the Trastuzumab after 1 freeze-drying
Curve be Trastuzumab after 2 freeze-dryings fluorescence immunoassay curve, the curve of up-side down triangle node be that Trastuzumab passes through 3
Fluorescence immunoassay curve after secondary freeze-drying.Figure 19 shows under specific concentration (9 μ g/mL), is freezed repeatedly by 3 times dry
After dry, Trastuzumab-POEGMA-1 maintains 79% activity, and Trastuzumab then only maintains 15% activity, compared with Trastuzumab,
Trastuzumab-POEGMA-1 is freeze-dried rear stability by 3 times and improves 4.3 times repeatedly.Figure 20 is shown in specific concentration
Under (9 μ g/mL), by 3 times repeatedly be freeze-dried after, Trastuzumab-PMPC-1 maintains 62% activity, compared with Trastuzumab,
Trastuzumab-PMPC is freeze-dried rear stability by 3 times and improves 3.1 times repeatedly.
Papain (papain) is commonly used to hydrolysis antibody, prepares Fab fragments and Fc segments.Figure 21 is shown
To Trastuzumab and Trastuzumab-POEGMA-1 under identical condition when small (0.08% papain hydrolyzed at 37 DEG C 16)
After (0.5mg/ml) is hydrolyzed, under specific concentration (9 μ g/mL), Trastuzumab-POEGMA-1 maintains 60% activity,
Trastuzumab then maintains 11% activity, and compared with Trastuzumab, the enzyme stability of Trastuzumab-POEGMA-1 improves 4.5 times.
Figure 22 is that the Trastuzumab SDS-PAGE (reduced form) front and rear with Trastuzumab-POEGMA-1 Papains enzymolysis is detected,
Swimming lane 1 be protein tag, swimming lane 2 be Trastuzumab (before enzymolysis), swimming lane 3 be Trastuzumab (after enzymolysis), swimming lane 4 for Trastuzumab-
POEGMA-1 (before enzymolysis), swimming lane 5 are Trastuzumab-POEGMA-1 (after enzymolysis).The analysis of Figure 22 again shows that Trastuzumab is big absolutely
Most heavy chains are digested as two peptide fragments (compared with swimming lane 2, two new bands occurs in swimming lane 3), and Trastuzumab-
There is no being digested, for two peptide fragments, (compared with swimming lane 4, there are very shallow two to POEGMA-1 overwhelming majority heavy chain in swimming lane 5
New band).It can be absolutely proved with reference to 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) that Trastuzumab-PMPC-1 is kept
34% activity, compared with Trastuzumab the enzyme stability of Trastuzumab-PMPC-1 improves 2.4 times.
Figure 24 is that the Trastuzumab SDS-PAGE (reduced form) front and rear with Trastuzumab-PMPC Papains enzymolysis is detected, swimming lane 1
For protein tag, swimming lane 2 is Trastuzumab (before enzymolysis), and swimming lane 3 is Trastuzumab (after enzymolysis), and swimming lane 4 is Trastuzumab-PMPC-1
(before enzymolysis), swimming lane 4 are Trastuzumab-PMPC-1 (after enzymolysis).It can be absolutely proved with reference to Figure 23 and Figure 24, compared with Trastuzumab
Trastuzumab-PMPC-1 has better enzyme stability.
Figure 25 is shown be incubated 3 days in 37 DEG C of waters after, under specific concentration Trastuzumab under (9 μ g/mL)-
POEGMA-1 maintains 63% activity, and Trastuzumab then maintains 61% activity.Compared with Trastuzumab, Trastuzumab-POEGMA-
1 thermal stability slightly improves.
Embodiment 2：
1st, rhodamine B derivative fluorescent monomer (Formulas I -6) is prepared
1) synthesis of -3 bromo propyl ester (Formula I -3) of methacrylic acid.
By methacrylic chloride (1.04g, 10mmol) and 3ml triethylamines be slowly dropped to 3- bromopropyl alcohols (1.39g,
In dichloromethane (20ml) and the mixed solution of DFM (1ml) 10mmol).After when reaction 12 is small at room temperature, by dilute salt of 0.1M
Slow acid is added drop-wise in reaction solution, until pH ＜ 7.0, (3 × 25mL) is extracted with ethyl acetate, organic phase is done with anhydrous magnesium sulfate
It is dry, concentrated after filtering, rapid column chromatography obtain Formula I -3 provided by the invention compound (colorless oil, 1.13g,
55%).
- 3 compound of Formula I is yellow oil.1H NMR (400MHz, CDCl3) δ 1.95 (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]+。
From the foregoing, it will be observed that above-claimed cpd structure is correct, it 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, addition sodium hydrate aqueous solution (1M,
150ml), it is sufficiently stirred.3 it is small when after, separate organic phase, water is mutually extracted twice with ethyl acetate (50ml).Merge organic phase, point
Organic phase, anhydrous magnesium sulfate drying are not washed with sodium hydrate aqueous solution and sodium-chloride water solution, filtering is concentrated to give the present invention
The compound of Formula I -4 (red foaming material, 5.13g, 84%) of offer.
Compound shown in Formula I -4 is red foaming material.1H NMR (400MHz, CD3OD) 1.27 (t, 12H, J=of δ
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]+。
From the foregoing, it will be observed that above-claimed cpd structure is correct, it is compound shown in Formulas 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 dropped to the two of piperazine (1.55g, 18mmol)
In chloromethanes (10ml) solution.The dichloromethane of I-4 compounds (2g, 4.5mmol) is slowly added dropwise after when stirring 1 is small at room temperature
(4ml) solution, is heated to seething with excitement, 24 it is small when after the dilute hydrochloric acid of 0.1M is slowly added dropwise.By above-mentioned reacting liquid filtering, dichloromethane is used
It is washed with the mixed solution of dichloromethane and methanol (4: 1).Residue is dissolved in dichloromethane after concentration filtrate, is filtered to remove not
Molten object, by residue in the mixed solution of dilute aqueous solution of sodium bicarbonate and ethyl acetate, water mutually to use ethyl acetate after layering
Extraction three times, removes responseless raw material.For remaining water mutually with sodium chloride saturation, it is faintly acid to add in 1M salt acid for adjusting pH,
It is extracted three times with the mixed solution of isopropanol/dichloromethane 2: 1, until the red in water phase disappears.Merge organic phase, anhydrous sulphur
Sour magnesium drying, concentrates, dry method loading, rapid column chromatography obtains Formulas I -5 provided by the invention compound (colorless oil after filtering
Shape object, 1.42g, 62%).
- 5 compound of Formulas I is atropurpureus solid.1H NMR (400MHz, CDCl3) δ 1.34 (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]+。
From the foregoing, it will be observed that above-claimed cpd structure is correct, it is compound shown in Formulas I -5.
4) synthesis of rhodamine B 4- (3- methacryloxypropyls) piperazine amide (RMPA) (Formula I -6).
3- bromopropionyl bromides (412mg, 2mmol) and N, N- diisopropyl ethyl amine (0.7ml) are slowly dropped to rhodamine B
In DFM (10ml) solution of piperazine amide (I-5), at 40 DEG C stir 24 it is small when after be cooled to room temperature, above-mentioned solution is poured into two
In the two-phase system of chloromethanes (25ml) and water (25ml), extracted three times with dichloromethane (25ml) after layering.It collects organic
Phase, anhydrous sodium sulfate drying, concentrates, dry method loading, rapid column chromatography is obtained described in Formula I -6 provided by the invention after filtering
Compound (atropurpureus solid, 92mg, 72%).
- 6 compound of Formulas I is atropurpureus solid.1H NMR (400MHz, CD3OD) δ 1.29 (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, CD3OD) δ 11.6,17.1,25.4,45.6,48.0,54.5,62.7,96.0,113.5,114.1,
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]+。
From the foregoing, it will be observed that above-claimed cpd structure is correct, it is compound shown in Formula I -6.
2nd, Trastuzumab-POEGMA- fluorescent derivatives and Trastuzumab-PMPC- fluorescent derivatives are prepared
CuCl (0.4mg) and hexamethyl trien (4mg) are dissolved in 1ml deionized waters.By 50mgOEGMA
(or 30mg PMPC) and fluorescent monomer (Formulas I -6,2.5mg, 5%) are dissolved in the TrisHCl of Trastuzumab-Br (10 μM, 5ml)
In (50mM, 150mM NaCl, pH=7.4) buffer solution.Argon gas 30 minutes (deoxygenation) is blasted into above-mentioned two solution respectively,
First is dissolved at room temperature to imported into second solution and (passes through bidirectional needle), it is backward when reaction 3 is small under an argon atmosphere
Air (termination polymerisation) is blasted in reaction solution.
Antibody-initiator combination triggers monomer OEGMA (or MPC) and fluorescent monomer that original position ATRP copolymerization occurs instead
Should, reaction process carries out tracing detection by polyacrylamide gel electrophoresis (SDS-PAGE) and gel permeation chromatography (GPC).
Figure 26 shows that the SDS-PAGE of Trastuzumab-POEGMA- fluorescent derivatives is detected, (left before coomassie brilliant blue staining
Side), after coomassie brilliant blue staining (right side).Swimming lane 1 is protein tag；Swimming lane 2 is Trastuzumab；Swimming lane 3 for Trastuzumab-
POEGMA- fluorescent derivatives.Figure 26 Trastuzumab-POEGMA- fluorescent derivatizations object locations (swimming lane 3) are red stripes, show red
Fluorescent monomer be copolymerized on high molecular main chain；SDS-PAGE pictures after coomassie brilliant blue staining can see anti-
Body-macromolecule combination fluorescent derivatization object location (swimming lane 3) has apparent blue bands, and corresponding molecular weight is more than antibody
(swimming lane 2) molecular weight of itself.SDS-PAGE pictures through ATRP in situ it is considered that be copolymerized before and after comparing coomassie brilliant blue staining
Antibody-initiator combination (Trastuzumab-Br) almost all is converted into Trastuzumab-POEGMA- fluorescent derivatives after reaction.
Figure 27 shows the gel permeation chromatography figure (GPC) of Trastuzumab-POEGMA- fluorescent derivatives in 280nn and 568nm
Locate UV absorption (absorption maximum of fluorescence molecule is at 568nm).Right side graph is Trastuzumab；Left side solid line for Trastuzumab-
POEGMA- fluorescent derivatives (280nm)；Left-hand broken line is Trastuzumab-POEGMA- fluorescent derivatives (568nm).By in albumen
The detection of the characteristic absorption peak 280nm of matter, it can be determined that the insertion of initiator and the yield (antibody-height of home position polymerization reaction
The ratio of molecule combination and unreacted antibody).After GPC (UV absorption of 280nm) detections find ATRP polymerization reaction, reaction
The retention time of mixture is less than the retention time of unreacted antibody.The retention time shortening of characteristic absorption peak shows through original position
ATRP polymerization reacts the antibody-macromolecule combination for generating molecular weight bigger.By being accumulated to characteristic absorption peak (280nm)
Point, it is more than 99% that can calculate the ratio after polymerisation shared by Trastuzumab-POEGMA- fluorescent derivatives.It is possible thereby to it pushes away
Break and the intercalation reaction of initiator and the efficiency of ATRP polymerization in situ reaction is all higher than 99%.Absworption peak is in 568nm (fluorescence lists
The maximal ultraviolet absorption of body) at GPC detections find ATRP polymerization reaction after, the retention time of reaction mixture exists with absworption peak
280nm's is identical, illustrates that fluorescent monomer has been copolymerized to really on the high polymer main chain of antibody-macromolecule combination, successfully prepares
Trastuzumab-POEGMA- fluorescent derivatives.
Figure 28 shows that the SDS-PAGE of Trastuzumab-PMPC- fluorescent derivatives is detected, (left before coomassie brilliant blue staining
Side), after coomassie brilliant blue staining (right side).Swimming lane 1 is protein tag；Swimming lane 2 is Trastuzumab；Swimming lane 3 for Trastuzumab-
PMPC- fluorescent derivatives.Figure 28 can directly be seen that in Trastuzumab-PMPC- fluorescent derivatizations object location (swimming lane 3) be red bar
Band shows that red fluorescent monomer has been copolymerized on high molecular main chain；SDS-PAGE pictures after coomassie brilliant blue staining can
To see that Trastuzumab-PMPC- fluorescent derivatizations object location (swimming lane 3) has apparent blue bands, corresponding molecular weight is more than conspicuous
Sai Ting (swimming lane 2) molecular weight of itself.SDS-PAGE pictures are it is considered that through ATRP in situ before and after comparing coomassie brilliant blue staining
Trastuzumab-Br almost all is converted into Trastuzumab-PMPC- fluorescent derivatives after copolyreaction.
Figure 29 shows the gel permeation chromatography figure (GPC) of Trastuzumab-PMPC- fluorescent derivatives at 280nm and 568nm
UV absorption.Right side graph is Trastuzumab；Left side solid line is Trastuzumab-PMPC- fluorescent derivatives (280nm absorptions)；Left side is empty
Line is Trastuzumab-PMPC- fluorescent derivatives (568nm absorptions).GPC (UV absorption of 280nm) detections find that ATRP polymerization is anti-
Ying Hou, the retention time of reaction mixture are less than the retention time of unreacted antibody.The retention time of characteristic absorption peak shortens table
Bright antibody-macromolecule combination that molecular weight bigger is generated through ATRP polymerization in situ reaction.By to characteristic absorption peak
(280nm) is integrated, and it is more than 99% that can calculate the ratio after polymerisation shared by Trastuzumab-PMPC- fluorescent derivatives.
After GPC detections of the absworption peak at 568nm (maximal ultraviolet absorption of fluorescent monomer) finds ATRP polymerization reaction, reaction mixture
Retention time and absworption peak in the identical of 280nm, illustrate that fluorescent monomer has been copolymerized to the height of antibody-macromolecule combination really
On molecular backbone, Trastuzumab-PMPC- fluorescent derivatives are successfully prepared.
To Trastuzumab-POEGMA- fluorescent derivatives and Trastuzumab-PMPC- fluorescent derivatizations after purified (desalination column purification)
Object has done further characterization.Trastuzumab-POEGMA- dyestuffs are configured to the about aqueous solution of 10nM, are measured at 25 DEG C
Its fluorescence excitation (selects 540nm to carry out emission spectrum scanning in 560nm~700nm as excitation wavelength with emission spectrum；Choosing
615nm is selected as launch wavelength, excitation spectrum scanning, Figure 30 are carried out in 500nm~605nm).Fluorescence spectrum show Trastuzumab-
It is 589nm that wavelength, which occurs, for the maximum fluorescences of POEGMA- fluorescent derivatives, Trastuzumab-PMPC- fluorescent derivatives and Trastuzumab-
The fluorescence spectrum of POEGMA- fluorescent derivatives is identical.
Each antibody is connected glimmering in Trastuzumab-POEGMA- fluorescent derivatives and Trastuzumab-PMPC- fluorescent derivatives
The number of optical molecule determines with the following method.
The concentration of antibody is measured by BCA methods first, secondly using the antibody of known gradient concentration as standard items, is used
BCA methods (the OD values of bioassay standard product and unknown sample at 607nm) measure Trastuzumab-POEGMA- fluorescent derivatives (or
Trastuzumab-PMPC- fluorescent derivatives) antibody concentration.As shown in figure 31, the working curve of standard items has linear well close
System.Then known gradient concentration fluorescence molecule standard items are measured at the uv-absorption maximum wavelength (568nm) of fluorescence molecule
OD values, using OD values as ordinate, the concentration of fluorescence molecule is abscissa drawing curve, and He Sai is measured by the working curve
The fluorescence molecule concentration of spit of fland-POEGMA- fluorescent derivatives (or Trastuzumab-PMPC- fluorescent derivatives).As shown in figure 32, it is glimmering
The working curve of optical molecule standard items has good linear relationship.Trastuzumab-POEGMA- fluorescent derivatives (or Trastuzumab-
PMPC- fluorescent derivatives) fluorescence molecule concentration and the molar ratio of antibody concentration be then Trastuzumab-POEGMA- fluorescent derivatives
The number for the fluorescence molecule that each antibody is connected in (or Trastuzumab-PMPC- fluorescent derivatives).
Table 1 shows the parameter of Trastuzumab-POEGMA-30R and Trastuzumab-PMPC-12.6R.As shown in table 1, it is average every
A Trastuzumab-POEGMA- fluorescent derivatives are connected with 30.0 fluorescence molecules, and average each Trastuzumab-PMPC- fluorescent derivatives connect
There are 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 that Trastuzumab-PMPC- fluorescent derivatives still maintain good work compared with Trastuzumab
Property.Figure 33 shows the EC of Trastuzumab-PMPC- fluorescent derivatives and antigen HER2 binding abilities50For 1.9 μ g/ml,
The EC that Herceptin is combined in itself with antigen HER250For 1.1 μ g/ml, curve above is the fluorescence immunoassay curve of Trastuzumab,
Following curve is the fluorescence immunoassay curve of Trastuzumab-PMPC- fluorescent derivatives.Figure 33 shows using specific site modification and original
The method of position polymerization, which prepares Trastuzumab-PMPC- fluorescent derivatives, can keep preferable activity.
By changing the ratio of OEGMA, fluorescent monomer and Trastuzumab-Br, can prepare containing different fluorescence molecules
Several Trastuzumab-POEGMA- fluorescent derivatives.
Figure 34 shows that Trastuzumab-POEGMA-2.9R (is connected with 2.9 fluorescence molecules, with lower class on average each Trastuzumab
Together), the SDS-PAGE of Trastuzumab-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R are examined
It surveys, before coomassie brilliant blue staining (left side), after coomassie brilliant blue staining (right side).Swimming lane 1 is protein tag；Swimming lane 2 is conspicuous
Sai Ting；Swimming lane 3 is Trastuzumab-POEGMA-2.9R；Swimming lane 4 is Trastuzumab-POEGMA-7.2R；Swimming lane 5 for Trastuzumab-
POEGMA-13.6R；Swimming lane 6 is Trastuzumab-POEGMA-30R.Figure 34 shows original Trastuzumab-POEGMA- fluorescent derivatives
Position (swimming lane 3~6) is red stripes；SDS-PAGE pictures after coomassie brilliant blue staining can see in antibody-high score
Sub- combination fluorescent derivatization object location (swimming lane 3~6) has apparent blue bands, and corresponding molecular weight is more than antibody (swimming lane
2) molecular weight of itself.SDS-PAGE pictures are it is considered that after ATRP copolyreaction in situ before and after comparing coomassie brilliant blue staining
Antibody-initiator combination (Trastuzumab-Br) almost all is converted into Trastuzumab-POEGMA- fluorescent derivatives.
Figure 35 shows the gel permeation chromatography (280nm) of different Trastuzumab-POEGMA- fluorescent derivatives.Left number first,
Second, third and Article 4 curve represent respectively Trastuzumab-POEGMA-2.9R, Trastuzumab-POEGMA-7.2R, Trastuzumab-
POEGMA-13.6R and Trastuzumab-POEGMA-30R, Article 5 curve represent Trastuzumab.Trastuzumab-POEGMA-2.9R, He Sai
The weight average molecular weight of spit of fland-POEGMA-7.2R, Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30R (Mw) are respectively
209.8kDa, 194.1kDa, 182.9kDa and 177.6kDa, corresponding molecular weight distribution (PDI) is respectively 1.3,1.3,1.2
With 1.2.
Fluorescence molecule is coupled on antibody and prepares antibody-fluorescent label (or derivative), is widely used for testing
Room and clinical detection.
In order to which Trastuzumab-POEGMA- fluorescent derivatives prepared by the present invention is (even directly by fluorescence molecule with conventional method
It is linked to antibody interchain disulfide bond) it prepares antibody-fluorescent label and compares, we are prepared for Trastuzumab-glimmering using conventional method
Signal object.Preparation method is as follows：
First, reduced form Trastuzumab (Trastuzumab-SH) is prepared according to preceding method.Then, by fluorescent marker TAMRA-
The DFM solution (20 times of equivalents) of C6- maleimides is added in the TrisHCl buffer solutions of above-mentioned Trastuzumab-SH, at 37 DEG C
Be incubated 14 it is small when, remove extra fluorescent marker TAMRA-C6- maleimides through desalting column and obtain antibody-fluorescent label
(Trastuzumab-TAM), and be determined that each antibody is average and be connected with 2.9 fluorescence molecules, such as Figure 36.
Figure 37 shows different Trastuzumab-POEGMA- fluorescent derivatives with Trastuzumab-TAM at same concentrations (27ng/mL)
Under relative intensity of fluorescence.As shown in the figure, first to the 5th block diagram represents Trastuzumab-TAM, Trastuzumab-POEGMA- respectively
2.9R, Trastuzumab-POEGMA-7.2R, the relative fluorescence of Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30.0R are strong
Degree.Fluorescence intensity increases with the increase of fluorescence molecule number in Trastuzumab-POEGMA- fluorescent derivatives, works as fluorescence molecule
Fluorescence intensity reaches highest 19.8 times of Trastuzumab-TAM (about) when number is 13.6, the fluorescence when fluorescence molecule reaches 30.0
Intensity is declined (about 17.4 times of Trastuzumab-TAM).
Fluorescence immunoassay experiment shows that Trastuzumab-TAM is still maintained preferably with Trastuzumab-POEGMA- fluorescent derivatives
Activity.First curve of upper number is the fluorescence immunoassay curve of Trastuzumab as shown in figure 38, and Article 2 curve is Trastuzumab-TAM's
Fluorescence immunoassay curve, third and fourth curve are respectively the fluorescence of Trastuzumab-POEGMA-2.9R and Trastuzumab-POEGMA-7.2R
Immune curve, the five, the six articles of curves are respectively that the fluorescence of Trastuzumab-POEGMA-13.6R and Trastuzumab-POEGMA-30.0R is exempted from
Epidemic disease curve.As shown in Figure 38, with Trastuzumab-POEGMA- fluorescent derivatives fluorescence molecule number increase, under activity is in
Drop trend.
Trastuzumab-POEGMA- fluorescent derivatives are compared with Trastuzumab-TAM to antigen by fluorescence immunoassay (direct method)
The fluorescence signal intensity of HER2 detections.
It is glimmering that Figure 39 shows that different Trastuzumab-POEGMA- fluorescent derivatives detect antigen HER2 with Trastuzumab-TAM
Curve is immunized in light.Upper several first, second, third and fourth curves represent respectively Trastuzumab-POEGMA-30R, Trastuzumab-
POEGMA-13.6R, Trastuzumab-POEGMA-7.2R and Trastuzumab-POEGMA-2.9R, Article 5 curve represent Trastuzumab-TAM.
Figure 39 shows the Trastuzumab-POEGMA- fluorescent derivatives of method preparation using the present invention compared with conventional method, to antigen
The fluorescence signal intensity of HER2 detections is significantly improved.
The dosage of antigen HER2 is chosen as 650ng/well, data mapping according to corresponding to Figure 40 (with Trastuzumab-
The number (n) of the fluorescence molecule connected in POEGMA- fluorescent derivatives is abscissa, with Trastuzumab-POEGMA- fluorescent derivatizations
The ratio of the fluorescence signal intensity of object and the fluorescence signal intensity of Trastuzumab-TAM is ordinate), as shown in figure 40, with fluorescence
The increase of Molecules, the fluorescence signal of the fluorescence signal intensity and Trastuzumab-TAM of Trastuzumab-POEGMA- fluorescent derivatives are strong
The ratio of degree gradually increases.When the fluorescence molecule number of Trastuzumab-POEGMA- fluorescent derivatives reaches 30, fluorescence signal is strong
Degree is 13.1 times of Trastuzumab-TAM.
Figure 41 shows Trastuzumab-POEGMA-13.6R and Trastuzumab-TAM to being overexpressed the immune glimmering of antigen HER2 cells
Light detects photo.(A and C) photo is opened for negative control group (A431, antigen HER2 low expressions cell) in left side two, and (B is opened on right side two
And D) photo be positive test group (SK-BR-3, antigen HER2 overexpressing cell).Two above (A and B) photos are respectively He Sai
Spit of fland-TAM to two groups of cell dyeings, below two (C and D) photos be respectively that Trastuzumab-POEGMA-13.6R contaminates two groups of cells
Color.As shown in Figure 41, Trastuzumab-TAM can selectively dye SK-BR-3, but fluorescence signal is relatively low；Trastuzumab-
POEGMA-13.6R equally can selectively dye SK-BR-3, while show very high fluorescence signal.
Figure 42 shows that Trastuzumab-POEGMA-13.6R and Trastuzumab-TAM examines the flow cytometer of SK-BR-3 cells
It surveys.Left first curve of number is negative control, and Article 2 curve is the cell of Trastuzumab-TAM dyeing, and Article 3 curve is He Sai
The cell of spit of fland-POEGMA-13.6R dyeing.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
12.2% cell can be dyed by Trastuzumab -2.9TAM, and the average fluorescence intensity for being each colored cell is 90.8；Have
48.0% cell can be dyed by Trastuzumab-POEGMA-13.6R, and the fluorescence intensity for being averagely each colored cell is
305.1, the fluorescence intensity adduction value by the cell of Trastuzumab-POEGMA-13.6R dyeing 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 provided by the invention for preparing antibody-macromolecule combination and its fluorescent derivative can at least by 10%,
20%th, the antibody of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% and ＞ 99% is connected with macromolecule, and
Each antibody can connect one or more (1~10) macromolecules.Antibody and high molecular connection site can be in chain
Disulfide bond, interchain disulfide bond and free sulfydryl (cysteine) or by chemical modification introduce disulfide bond (or
Person's free sulfhydryl group) etc. sites.
Antibody prepared by the present invention-macromolecule combination and its fluorescent derivative can have multiple different purposes.For example,
When selection has the antibody of therapeutic efficiency, combination can improve acceptable antibody and be controlled in the assemble index of disease site thus raising
Therapeutic effect.Compared with simple antibody, antibody-macromolecule combination and its fluorescent derivative can improve antibody water solubility,
Stability, pharmacokinetics and bio distribution simultaneously reduce its immunogenicity.
Antibody, which is combined (antibody-macromolecule combination) with macromolecule, can effectively improve antibody in vivo or in blood
Half-life period.The raising of half-life period can reach 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
200%th, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or even 20,30,40,50 times etc..It is anti-
Body, which is combined (antibody-macromolecule combination) with macromolecule, can effectively improve the dissolubility of antibody in aqueous solution, deliquescent
Raising can reach 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, even more.Antibody and high score
Son can effectively improve the pharmacokinetics of antibody with reference to (antibody-macromolecule combination).For example, antibody-macromolecule combination
The metabolism of active material can be slowed down or stimulate inert matter that metabolism is accelerated to form metabolite so as to reduce
The metabolism of active material.Antibody is combined (antibody-macromolecule combination) with macromolecule can be by reducing antibody exempting from vivo
Epidemic disease response is so as to effectively reduce the immunogenicity of antibody.The reduction of immunogenicity can reach 10%, 20%, 30%, 40%,
50%th, 60%, 70%, 80%, 90%, 100%, it is even more.
Without departing from the inventive concept of the premise, the protection that any possible change or replacement belong to the present invention is carried out
Scope.
Claims (16)
1. a kind of method for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, the antibody includes at least one
Disulfide bond or free sulfydryl, the macromolecule by be connected to the antibody disulfide bond or free sulfydryl to be attached to
It states on antibody, the described method includes：
A1 the combination of antibody-initiator) is prepared, wherein the initiator is attached to the disulfide bond of the antibody or free mercapto
On base；
B1) antibody-initiator combination in buffer solution with high polymer monomer or with high polymer monomer and fluorescence list
Body mixes, and the high polymer monomer is triggered to polymerize under catalyst action and prepares the antibody-macromolecule combination or anti-
The fluorescent derivative of body-macromolecule combination,
In step a1) in, the disulfide bond of the antibody is reduced into free sulfydryl, and then the initiator is connected to the mercapto
On base,
The initiator molecule is at least one in chemical formula 1, chemical formula 2 or chemical formula 3：
In chemical formula 1~3,
R1For the functional group of atom transition free radical polymerization reaction or its functional group derivant can be triggered；
Wherein, the functional group of atom transition free radical polymerization reaction can be triggered to be selected from 2- bromo -2- methyl propanamides, 2- chlorine
Generation -2- methyl propanamides, 2- bromos propionamide, 2- chloros propionamide, 2- bromos -2 Methylpropionic acid ester, 2- chloro -2- methyl-props
Acid esters, 2- ester bromopropionylaminos, 2- chloropropionic acid esters；
R2And R3It is identical or different, R2And R3It is each independently selected from easily spreading out by the functional group of nucleophilic displacement of fluorine and the functional group
Biology, it is described that H, I, Br, Cl, C are easily selected from by the functional group of nucleophilic displacement of fluorine6H5S、CH3C6H5S, to toluene ring sulfonyl extremely
It is few a kind of, R in chemical formula 22And R3Cannot be H simultaneously；
X and Y are identical or different, and X and Y are each independently selected from NH, O, S, Se atom；
Z includes N or CH,
The high polymer monomer in acrylate, methacrylate, acrylamide, Methacrylamide at least one
Kind,
The fluorescent monomer is selected from least one of acrylate, methacrylate, acrylamide, Methacrylamide,
The fluorophor of the fluorescent monomer is selected from fluorescein and its derivative, rhodamine and its derivative, flower cyanine fluorochrome
At least one of material, Fluorescent Brightening agents based on Coumarin, the glimmering fluorochrome of fluorine boron, phthalocyanine fluorochrome,
Wherein, the antibody is selected from medicine, agricultural, scientific research and the relevant antibody of other industrial circles.
2. a kind of method for preparing antibody-macromolecule combination and its fluorescent derivative, including：
A2) initiator triggers high polymer monomer or high polymer monomer and fluorescent monomer to polymerize under catalyst action, generation
The macromolecule or high molecular fluorescent derivative；
B2) macromolecule is coupled by the disulfide bond or free sulfydryl of the initiator and the antibody, is prepared described anti-
The fluorescent derivative of body-macromolecule combination or antibody-macromolecule combination,
In step b2) in, the disulfide bond of the antibody is reduced into free sulfydryl, and then the macromolecule passes through the initiation
Agent is connected on the sulfydryl, prepares the fluorescent derivatization of the antibody-macromolecule combination or antibody-macromolecule combination
Object,
The initiator molecule is at least one in chemical formula 1, chemical formula 2 or chemical formula 3：
In chemical formula 1~3,
R1For the functional group of atom transition free radical polymerization reaction or its functional group derivant can be triggered；
Wherein, the functional group of atom transition free radical polymerization reaction can be triggered to be selected from 2- bromo -2- methyl propanamides, 2- chlorine
Generation -2- methyl propanamides, 2- bromos propionamide, 2- chloros propionamide, 2- bromos -2 Methylpropionic acid ester, 2- chloro -2- methyl-props
Acid esters, 2- ester bromopropionylaminos, 2- chloropropionic acid esters；
R2And R3It is identical or different, R2And R3It is each independently selected from easily spreading out by the functional group of nucleophilic displacement of fluorine and the functional group
Biology, it is described that H, I, Br, Cl, C are easily selected from by the functional group of nucleophilic displacement of fluorine6H5S、CH3C6H5S, to toluene ring sulfonyl extremely
It is few a kind of, R in chemical formula 22And R3Cannot be H simultaneously；
X and Y are identical or different, and X and Y are each independently selected from NH, O, S, Se atom；
Z includes N or CH,
The high polymer monomer in acrylate, methacrylate, acrylamide, Methacrylamide at least one
Kind,
The fluorescent monomer is selected from least one of acrylate, methacrylate, acrylamide, Methacrylamide,
The fluorophor of the fluorescent monomer is selected from fluorescein and its derivative, rhodamine and its derivative, flower cyanine fluorochrome
At least one of material, Fluorescent Brightening agents based on Coumarin, the glimmering fluorochrome of fluorine boron, phthalocyanine fluorochrome,
Wherein, the antibody is selected from medicine, agricultural, scientific research and the relevant antibody of other industrial circles.
3. the method that any one of them such as claim 1 to 2 prepares antibody-macromolecule combination and its fluorescent derivative,
Wherein, the disulfide bond is selected from disulfide bond, at least one of the disulfide bond of interchain in chain.
4. the method as claimed in claim 3 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, it is described anti-
Body is included to be drawn by being selected from gene mutation, introducing alpha-non-natural amino acid, redox, the chemistry of enzymatic or biological modification method
The disulfide bond or free sulfydryl entered.
5. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, it is described
Polymerization is selected from atom transition free radical polymerization reaction.
6. the method as claimed in claim 5 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, for drawing
The catalyst for sending out atom transition free radical polymerization reaction described is selected from copper ion, second bipyridine and its derivative ligand, π receptors spread out
At least one of bio-ligand, nitrogen-atoms cheland and fat polyamine class ligand.
7. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, it is described
It is aggregated under hypoxemia or atmosphere of inert gases and carries out, the reaction time is 0.1 to 48 hour, and reaction temperature is 0 to 80 DEG C.
8. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, it is described
Initiator triggers initiation group of the group selected from the initiator for atom transition free radical polymerization reaction.
9. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, it is described
Macromolecule in antibody-macromolecule combination is selected from homopolymer, more heteropolymers, block polymer, copolymer, terpolymer
At least one.
10. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, institute
The side group for stating high polymer monomer is selected from carboxylic acid beet base, sulphonic acid betaine base, oligomeric ethylene glycol, polyethylene glycol, phosphatidic acid courage
Alkali, temperature-responsive group, pH responses group, optical Response group, monose or polysaccharide group.
11. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, institute
It states high polymer monomer and further comprises one or more reactive groups being embedded into when polymerisation occurs in high molecular skeleton
Group.
12. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, institute
The high molecular degree of polymerization is stated as 1 to 100,000.
13. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, institute
Macromolecule is stated with side chain, the side chain is in betaine side chain, phospholipid acid choline side chain, carboxyl betaine side chain, sulfuryl
Ammonium salt side chain, oligomeric ethylene glycol side chain, side-chain of polyelycol, temperature-responsive side chain, pH responses side chain, optical Response side
At least one of chain, monose or polysaccharide side chain.
14. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, often
One antibody-macromolecule combination and its fluorescent derivative have at least one macromolecular chain.
15. the method as claimed in claim 1 or 2 for preparing antibody-macromolecule combination and its fluorescent derivative, wherein, institute
It states antibody and is selected from vitro detection monoclonal antibody or mostly anti-, such as pathogenic pathogen detection antibody, drug targets for therapy detection is anti-
Body, tumor-marker analyte detection antibody, endocrine detection antibody, the one or more of cytokines measurement antibody；In-vivo imaging and
Monoclonal antibody or mostly anti-is treated, such as infliximab, Rituximab, bevacizumab, adalimumab, Cetuximab, Pa Li
Pearl monoclonal antibody, Gemtuzumab ozogamicin, the one or more of ibritumomab tiuxetan and Trastuzumab.
16. a kind of antibody-macromolecule combination and its fluorescent derivative, the antibody-macromolecule combination and its fluorescent derivatization
It is prepared by the method for any one that object passes through claim 1 to 15.
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| CN113248466B (en) * | 2021-04-16 | 2023-08-08 | 海昌隐形眼镜有限公司 | Rhodamine B dye, preparation method thereof and application thereof in preparing contact lenses for correcting red-green color blindness |
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| 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 |
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