CN108610415A - A kind of preparation method of antibody complex - Google Patents
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a kind of preparation method of antibody complex.The present invention by realizing the divalent of C-terminal C-terminal connection or the preparation of bispecific antibody complex in ice conditions, and mild condition, reaction speed is fast, and yield is higher.The divalent or the bispecific antibody complex present invention connected compared to the C-terminal N-terminal that conventional method is produced can obtain higher affinity, compared to the divalent of existing preparation C-terminal C-terminal connection or the disulphide bridges connection or click chemistry method of bispecific antibody, the present invention has preferably specificity and stability, step is less simultaneously, show that only the aldehyde radical that is generated in C-terminal is attached, the hydrazone bond or oxime key of generation stabilization and irreversible after being restored with reducing agent in physiological conditions.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of divalent that C-terminal-C-terminal (c-terminus-c-terminus) connects or double spies
The preparation method of the opposite sex and multivalence or multi-specificity antibody compound, the especially preparation method of nano antibody compound.
Background technology
Up to the present, U.S. FDA has been approved by more than 50 monoclonal antibody drugs and enters market, these drugs are in cancer
Disease is infected, and huge effect has been played in the treatment of the illnesss such as autoimmune disease.According to statistics, global antibody medicine in 2013
The sales volume of object reaches 75,000,000,000 dollars, occupies the half of all biological sales amount of medicine.In addition to the monoclonal antibody listed
Outside, 300 antibody are also had more than to be in research and development.In these antibody, multivalence, the work of polyspecific are synthesized with antibody fragment
Engineered antibody is since more preferably characteristic is occupying higher and higher proportion (Holliger, P.and P.J.Hudson for it
(2005)."Engineered antibody fragments and the rise of single domains."Nature
Biotechnology 23(9):1126-1136).Multivalence or multi-specificity antibody have following several sides for comparing antibody monomer
The advantage in face.First, because having more antigen-binding sites, multivalence or multi-specificity antibody have higher affinity, so
To combine target molecules more quickly and stable in vivo or in vitro.Second, since molecular weight increases, followed in vivo to extend
The half-life period of ring so that drug treating time is elongated.Third, multi-specificity antibody can identify different antigen, this imparting
Conventional antibody unexistent new function.Such as applied to specific T-cells raise (bispecific T cell engager,
BiTE) with killing tumor cell, wherein with Single chain antibody (Single chain fragment variable, scFv)
The bispecific antibody (Blinatumomab) constructed as unit can effectively treat leukaemia and non-Hodgkin lymphoma,
Ratify to list by U.S. FDA in the end of the year 2014.In addition to this polyspecific is also extensively used for treatment a variety of toxin in vivo
Or the fields such as pathogen and in-vitro diagnosis, it has broad application prospects and market.
Existing preparation divalent or bispecific and multivalence or multi-specificity antibody are most of using recombination table
It up to technology, is embodied in and the gene order of antibody fragment is linked in sequence, flexible peptide is inserted between antibody gene sequences
The gene such as sequence of glycine serine repetitive sequence or natural antibody hinge region of section, to improve the degree of freedom of antibody units
(Cuesta, A.M., et al. (2010), Trends in Biotechnology 28 (7):355-362).This routine side
Although method has been widely used, its technical attributes determines that it can only synthesize C-terminal-N-terminal (c-terminus-aminoterminal) connection
Multispecific antibody compound.Since antibody activity region is mostly near N-terminal, so the mode of C-terminal-N-terminal connection be easy to cause N-terminal
Steric interference antibody activity (van Lith, S.A.M., et al. (2017), Bioconjug Chem 28 (2):
539-548).Therefore, develop the mode that the technology that C-terminal-C-terminal connects is ideal.The technology of existing C-terminal-C-terminal connection is adopted more
With disulfide bond bridge joint and click chemistry method, how unstable disulfide bridge connection is and is easily influenced by local disulfide bond, and clickization
More and complicated (Witte, M.D., et al. (2012), Proceedings of the National of method process
Academy of Sciences of the United States of America 109(30):11993-11998), therefore
It is badly in need of developing a kind of C-terminal-C-terminal interconnection technique of the stabilization of simplicity.
The construction unit of existing multivalence or multi-specificity antibody mainly has scFv and nano antibody (nanobody, single
Domain antibody, sdAb) two kinds.Nano antibody is also referred to as single domain antibody, is a kind of antibody point being found in Camelidae body
Son.Its size only has 1/10th of conventional monoclonal antibody, but has and its comparable antigen binding capacity；In stability,
Prokaryotic expression, it is even better in conjunction with hiding epitope etc..There is smaller structure due to comparing scFv, so nanometer is anti-
Body is more suitable for the construction unit as multivalence or multi-specificity antibody, has broad application prospects and market
(Muyldermans,S.(2013).Vol 82 82:775-797)。
Due to divalent or bispecific, the important function of multivalence or multi-specificity antibody has C-terminal-N-terminal and connects synthesis side
Formula needs to improve, and the simply stable C-terminal of development-C-terminal connection is a kind of optimal synthesis mode.
Invention content
To solve the above-mentioned problems, the present invention provides for being synthesized altogether as construction unit using single domain antibody or nano antibody
The method of the bivalent antibody compound of the C-terminal of valence-C-terminal connection.
A kind of preparation method for the bivalent antibody or bispecific antibody complex that C-terminal-C-terminal connects, includes the following steps：
(1) FGE is merged in the C-terminal of antibody by genetic recombination and identifies amino acid sequence, carried out by the way that FGE is added in vitro
Catalysis, obtains C-terminal and pinpoints aldehyde group modified antibody；
(2) C-terminal is pinpointed into aldehyde group modified antibody and the same bifunctional linker of aldehyde radical reactivity, in ice conditions instead
It answers, obtains bivalent antibody compound, wherein the C-terminal pinpoints aldehyde group modified antibody with aldehyde radical reactivity with bifunctional linker
Molar ratio is 1:0.4~1.2；
(3) C-terminal is pinpointed into aldehyde group modified antibody and the same bifunctional linker of aldehyde radical reactivity, in ice conditions instead
It answers, the antibody of single same bifunctional linker connection is obtained, wherein the C-terminal pinpoints aldehyde group modified antibody and aldehyde radical reactivity is same
The molar ratio of bifunctional linker is 1:5~15；
(4) antibody and C-terminal that step (3) obtains are pinpointed into aldehyde group modified antibody, reacted in ice conditions, obtained double
The variable region that the antibody that specific antibody complex, wherein step (3) obtain and C-terminal pinpoint aldehyde group modified antibody is different.
Further, in the above-mentioned technical solutions, in step (2), step (3) and step (4), the freezing conditions
It is -5 DEG C~30 DEG C, preferably -10 DEG C~25 DEG C, more preferably -20 DEG C.
Further, in the above-mentioned technical solutions, described in frost item in step (2), step (3) and step (4)
Reaction refers to 2~48h of reaction at a temperature of -5 DEG C~-30 DEG C under part, and the temperature is -10 DEG C~-25 DEG C preferred, more preferably
It it is -20 DEG C, the reaction time is preferably 10-30h, more preferably for 24 hours.
Further, in the above-mentioned technical solutions, described in frost item in step (2), step (3) and step (4)
The pH value for the reaction system reacted under part is 4.0~7.5, and pH value is preferably 4-5, more preferably 4.
Further, in the above-mentioned technical solutions, in step (1), the antibody be nano antibody, single-chain antibody scFV or
Variable region monoclonal antibody.
Further, in the above-mentioned technical solutions, in step (1), FGE identification amino acid sequences are to contain half Guang
Propylhomoserin-X- proline-X- arginine, the X are arbitrary natural amino acid, and preferably FGE identifies that amino acid sequence is LCTPSR.Into
One step, in the above-mentioned technical solutions, in step (2), step (3), the aldehyde radical reactivity is R-L- with bifunctional linker
R, wherein R are the aldehyde radical reactive group for including amino, hydrazide group, oxygen amino, phenylhydrazino or pyridine diazanyl, L be with-
(CH2CH2- O) n and/or-(O-CH2CH2) polymer of the n as Component units, wherein the integer that n is 1 to 100, n is preferably 1
To 50, more preferably 10 to 30.
Further, in the above-mentioned technical solutions, the gene coded sequence of the FGE derives from mycobacterium tuberculosis or people
Class.
Further, in the above-mentioned technical solutions, in step (3), the reaction product after reacting in ice conditions is led to
Size exclusion chromatography separation is crossed, the antibody of single same bifunctional linker connection is obtained.
Further, in the above-mentioned technical solutions, in step (4), the antibody and C-terminal that the step (3) obtains are fixed
The molar ratio of the aldehyde group modified antibody of point is 1:1~3.
In the above-mentioned technical solutions, it is additionally added in the reaction system described in step (2), step (3) and step (4)
Former agent, the C=N double bonds of generation, which are reduced into C-N singly-bounds, can increase hydrazone bond or the stability of oxime key, for the reducing agent with
And its addition is not specially limited, those skilled in the art can select reducing agent appropriate and addition according to routine techniques
Amount, it is preferable that it is need reducing substances mole 10 times that sodium borohydride, sodium cyanoborohydride etc., addition, which can be used, in reducing agent
More than.
In the present invention, the FGE is that formylglycine generates enzyme, and the bivalent antibody of the C-terminal-C-terminal connection is compound
Object refers to dimer obtained from two identical antibody are connect each by c-terminus with same bifunctional linker, the C-terminal-C
The bispecific antibody complex of end connection refers to two antibody with different variable regions each by c-terminus and same double officials
Antibody complex with bispecific obtained from energy connector connection.The antibody of the single same bifunctional linker connection refers to
With the compound for only having one end to be connected to antibody in bifunctional linker both ends and being formed.
In currently preferred technical solution, the C-terminal pinpoints aldehyde group modified antibody, can be as follows
It is prepared：
1. the gene order that formylglycine generates the identification peptide fragment of enzyme (FGE) is added at the end of gene order 3 ' of antibody,
The recombination is connected to carrier, it is thin to be transferred to the hosts such as Bacillus coli expression host strain or Pichia pastoris or mammalian cell
It is recombinantly expressed in born of the same parents, the antibody of expression is extracted, purifies to obtain 3 ' end (ends C-) fusions and have the weights of FGE identification amino acid sequences
The method that this field routine may be used in group antibody, the extraction and purifying carries out, as immobilized metal ion affinity can be used in purifying
With chromatography preliminary purification, then through size exclusion chromatography polishing purification etc.；The wherein preferred LCTPSR of the FGE identification peptide fragments, it is described
Plasmid preferred pET21a or pET23a；
2. in vitro, the recombinant antibodies and a certain amount of FGE are mixed, buffer conditions appropriate and at a temperature of FGE
It is catalyzed the label converting of the ends C- of recombinant antibodies and pinpoints aldehyde with aldehyde groups to get to C-terminal of the present invention for side chain
The antibody of base modification, wherein the addition of the recombinant antibodies and FGE preferably in molar ratio 1:5~10, buffer solution preferably three
1-5mM mercaptoethanols, ionic strength 50-150mM chlorinations are added in buffer solution by ethanol amine-hydrochloride buffer, pH 7.0-9.0
Sodium, preferably 18 DEG C to 30 DEG C of catalytic temperature.
In currently preferred technical solution, the bivalent antibody compound of the C-terminal-C-terminal connection can be made as follows
It is standby to obtain：C-terminal obtained above is pinpointed into aldehyde group modified antibody and the same bifunctional linker of aldehyde radical reactivity, according to molar ratio
1:0.5~1.0 mixing, reacts 2-24h under conditions of -5 DEG C~-30 DEG C, pH 4-5, can complete aldehyde radical reactivity with difunctionality
The connection of connector and antibody C-terminal aldehyde radical, the bivalent antibody compound that C-terminal-C-terminal to form covalence stablility connects, finally
Yield is 50% or so.
In currently preferred technical solution, the bispecific antibody complex of the C-terminal-C-terminal connection can be by such as lower section
Method is prepared：
(1) C-terminal obtained above is pinpointed into the same bifunctional linker of aldehyde group modified antibody and aldehyde radical reactivity, according to rubbing
That ratio 1:5-15 is mixed, and reacts 2-24h under conditions of -5 DEG C~-30 DEG C, pH 4-5, can complete aldehyde radical reactivity with difunctionality
The connection of connector and antibody C-terminal aldehyde radical obtains the antibody of single same bifunctional linker connection；Wherein it is preferred to reaction product
It can be detached through size exclusion chromatograph, the antibody of the single same bifunctional linker connection of purifying be obtained, for reacting in next step.
(2) antibody for the single same bifunctional linker connection that above-mentioned steps (1) obtain is pinpointed with C-terminal aldehyde group modified
Antibody is according to molar ratio 1:1~3 mixing, reacts 2-24h under conditions of -5 DEG C~-30 DEG C, pH 4-5, wherein single with double
It is different that the antibody of function connector connection pinpoints the variable region of aldehyde group modified antibody from C-terminal, is connected to which C-terminal-C-terminal can be obtained
Bispecific antibody complex, ultimate yield be 30% or so.
The present invention has the following advantages compared with the prior art and effect：
The present invention by realizing the divalent or bispecific antibody complex of the connection of C-terminal-C-terminal, condition in ice conditions
Mildly, reaction speed is fast, and yield is higher.The divalent or bispecific antibody of the C-terminal produced compared to conventional method-N-terminal connection
The compound present invention can obtain higher affinity (such as Fig. 1), and the affinity is surveyed based on surface plasma body resonant vibration
Obtain equilibrium dissociation constant.Compared to existing preparation C-terminal-divalent of C-terminal connection or disulphide bridges connection or the click of bispecific antibody
Chemical method, the present invention has preferably specificity, while step is less, shows that the aldehyde radical only generated in C-terminal is attached, raw
At hydrazone bond or oxime key stablize in physiological conditions and after being restored with reducing agent it is irreversible, simultaneously because being added to polyethylene glycol
Connector, blood halflife can extend.
Description of the drawings
Fig. 1 is the structure of a nano antibody, prepares the divalent or bispecific nano antibody of C-N connections by conventional method
Structure and the divalent of C-C connections or the schematic diagram of bispecific nano antibody structure are prepared through the invention.
Fig. 2A is to modify nano antibody to aldehyde radicalization using fluorescent molecular to be marked, and is detached to it on SDS-PAGE
As a result, Fig. 2 B are to be combined point spraying ionization path ion trap mass spectrometry in high performance liquid chromatography to modify nano antibody essence to aldehyde radicalization
Determine that amount, shown collection of illustrative plates are molecular weight-abundance figure after deconvoluting.
Fig. 3 be added bishydrazide functionalization PEG400 connectors, different pH value and at a temperature of react after after 24 hours
Separating resulting of the product on SDS-PAGE.
Fig. 4 A are that high effective liquid chromatography for measuring bivalent antibody yield changes with time under -20 DEG C of freezing temperatures, are schemed
4B is that divalent nano antibody yield changes with time at different temperatures.
Fig. 5 A are the separating resulting for being separately added into the divalent nano antibody of different length connector on SDS-PAGE, Fig. 5 B
It is size exclusion chromatography to being isolated and purified containing different length connector divalent nano antibody.
Fig. 6 is to be detached to the nano antibody B monomer for being connected to same bifunctional linker using size exclusion chromatography
Specific implementation mode
Following non-limiting embodiments can make those skilled in the art be more fully understood the present invention, but not with
Any mode limits the present invention.In following embodiments, unless otherwise specified, used experimental method is conventional method, institute
It can be bought from biological or chemical company with material, reagent etc..
Material as used in the following examples：
Nano antibody A：Coding gene sequence such as SEQ ID NO.1, wherein 511-528 sequences
Ctgtgcaccccgtctcgt is that FGE identifies sequence；
Nano antibody B：Coding gene sequence such as SEQ ID NO.2, wherein 499-516 sequences
Ctgtgcaccccgtctcgt is that FGE identifies sequence.
The C-terminal pointed decoration of 1 nano antibody of embodiment
The coding gene sequence of nano antibody A is cloned into expression vector plasmid pET23a, Escherichia coli are imported
It is expressed in T7Shuffle (DE3), collects thalline, and clasmatosis, it is affine using metal-chelating from clasmatosis supernatant
HisTrap HP 5mL (GE Healthcare) purified nanotubes antibody is chromatographed, ultimate output is on 100 milligrams per liter of zymotic fluid left sides
It is right.By the nano antibody of purifying through be concentrated by ultrafiltration change liquid to Triethanolamine buffer (25mM TEAM, pH9.0,150mM NaCl,
1mM mercaptoethanols), nano antibody concentration 5mg/mL, it is that nano antibody mole about 1/10th is that ultimate density, which is added,
The purity of 1mg/mL is>90%FGE enzymes (FGE enzyme molecular weights are about 33kDa, and nano antibody molecular weight is about 18kDa), at 18 DEG C
Under the conditions of mildly concussion catalysis reaction 20 hours, after reaction centrifugation removal albumen precipitation, obtain c-terminus fixed point aldehyde radicalization modification
Nano antibody A, be named as nano antibody A0.
Aldehyde group modified efficiency is accurately detected with high performance liquid chromatography combination high-resolution electrospray ionization mass spectrometry, Fig. 2 B are shown
Molecular weight-abundance figure after proteomic image figure deconvolutes, (nanometer is anti-for the protein in collection of illustrative plates before visible modification and after modification
Body) spectral peak, after aldehyde radicalization modification, the component of albumen about reduces 18Da, and egg can be determined by comparing the integrated peak areas of the two
White aldehyde radicalization modifies efficiency.Protein solution after reaction can also be changed liquid to acidic buffer (such as 0.1M through being concentrated by ultrafiltration
4.0 NaCl containing 150mM of acetate buffer solution pH), the fluorescent molecular that final concentration of 500 μm of ol/L carry hydrazides group is then added
Aldehyde radical is marked in Lucifer Yellow CH lithium slat (Thermo Fisher), the analysis through SDS-PAGE
Also can be tentatively quantitative to the aldehyde group modified efficiency of C-terminal, as shown in Figure 2 A, due to marking upper Lucifer Yellow fluorescent moleculars,
Albumen visible apparent fluorescence under ultraviolet imagery, while albumen is due to marking a upper fluorescent molecular that molecular weight is caused to increase
Mobility reduction is shown as on SDS-PAGE, is compared and is migrated upwards without the protein band on label, it can be thick by gray scale scanning
Slightly aldehyde radicalization modification is quantified.
Embodiment 2 is realized in ice conditions prepares C-C connection divalent nano antibodies
Nano antibody A (nano antibody A0) ultrafiltration for the c-terminus fixed point aldehyde radicalization modification that embodiment 1 is prepared is dense
Contracting changes liquid to acidic buffer, 0.1M acetate buffer solutions pH 4.0 or 0.1M MES pH of buffer 5.5 or neutral buffered liquid
A concentration of 2mg/mL i.e. 100 μm ol/L of 0.2M PBS pH7.4, nano antibody A0.Take a certain amount of nano antibody configured
Bifunctional linker HZ-PEG-HZ 400 (bishydrazide polyethylene glycol-400) and reducing agent sodium cyanoborohydride is added in A0, wherein
The final concentration of HZ-PEG-HZ 400 and sodium cyanoborohydride in the reaction system is respectively 50 μm of ol/L and 1mmol/L, that is,
The molar ratio of PEG and nano antibody is 1 in reaction system:2, the molar ratio of sodium cyanoborohydride and nano antibody is 10:1.It will
The reaction mixture being added, is put into cryostat, and is cooled to -30 DEG C, so that reaction mixture is become freezing state, then adjusts
To different temperatures, respectively -30 DEG C, -20 DEG C, -10 DEG C and -5 DEG C, 0~25h is reacted.Wherein, reaction mixture is become into ice
Then jelly state is adjusted to each reaction temperature, can shorten the time of sample icing in this way, so as to shorten reaction time, the present invention
It is middle the reaction mixture is directly placed into relevant temperature can also.In addition, being additionally provided with 37 DEG C and -80 DEG C of reaction conditions, i.e., such as
It is upper described, it changes in liquid to the nano antibody of different acidic buffers, HZ-PEG-HZ 400 and reducing agent is added (addition is same as above)
Afterwards, reacting liquid temperature is adjusted to 37 DEG C and -80 DEG C, the reaction was continued 0~25h.Fig. 3 be different pH value and at a temperature of react
Electrophoretogram of the reaction product on SDS-PAGE after 24 hours, it is seen that visible apparent under conditions of degree is with 4.0 pH at -20 DEG C
Divalent nano antibody band.
It, then will be after reaction by high performance liquid chromatography by the solution after reaction with sodium hydroxide adjusting pH to neutrality
Mixture detach analysis and compare respective peaks integral area that reaction yield can be calculated.Fig. 4 A are high effective liquid chromatography for measuring
Bivalent antibody yield changes with time under -20 DEG C of freezing temperatures, and Fig. 4 B are divalent nano antibody yield at different temperatures
It changes with time.As it can be seen that can reach maximum reacting 24 hours or so yields, -30 DEG C and -5 DEG C are reacted relatively slow.- 10
DEG C to reacting and can comparatively fast occur in -20 DEG C of sections.At -10 DEG C arrive -20 DEG C of sections, divalent nanometer yield be 30% to 50% it
Between.
Fig. 5 A are electrophoretogram of the above-mentioned reaction product for being separately added into different length connector on SDS-PAGE, wherein swimming
Road M is albumen Marker, 1-4 swimming lane Wei not be added bifunctional linker (eventually in nano antibody A0 (100 μm of ol/L of final concentration) successively
50 μm of ol/L of concentration), the reaction production after O-linker, HZ-PEG-HZ 400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000
Object, 5-8, which is followed successively by nano antibody B0 (100 μm of ol/L of final concentration), is added bifunctional linker (50 μm of ol/L of final concentration), O-
Reaction product after linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000.As it can be seen that swimming lane in Fig. 5 A
There is a certain amount of divalent nano antibody band in 40kDa or so in 1-8, shows for nano antibody A0 and B0, is added not
With the bifunctional linker of length, a certain amount of divalent nano antibody can be generated after reacting in ice conditions.Fig. 5 B are upper
It states and is separately added into the result chromatogram that the reaction product of different length connector is isolated and purified by size exclusion chromatography.In Fig. 5 B
As it can be seen that can also remain monomer in solution after reaction, it is connected to the monomer and bivalent antibody of connector, wherein bivalent antibody can be very
Good being separated carries out the research of next step.
3 freezing method of embodiment prepares the bispecific nano antibody of C-C connections
(1) coding gene sequence of nano antibody B is cloned into expression vector plasmid pET23a, imports Escherichia coli
It is expressed in T7Shuffle (DE3), collects thalline, and clasmatosis, it is affine using metal-chelating from clasmatosis supernatant
HisTrap HP 5mL (GE Healthcare) purified nanotubes antibody is chromatographed, ultimate output is about 100 milligrams per liter of zymotic fluids.
By the nano antibody of purifying through be concentrated by ultrafiltration change liquid to Triethanolamine buffer (25mM TEAM, pH 9.0,150mM NaCl,
1mM mercaptoethanols), nano antibody concentration 5mg/mL, it is that nano antibody mole about 1/10th is that ultimate density, which is added,
The purity of 1mg/mL is>90%FGE enzymes (FGE enzyme molecular weights are about 33kDa, and nano antibody molecular weight is about 18kDa), at 18 DEG C
Under the conditions of mildly concussion catalysis reaction 20 hours, after reaction centrifugation removal albumen precipitation, obtain c-terminus fixed point aldehyde radicalization modification
Nano antibody B, be named as nano antibody B0.
(2) nano antibody B0 ultrafiltration concentrations obtained by previous step are changed into liquid to acidic buffer (0.1M acetate buffer solutions pH
4.0, NaCl containing 150mM), final concentration of 2mg/mL i.e. 100 μm ol/L.The a certain amount of nano antibody B0 configured is taken, is added respectively
Enter different length bifunctional linker, O-linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, double work(
The final concentration of 1mmol/L of energy connector in the reaction system, that is, mole of bifunctional linker and nano antibody in the reaction system
Than being 10:1, additionally incorporate the sodium cyanoborohydride of final concentration 1mmol/L.By the reaction mixture, under -20 DEG C of freezing conditions
Reaction 24 hours.
The size exclusion chromatography separating step is as follows：By reaction product, room temperature is placed, 1M is added after melting
It is neutrality that NaOH solution, which adjusts pH value, then passes through size exclusion chromatograph column Superdex 75Increase 10/300GL (GE
Healthcare), use 20mM phosphate 150mM NaCl pH7.4 buffer solutions as operation solution with the speed of 0.6mL/min into
Row separation, collects each component, band is verified by SDS-PAGE.Finally retain the nano antibody for being connected to same bifunctional linker
B monomer, as shown in Figure 6.Fig. 6 A are the chromatography that the product after different connectors are excessively added isolates and purifies on size exclusion chromatography
Peak figure, Fig. 6 B are SDS-PAGE identifications (the respectively excessive addition bifunctional linker O- of swimming lane 0,3,6,10 of different chromatographic peaks
Reaction product after linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, remaining swimming lane are in Fig. 6 A
Corresponding chromatographic peak component).As it can be seen that a certain amount of nano antibody for being connected to connector can effectively be isolated and purified for
The research of next step.
(3) the nano antibody B for being connected to same bifunctional linker obtained by previous step and equimolar than embodiment 1 in aldehyde
Baseization modifies nano antibody A mixing, and reaction solution is adjusted to pH 4.0 with acetic acid, additionally incorporates the cyanogen of final concentration 1mmol/L
Base sodium borohydride then reacts at -20 DEG C at least 24 hours, and product passes through size exclusion chromatograph column after gained reaction
Superdex 75Increase 10/300GL separation can obtain the bispecific nano antibody A-B of C-C connections.Yield arrives for 20%
40%.
4 divalent of embodiment and the affinity costant of bispecific nano antibody measure
Determine that the combination of divalent or bispecific nano antibody and antigen is affine by surface plasma body resonant vibration (SPR)
Power and kinetic parameter, the surface plasma body resonant vibration are using CM5 sensor chips and HBS-EP (10mM HEPES (pH
7.4), 150mM NaCl, 3mM EDTA, 0.05%v/v P20) running buffer Biacore T200 on carry out.Pass through
EDC/NHS methods by antigen β2-microglobulin by amino coupled in chip surface, final supported quantity is about 700Ru.Each cycle
Measurement comprises the steps of：First with 120 seconds injection divalent or bispecific nano antibody, dissociation 180 seconds is then monitored, often
Injected after a cycle 60 seconds glycine-hcl buffer solutions (10mM, pH 1.5) with regenerate.Software is assessed using Biacore, is passed through
The sensing figure of gained is with standard 1：The 1 binding model overall situation is fitted, and determines kinetic parameter.As shown in Table 1, A0 is C-terminal aldehyde radical
Change the nano antibody A of modification, B0 be monomer the nano antibody B, A1-A4 of C-terminal aldehyde radicalization modification be use the method for the present invention with
A0 is the C-C connection divalent nano antibodies that unit is constructed, and A1 is used using the connector that both ends are oxygen amino, i.e. O-linker, A2
Both ends are the PEG400 connectors i.e. HZ-PEG-HZ 400 of hydrazide group, and A3 is using the PEG1000 connectors i.e. HZ- that both ends are hydrazide groups
PEG-HZ 1000, A4 are using the PEG2000 connectors i.e. HZ-PEG-HZ 2000 that both ends are hydrazide groups, wherein connection divalent nanometer
Antibody response temperature is -20 DEG C, and the reaction time is pH value of reaction system 4.0 for 24 hours.B1-B5 be use the method for the present invention with
The C-C connection divalent nano antibodies that B0 constructs for unit, the specific same A1-A5 of meaning, reaction condition are same as above.C1-C4 is to use
The bispecific nano antibody that the method for the present invention is connected with B0 as the C-C that unit is constructed using nano antibody A0, C1 are using both ends
Connector, that is, O-linker of oxygen amino, C2 use two using the PEG400 connectors i.e. HZ-PEG-HZ 400 that both ends are hydrazide groups, C3
End is the PEG1000 connectors i.e. HZ-PEG-HZ 1000 of hydrazide group, and C4 is using the PEG2000 connectors i.e. HZ- that both ends are hydrazide groups
PEG-HZ 2000, wherein connection antibody response temperature is -20 DEG C, and the reaction time is pH value of reaction system 4.0 for 24 hours.A5
It is that DNA recombinant expression C-N connects divalent nano antibody with B5, C5 is that DNA recombinant expression C-N connects bispecific nanometer with C6
Antibody.The C-N connection divalent or bispecific nano antibody are that this field conventional method is prepared, i.e., by two sections it is identical or
Different nano antibody encoding genes is connected by chemical synthesis or round pcr, and the connection between two sections of genes is that coding is flexible
The gene of amino acid sequence, it is flexible using Gly-Gly-Gly-glycine-serine of 3 sections of repetitions in of the invention
Then gene after synthesis is transferred in expression plasmid pET21a by amino acid sequence as connector, carried out in Escherichia coli intracellular
Expression.It is SEQ ID NO.4, C5 sequence be SEQ ID NO.5, C6 sequence is SEQ that A5 sequences, which are SEQ ID NO.3, B5 sequence,
ID NO.6.C5 is to connect divalent nano antibody using nano antibody A0 with the DNA recombinant expression C-N that B0 is constructed as unit with C6,
The C5 orders of connection are A0-B0, and the order of connection of C6 is B0-A0, both uses the sweet ammonia of Gly-Gly-of 3 sections of repetitions
Acid-glycine-serine flexible amino acid sequence is as connector.Table 1 is that as previously discussed utilizes surface plasma body resonant vibration
Technology connects different length C-terminal-C-terminal the C-terminal-of divalent nano antibody, bispecific nano antibody and conventional method production
N-terminal connects divalent nano antibody, the association rate constant K of bispecific nano antibodya, dissociation rate constant KdAnd it is obtained
Affinity constant KD。
Table 1.
The result of table 1 is shown：It is detected with surface plasma body resonant vibration, C-N structures and C-C structures divalent nano antibody
Or bispecific antibody has different degrees of promotion, the A1 of wherein C-C connections to compare C-N with A2 in affinity compared to monomer
The A5 affinity of connection is higher by 20 times to 30 times, likewise, B3, which compares B5 affinity with B4, is also higher by 20 to 30 times.C-C connections
Bispecific antibody C1-C4 affinity to be higher by about 40 to 50 times of the C5 and C6 of C-N connections.In conclusion the two of C-C connections
Valence or bispecific nano antibody, are better than the antibody of C-N connections in antigen binding capacity.
Finally it should be noted that：Obviously, above-described embodiment is only intended to clearly illustrate the application example, is excellent
The embodiment of choosing.And it does not limit the embodiments.For those of ordinary skill in the art, in above description
On the basis of can also make other variations or changes in different ways.There is no need and unable to give thoroughly all embodiments
It lifts.And among thus changes and variations that derived from are still in the protection domain of the application type.
Sequence table
<110>Dalian University of Technology
<120>A kind of preparation method of antibody complex
<130> 2011
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 537
<212> DNA
<213>Artificial synthesized ()
<400> 1
catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60
ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120
cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180
atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240
acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360
tattggggcc aggggaccca ggtcaccgtg agctcagaac ccaagacacc aaaaccacaa 420
ccacaaccac aaccacaacc ccaaccggat cctacaacag aaggaggcgg tgggagccac 480
caccaccacc accacggagg cggtgggagc ctgtgcaccc cgtctcgtta actcgag 537
<210> 2
<211> 525
<212> DNA
<213>Artificial synthesized ()
<400> 2
catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60
ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120
cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180
acctactacg cagatagcgt taaaggccgc tttaccttca gccaggataa cgcgaaaaac 240
accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300
accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360
tggggtcaag gtacccaagt taccgtgagc tcagaaccca agacaccaaa accacaacca 420
caaccacaac cacaacccca acccaatcct acaacagaag aattccacca tcaccaccat 480
catggtggcg gtggttcgct gtgcaccccg tcccgttgac tcgag 525
<210> 3
<211> 1017
<212> DNA
<213>Artificial synthesized ()
<400> 3
catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60
ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120
cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180
atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240
acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360
tattggggcc aggggaccca ggtcaccgtg gaacccaaga caccaaaacc acaaccacaa 420
ccacaaccac aaccccaacc ggatcctaca acagaagaat tcggtggtgg aggctccggc 480
ggagggggta gtggcggcgg tggaagtaag cttgcccagg tgcagctcgt ggagtctggg 540
ggagggttgg tgcaggctgg ggggtcactg agactctcct gtgcagcctc tggatccact 600
ttggattctt attacatagg ctggttccgc caggccccag gcaaagagcg cgagggggtc 660
tcatgtatta gtagtagtgg taatagcata cgttatgtag attccgtgaa ggaccgattc 720
accatctcta gagacaacgg caagaacacg gcctatctcc acatcaacag cctgaaacct 780
gaggacacgg ccgtttatta ctgtgcagcg agtcgtcgag ggcgcatacc gggcctacct 840
tgtagtttag tacgtgaacg ctatgcctat tggggccagg ggacccaggt caccgtgagc 900
tcagaaccca agacaccaaa accacaacca caaccacaac cacaacccca accggatcct 960
acaacagaag gaggcggtgg gagccaccac caccaccacc accaccacta actcgag 1017
<210> 4
<211> 1005
<212> DNA
<213>Artificial synthesized ()
<400> 4
catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60
ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120
cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180
acctactacg cagatagcgt taaaggccgc tttaccttca gccaggataa cgcgaaaaac 240
accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300
accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360
tggggtcaag gtacccaagt taccgtggaa cccaagacac caaaaccaca accacaacca 420
caaccacaac cccaacccaa tcctacaaca gaagaattcg gaggcggtgg gagcggaggc 480
ggtgggagcg gaggcggtgg atccgcccaa gttcaactgc aagaatctgg cggcggttct 540
gttcaagcag gcggtagtct gcgtctgagt tgtgcagcaa gcggttatac cgattcccgc 600
tattgtatgg cctggtttcg tcaagctccg ggtaaagaac gcgagtgggt tgcacgtatc 660
aacagcggtc gcgatatcac ctactacgca gatagcgtta aaggccgctt taccttcagc 720
caggataacg cgaaaaacac cgtctacctg cagatggata gtctggaacc ggaagatacc 780
gcgacctatt attgcgcaac cgatatcccg ctgcgttgtc gcgatattgt agcaaaaggc 840
ggcgacggtt ttcgttattg gggtcaaggt acccaagtta ccgtgagctc agaacccaag 900
acaccaaaac cacaaccaca accacaacca caaccccaac ccaatcctac aacagaagga 960
ggcggtggga gccaccacca ccaccaccac caccactaac tcgag 1005
<210> 5
<211> 1008
<212> DNA
<213>Artificial synthesized ()
<400> 5
catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60
ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120
cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180
atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240
acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360
tattggggcc aggggaccca ggtcaccgtg gaacccaaga caccaaaacc acaaccacaa 420
ccacaaccac aaccccaacc ggatcctaca acagaagaat tcggaggcgg tgggagcgga 480
ggcggtggga gcggaggcgg tggatccgcc caagttcaac tgcaagaatc tggcggcggt 540
tctgttcaag caggcggtag tctgcgtctg agttgtgcag caagcggtta taccgattcc 600
cgctattgta tggcctggtt tcgtcaagct ccgggtaaag aacgcgagtg ggttgcacgt 660
atcaacagcg gtcgcgatat cacctactac gcagatagcg ttaaaggccg ctttaccttc 720
agccaggata acgcgaaaaa caccgtctac ctgcagatgg atagtctgga accggaagat 780
accgcgacct attattgcgc aaccgatatc ccgctgcgtt gtcgcgatat tgtagcaaaa 840
ggcggcgacg gttttcgtta ttggggtcaa ggtacccaag ttaccgtgag ctcagaaccc 900
aagacaccaa aaccacaacc acaaccacaa ccacaacccc aacccaatcc tacaacagaa 960
ggaggcggtg ggagccacca ccaccaccac caccaccact aactcgag 1008
<210> 6
<211> 1008
<212> DNA
<213>Artificial synthesized ()
<400> 6
catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60
ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120
cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180
acctactacg cagatagcgt taaaggccgc tttaccttca gccaggataa cgcgaaaaac 240
accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300
accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360
tggggtcaag gtacccaagt taccgtggaa cccaagacac caaaaccaca accacaacca 420
caaccacaac cccaacccaa tcctacaaca gaagaattcg gaggcggtgg gagcggaggc 480
ggtgggagcg gaggcggtgg atccgcccag gtgcagctcg tggagtctgg gggagggttg 540
gtgcaggctg gggggtcact gagactctcc tgtgcagcct ctggatccac tttggattct 600
tattacatag gctggttccg ccaggcccca ggcaaagagc gcgagggggt ctcatgtatt 660
agtagtagtg gtaatagcat acgttatgta gattccgtga aggaccgatt caccatctct 720
agagacaacg gcaagaacac ggcctatctc cacatcaaca gcctgaaacc tgaggacacg 780
gccgtttatt actgtgcagc gagtcgtcga gggcgcatac cgggcctacc ttgtagttta 840
gtacgtgaac gctatgccta ttggggccag gggacccagg tcaccgtgag ctcagaaccc 900
aagacaccaa aaccacaacc acaaccacaa ccacaacccc aaccggatcc tacaacagaa 960
ggaggcggtg ggagccacca ccaccaccac caccaccact aactcgag 1008
Claims (10)
1. a kind of C-terminal-bivalent antibody of C-terminal connection or the preparation method of bispecific antibody complex, include the following steps：
(1) FGE is merged in the C-terminal of antibody by genetic recombination and identifies amino acid sequence, urged by the way that FGE is added in vitro
Change, obtains C-terminal and pinpoint aldehyde group modified antibody；
(2) C-terminal is pinpointed into aldehyde group modified antibody and the same bifunctional linker of aldehyde radical reactivity, reacts, obtains in ice conditions
Bivalent antibody compound, wherein the C-terminal pinpoints mole of the aldehyde group modified antibody with aldehyde radical reactivity with bifunctional linker
Than being 1:0.4~1.2；
(3) C-terminal is pinpointed into aldehyde group modified antibody and the same bifunctional linker of aldehyde radical reactivity, reacts, obtains in ice conditions
The antibody of single same bifunctional linker connection, wherein the C-terminal pinpoints aldehyde group modified antibody with aldehyde radical reactivity with double officials
The molar ratio of energy connector is 1:5~15；
(4) antibody and C-terminal that step (3) obtains are pinpointed into aldehyde group modified antibody, reacted in ice conditions, obtained double special
Property antibody complex, the variable region that the antibody that wherein step (3) obtains and C-terminal pinpoint aldehyde group modified antibody is different.
2. preparation method according to claim 1, which is characterized in that in step (2), step (3) and step (4),
The freezing conditions are -5 DEG C~-30 DEG C.
3. preparation method according to claim 1, which is characterized in that in step (2), step (3) and step (4),
The reaction in ice conditions refers to 2~48h of placement at -5 DEG C~-30 DEG C.
4. according to any one of them preparation method of claims 1 to 3, which is characterized in that step (2), step (3) and
In step (4), the pH value of reaction system reacted in ice conditions is 4.0~7.5.
5. preparation method according to claim 1, which is characterized in that in step (1), the antibody be nano antibody,
Single-chain antibody scFV or variable region monoclonal antibody.
6. preparation method according to claim 1, which is characterized in that in step (1), the FGE identifies amino acid
Sequence is containing cysteine-X- proline-X- arginine, and the X is arbitrary natural amino acid.
7. preparation method according to claim 1, which is characterized in that in step (2) and step (3), the aldehyde radical
Reactivity is R-L-R with bifunctional linker, and wherein R is the aldehyde comprising amino, hydrazide group, oxygen amino, phenylhydrazino or pyridine diazanyl
Base reactive group, L are with-(CH2CH2- O) n and/or-(O-CH2CH2) polymer of the n as Component units, wherein n is
1 to 100 integer.
8. preparation method according to claim 1, which is characterized in that the gene coded sequence of the FGE derives from tuberculosis
Mycobacteria or the mankind.
9. preparation method according to claim 1, which is characterized in that in step (3), after reacting in ice conditions
Reaction product, detached by size exclusion chromatography, obtain the antibody of single same bifunctional linker connection.
10. preparation method according to claim 1, which is characterized in that in step (4), the step (3) obtains
The molar ratio that antibody pinpoints aldehyde group modified antibody with C-terminal is 1:1~3.
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| PCT/CN2018/103120 WO2019218533A1 (en) | 2018-05-15 | 2018-08-30 | Method for preparing antibody complex |
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| CN110698615A (en) * | 2019-10-16 | 2020-01-17 | 大连理工大学 | Protein functionalized ice gel material, preparation method and application thereof |
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