CN106146663A - Novel antibodies-the drug conjugates of alpha-non-natural amino acid labelling and preparation thereof - Google Patents
Novel antibodies-the drug conjugates of alpha-non-natural amino acid labelling and preparation thereof Download PDFInfo
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Abstract
The invention provides the Mabthera through rite-directed mutagenesis containing alpha-non-natural amino acid labelling.Additionally provide rite-directed mutagenesis and the method pinpointing little molecule coupling Mabthera, described method includes using gene codon expansion technique to introduce in Mabthera gene by alpha-non-natural amino acid fixed point, by alpha-non-natural amino acid and dressing agent, as poly bis function linking arm DIBO-DOTA fixed point connects, Mabthera and radiosiotope, such as Cu is realized further by DOBO-DOTA64Site-directed coupling.The invention further relates to the application of site-directed coupling little molecule Mabthera, as imaging, spike, the purposes of therapeutic type radioimmunity conjugate.
Description
Technical field
The invention belongs to field of biological pharmacy, the fixed point of the alpha-non-natural amino acid relating to protein is inserted
Enter and the site-directed coupling method of small-molecule drug.Described method includes extending based on gene codon
Alpha-non-natural amino acid fixed point is introduced protein by technology, and by alpha-non-natural amino acid and effect
Property little molecule fixed point connect.
Background technology
The background technology of the present invention is described below as a example by Rituximab, but the description below without
Opinion is the most all it is not intended that it is recognizing of prior art, and also nothing else all must not believe that this
That invents is suitable only for Rituximab.
(1) Rituximab
Bone-marrow-derived lymphocyte antigens c D20 developed by molecule is in from late pro-B-cells to mature B cell
All B cell surfaces (Loken et al., 1987, Blood 70,1316-1324) it 90% with
On B cell lymphoma in stable express, so being highly suitable as the target spot of targeted therapy
(McLaughlin et al.,1998,Journal of clinical oncology:official journal of
the American Society of Clinical Oncology 16,2825-2833).CD20 Dan Ke
Grand antibody achieves in treatment B cell lymphoma and chronic lymphocytic leukemia and makes us
Promising result.The anti-CD-20 monoclonal antibody that American I DEC is prepared for B cell lymphoma
Rituximab (C2B8)-Rituximab, i.e. Mabthera, is first of FDA approval
For treating the antibody of B cell lymphoma, it is the targeting lymphoma of first listing and white blood
People's Mus chimeric mAb of sick B cell surface C D20.It is IgG1 κ immunoglobulin,
The variable region of light chain and heavy chain is Mus source, constant region behaviour source (DrugBank DB0073).
The heavy chain of Rituximab is made up of 451 aminoacid, and light chain is made up of 213 aminoacid,
Its sequence of light chain such as SEQ ID NO:1, its nucleotide sequence is as shown in SEQ ID NO:2；Weight
Chain-ordering is as shown in SEQ ID NO:3, and its nucleotide sequence is as shown in SEQ ID NO:4.
Its main mechanism includes antibody-mediated killing functions of immunocytes, the cell killing of complement-mediated
Effect and the apoptotic effect of inducing tumor cell.
Although Mabthera has shown good curative effect in clinical treatment, but overall reaction rate
All about 50%, complete cure rate is less than 20%, and recurrence and drug resistance phenomenon often have generation
(McLaughlin et al.,1998,Journal of clinical oncology:official journal of
the American Society of Clinical Oncology 16,2825-2833；Coiffier et al.,
2002,The New England journal of medicine 346,235-242；Maloney et al.,
1997,Blood 90,2188-2195).Therefore, with CD20 as target spot, research and development are more effectively
Antibody drug has the biggest market value.
(2) antibody coupling medicine
Radiotherapy chemotherapy is quite varied in clinical practice as the Main Means of oncotherapy, and it is main
By causing DNA double chain interruption, suppress cell mitogen, suppression DNA and relevant egg
Tumor cell is killed in white synthesis.But, normal cell is had and kills more greatly by traditional chemicotherapy
Wound effect, therefore by isotope or chemotherapeutics are improved medicine with target antibody coupling
Thing curative effect also reduces the research of side effect and is paid close attention to the most widely.
Antibody coupling medicine is a part emerging in pharmaceutical grade protein, also referred to as immunoconjugates
Thing.Immunoconjugates medicine is by monoclonal antibody component and cell killing component two parts structure with targeting
Becoming, according to the difference of cell killing component, (coupling is same to be broadly divided into radioimmunotherapy medicine
Position element), antibody drug conjugates (coupling small-molecule drug), immunotoxin (coupling antibacterial
Toxin) and enzyme len antibody prodrug medicine (conjugate enzyme) four class (Weiner et al., 2012,
Cell 148,1081-1084).By medicine and monoclonal antibody coupling, the targeting utilizing antibody will
Medicine is transported to target cell accurately, and the medicine that can effectively improve target cell local is dense
Degree, reduces the distribution of medicine normal cell in vivo, thus realizes the treatment effect of medicine high-efficiency low-toxicity
Really.
As a example by radioimmunity coupling drug, it is extensive in clinical practice at present.Radioimmunity shows
As medicine (radioimmunoimaging, RII) utilizes monoclonal antibody that diagnostic nucleic is transported to target portion
Position, carries out tomography by ECT and Computerized three-dimensional reconstructs, in order to show this position
The size of tumor body, position etc.；Radioimmunotherapy treatment medicine (radiommunotherapy, RIT),
I.e. utilize antibodies as carrier, therapeutic nuclides is directed to tumor locus and carries out controlling of internal radiation
Treatment method, obtains prominent clinical effectiveness in terms of lymphoma.Two radioimmunities are wherein had to control
Treat medicine and pass through the drug approval of FDA.The two medicine is all that targeting CD20 treatment is multiple
Send out or the follicular lymphoma of drug resistance: one is 90Y-ibritumomab tiuxetan
(Zevalin), another be 131I-tositumomab (Bexxar 2014 because of and
90Y-ibritumomab tiuxetan effect is identical and removes city).Wherein patient is for Zevalin
Overall reaction rate reached 80%, complete cure rate be about 30% (Goldsmith et al., 2010,
Seminars in nuclear medicine 40,122-135)。
(3) antibody drug coupling technology
Traditional antibody coupling matter is generally with the amino of lysine side-chain on antibody, or disulfide bond
Sulfydryl after reduction is the little molecule of functional group coupling.Antibody surface has the bad ammonia of about 80
Acid and the cysteine of about 30, so traditional method of modifying is non-fixed point non-quantitation
's.And the conjugate of the little molecule of antibody lacks the means of efficiently separating, therefore traditional coupling method
It is not appropriate for quality control prepared by large-scale production.Antibody surface has a lot of functional areas,
Even destroying the disulfide bond of antibody, random coupling may have influence on the combination of antibody antigen,
Reduce the antibody half-life in vivo, affect antibody-mediated killing functions of immunocytes and complement is situated between
The killing functions of immunocytes led, the too much little molecule of coupling also can increase the immunogen of antibody drug
Property.
(4) gene codon expansion technique
Genetic code expansion technique quickly grows in recent years, utilizes Amber stop codon for having
Justice coding, by introducing corresponding orthogonal tRNA and aminoacyl tRNA synthetase, finally
The alpha-non-natural amino acid designed can be introduced in protein.According to alpha-non-natural amino acid
Character, the function that protein is special can be given.Up to the present, this technology is
Express at protein surface through tens kinds of alpha-non-natural amino acids are successfully pinpointed, relate to
With alkynyl and nitrine etc. in alpha-non-natural amino acid, utilize these bio-orthogonal groups, just
Specifically protein can be carried out pointed decoration.
The problem modified in the face of existing antibody little molecule coupling drug unfixed point non-quantitative, by gene
Codon expansion technique is applied in the preparation of existing antibody coupling matter, will be effectively promoted antibody
The development of conjugate.
Summary of the invention:
Inventor passes through the thinking to prior art and research, utilizes ancient methanosarcina
tRNA(tRNAPyl) and pyrrolysyl-tRNA synthetase (tRNAPyl/PylRS)
Protein translation system make alpha-non-natural amino acid fixed point be inserted into protein surface, thus
Destination protein to rite-directed mutagenesis.Then using the albumen of described rite-directed mutagenesis as being entered
The raw material of one one-step site coupling, molecule coupling little with linking arm or effect, the coupling obtained
Thing molecule little with effect, such as radionuclide, coupling further, and then obtain single
The product of site site-directed coupling, especially, described albumen is Rituximab.
Compared to other method, advantages of the present invention may be embodied in following one or several
Aspect:
1. can introduce alpha-non-natural amino acid in any site of protein, thus creation can be only
This site is carried out the material protein of specificity modification；
2. utilize distinctive active group on alpha-non-natural amino acid, it is possible to achieve efficiently, special
The modification purpose of property；
3. the site-directed coupling of specific site can bring more preferable drug effect.Selection is coupled at NOT function
Energy district, can effectively reduce drug coupling to protein active, stability, half-life
The impact brought；Selection is coupled at non-exposed region, the modification quantity of fixing little molecule, can
To reduce potential immunogenicity, reduce internal zymolysis, thus reduce conjugate
The possibility missed the target；
4., by modifying the optimization of condition, utilize the reaction of Click without copper that cyclooctyne mediates,
Can realize efficiently, harmless to albumen, simple coupling reaction.
Specifically, in a specific embodiment of the present invention, it is provided that introduce non-
The destination protein of natural amino acid, such as Rituximab, main by two steps:
(1) build containing the coding purpose egg on selected site with amber codon sudden change
In vain, the such as carrier of Rituximab gene, (2) build and obtain pXH-N3Plasmid,
By step (1) and (2) coexpression in suitable host cell, and in the medium
Add alpha-non-natural amino acid, it is thus achieved that introduce the destination protein of sudden change, such as Rituximab.
The principle of this abruptly-changing system is: the tRNA of saltant typePyl/ PylRS meets following pass
System: (1): the tRNA of saltant typePylThe lysyl tRNA of host cell can not be utilized
Synzyme, can only be acylated by the PylRS of saltant type；(2): the PylRS of saltant type is only
Can acylated tRNAPyl, it is impossible to other tRNA acylated, therefore, saltant type tRNAPyl and
Relation between PylRS is orthogonality, i.e. the PylRS of saltant type can only be acylated sudden change
Type tRNAPyl, the tRNA of simultaneous mutation typePylCan only be acylated by the PylRS of saltant type,
The tRNA of the saltant type in the most same plasmidPylWith PylRS be absolute the most specially
One.The enzyme of this orthogonality and be that the most this enzyme can be alpha-non-natural amino acid acyl
Change on this orthogonal tRNA, and this tRNA can only be acylated, and can not be acylated
Other tRNA.The orthogonal lysyl tRNA synthase/tRNA system obtained, makes non-20
Plant the Lys-azido of common amino acid, it is possible to be referred to as: NAEK, with amber codon phase
Correspondence, thus alpha-non-natural amino acid fixed point is incorporated into destination protein, such as rituximab list
In Kang.
In a specific embodiment of the present invention, by will be with amber codon
Rituximab gene be inserted in carrier such as pEF-1b, the load that obtains after inserting
Body and described pXH-N3Transient transfection Freestyle293F cell line, then may be used together
By adding alpha-non-natural amino acid such as NAEK in the medium, finally from culture medium
Clear liquid obtains the destination protein of rite-directed mutagenesis, such as saltant type Rituximab.
In a specific embodiment of the present invention, the invention provides a kind of food in one's mouth
Breast zooblast in the amino acid whose mutated recombinant proteins of fixedpoint introduction of non-natural or peptide, as
Specific site introduces alpha-non-natural amino acid NAEK, and its distinctive azido group can be special
Property ground and conjugate, the most difunctional linking arm, there is Click reaction, thus by purpose
Little molecule site-directed coupling is on destination protein.
In the other embodiments of the present invention, it is provided that a kind of difunctional linking arm
DIBO-DOTA.This linking arm one end, with cyclooctyne (DIBO), is available for specificity and knows
Other azido group is also coupled；The other end is with chelation group
Isosorbide-5-Nitrae, 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),
This group can firmly chelate Y90, Lu177Deng metal isotope.Such as saltant type rituximab
Monoclonal antibody and the little molecule of site-directed coupling thereof,
In the other embodiments of the present invention, by the NAEK rite-directed mutagenesis mesh of purification
Albumen or peptide, such as Rituximab and DIBO-DOTA carry out the Click that is catalyzed without copper
Reaction so that DIBO-DOTA is by the non-sky containing azido group on Rituximab
So aminoacid and carry out fixed point and quantitative coupling, due to the high efficiency of reaction, Jing Guojian
Single desalination operation i.e. can get the little molecule of site-directed quantitative coupling, such as DIBO-DOTA
Destination protein or peptide.Through experiment in vitro preliminary proof, through alpha-non-natural amino acid replace with
And the Rituximab of the little molecule of site-directed coupling still has biologic activity.
In one embodiment of the invention, coupling is had linking arm DIBO-DOTA
Destination protein or peptide, such as Rituximab, react with radiosiotope, and then
Being coupled to radiosiotope site-directed quantitative in destination protein or peptide, such as rituximab
Monoclonal antibody.Obtained site-directed coupling has radioisotopic antibody, can be more effective
Carry out Radionuclide imaging, radioactivity targeted therapy etc..
In one embodiment of the invention, the present invention have chosen mammalian cell hang
Floating expression system, used by this system, cell is through the domestication that suspends, both can be with instantaneous big scale
Reach destination protein, large-scale fermentation culture can be carried out by building stability series again.
More specifically, the invention provides:
1. the polypeptide of sudden change, the aminoacid on its at least 1 site is sported non-natural ammonia
Base acid, described alpha-non-natural amino acid is:
Shown Lys-azido (NAEK),
Or other contains the alpha-non-natural amino acid of nitrine structure.
2. the polypeptide of the sudden change as described in item 1, described polypeptide is Rituximab.
3. the polypeptide of the sudden change as described in item 2, described sudden change be positioned at SEQ ID NO:2 and
/ or SEQ ID NO:4 in arbitrary one or more sites.
4. the polypeptide of the sudden change as described in item 3, described mutational site is selected from: be shown in SEQ
ID NO:2 K168 position, the A122 position of SEQ ID NO:4, or SEQ ID NO:
Less to activity influence to other on 2 or SEQ ID NO:4 one or many in site
Individual.
5. the polypeptide of the sudden change as described in item 1, its mutating acid is N amino acids,
Described mutating acid connected mode in described polypeptide is shown below:
Wherein, by R1To R2The N-terminal that direction is aminoacid sequence to C-terminal side
To, R1For the 1st to N-1 amino acids residue in polypeptide,
R2For the amino acid residue of the N+1 position in polypeptide to C-terminal, R4For
6. the polypeptide of the sudden change as described in item 5, the polypeptide of described sudden change is that the profit of sudden change is appropriate
Former times monoclonal antibody.
7. the polypeptide of the sudden change as described in item 6, described N amino acids is SEQ ID
Aminoacid on any one or more sites in NO:2 or SEQ ID NO:4.
8. the polypeptide of the sudden change as described in item 7, described mutational site is selected from: be shown in SEQ ID
NO:2 K168 position, the A122 position of SEQ ID NO:4, or SEQ ID NO:2
Or one or more in the site that on SEQ ID NO:4, other are less to activity influence.
9. the polypeptide of the sudden change as described in item 1, its comprise be available for coupling nitrine and metal with
The difunctional linking arm of position element, described linking arm one end contains cyclooctyne (DIBO) group,
The other end contains DOTA, and described difunctional linking arm structure is shown below:
10. the polypeptide of the sudden change as described in item 9, the polypeptide of wherein said sudden change is sudden change
Rituximab.
The polypeptide of the sudden change in 11. aforementioned any one, its heavy chain amino acid sequence is SEQ ID
NO:10。
The polypeptide of the sudden change in 12. aforementioned any one, its light-chain amino acid sequence is SEQ ID
NO:8。
The polypeptide of 13. sudden changes as described in item 1 after site-directed coupling, its structure is as follows
Shown in formula:
Wherein, wherein n-th aminoacid is undergone mutation, and R1 is the 1st to N-1 position
Amino acid residue, R2 is N+1 position to the amino acid residue of C-terminal, and R3 is DOTA
Or other directly or indirectly provide the little molecule of function.
The polypeptide of 14. sudden changes after site-directed coupling as described in item 13, wherein suddenlys change
Before polypeptide comprise heavy chain that aminoacid sequence is SEQ ID NO:2 and/or sequence is SEQ
The light chain of ID NO:4.
The polypeptide of 15. sudden changes after site-directed coupling as described in item 14, R therein3
For DOTA, it chelates radiosiotope.
The polypeptide of 16. sudden changes after site-directed coupling as described in item 15, described radiation
Property isotope selected from Lu177, Cu64Or Y90。
17. sudden changes after site-directed coupling as according to any one of item 13-16 are many
Peptide, the R in described conjugate3Auspicious selected from amycin (doxorubicin), monomethyl Australia
Statin (Monomethyl auristatin E, MMAE).
The nucleic acid molecules of the polypeptide of the sudden change in 18. coding item 1-12.
19. carriers comprising the nucleic acid molecules described in item 18.
20. 1 kinds of carriers, it is pXH-N3.
21. 1 kinds of pharmaceutical compositions, it contains the institute any one of the item 1-12 of effective dose
The polypeptide of the sudden change stated, or the sudden change after site-directed coupling described in item 13-17 is many
Peptide.
22. pharmaceutical compositions as described in item 21, in preparation for Radionuclide imaging, treatment
Purposes in the medicine of refractory malignancies or tumor recurrence.
23. 1 kinds of microorganisms, it contains the carrier of item 19 or 20.
24. microorganisms as described in item 23, it is escherichia coli.
25. 1 kinds of zooblasts, it contains the carrier of item 19 or 20.
26. zooblasts as described in item 25, it is Freestyle293 cell line.
Accompanying drawing illustrates:
The mammalian cell expression of Fig. 1: Mabthera and activity checking
A:Western Blot verifies antibody expression: first is that empty carrier converts comparison,
Second is the vector expression inserting Mabthera gene.Diagram antibody is successfully made expression；
B: the different expression vector impacts on expression: be respectively from left to right
PcDNA3.1, pEF-1b, pBudce4.1；Diagram pEF-1b carrier is most beneficial for the table of antibody
Reach；
Antibody activity is expressed in the checking of C: fluorescence co-focusing: from left to right, antibody acts on respectively
Jurket, Raji, Ramos RA1 cell, wherein Jurkat cell is that CD20 is negative thin
Born of the same parents, as negative control, Raji and Ramos RA1 cell is CD20 positive cell；
Antibody activity is expressed in the checking of D: flow cytometry: be CD20 in M1 region
The antibodies response value of negative cells Jurket, is CD20 positive cell in M2 region
The antibodies analog value of Raji and Ramos RA cell；
Fig. 2: pXH-N3The optimization of carrier and structure
A:pXH-N3Carrier schematic diagram；
B:Western Blot verifies that all types of promoteres start tRNA to non-natural amino
The impact of efficiency is inserted in acid, and promoter title is respectively as follows: U1, mu6, H1 from left to right,
hu6,7sk；Diagram 7sk promoter is relative to remaining promoter non-natural amino insert type albumen
Expression efficiency is the highest；
C: on cellular level, green fluorescent protein verifies that each promoter alpha-non-natural amino acid is inserted
Enter efficiency；It is consistent with Fig. 2 B result；
Termination codon TAG is readed over efficiency impact by D: different promoters, from left to right
It is respectively U1, mu6, H1, hu6,7sk promoter, reads over efficiency height and be equivalent to non-natural
Aminoacid insertion efficiency efficient；
Termination codon TAG is readed over effect by different number tRNA of E:7sk promoter series connection
The impact of rate, the most respectively 1,2,3,4 and 5 7sk-tRNA series connection
Unit, as seen from the figure under 7sk promoter, the alpha-non-natural amino acid of 4 tRNA of series connection
Insert efficiency the highest；
Fig. 3: the expression of saltant type Mabthera
A. Mabthera variable region and CD20 bound short peptide crystal structure schematic diagram (Ding
Jianping et al.J.Biol.Chem.2007,282:15073-15080)；Shown in figure, select
There is certain surface degree of exposure, and do not affect three the site LC-P15 being combined with receptor,
LC-K168 and HC-A122 is as potential alpha-non-natural amino acid insertion point；
B.Western Blot checking mutein is expressed, and the most respectively WT is beautiful
Luo Hua (without alpha-non-natural amino acid insert type Mabthera)；LC-P15 site mutation Mabthera
It is not added with alpha-non-natural amino acid (UAA), adds UAA；HC-A122 site mutation Mabthera
It is not added with UAA, adds UAA；LC-K168 site mutation Mabthera is not added with UAA, adds UAA；
HC-A122 and LC-K168 site is successively inserted into alpha-non-natural amino acid as seen from the figure；
The condition optimizing that Fig. 4: saltant type Mabthera is expressed
A. different cell lines on and the transfection reagent impact on expression condition: divide from left to right
Do not transfect for Freestyle293 cell line 25kDa straight chain PEI, Freestyle293 cell
It is 40kDa branched chain PEI transfection, Freestyle Chinese hamster ovary celI system 25kDa straight chain PEI
Transfection, Freestyle Chinese hamster ovary celI system 40kDa branched chain PEI transfects；Wink as seen from the figure
In the case of Zhuaning, 293 cell lines relatively Chinese hamster ovary celI system expression is significantly increased, and 40kDa
Branched chain PEI relatively 25kDa straight chain PEI has more preferable expression effect；
B. transfection efficiency is affected by transfection DNA total amount with PEI ratio (mass ratio), by
During the visible DNA:PEI=1:4 of figure, expression efficiency is higher；
C. fixed dna total amount, during the expression of HC-A122 mutein,
Transfected plasmids light chain, heavy chain and pXH-N3The ratio impact on expression efficiency；
D: additive (VPA and NaBut) whether adds and expression time is to expression
The impact of efficiency；
Fig. 5: saltant type Mabthera site-directed coupling DIBO-DOTA
A. the ESI-MS collection of illustrative plates of 2 DIBO-DOTA of complete antibody site-directed quantitative coupling；
B., after reduction treatment antibody samples, site-directed quantitative is heavy chain coupling 1
The ESI-MS collection of illustrative plates of DIBO-DOTA；
C. the ESI-MS collection of illustrative plates of complete antibody non-site-directed coupling DOTA-SCN；
D., after reduction treatment antibody samples, non-fixed point, non-quantitation are even at light chain and/or heavy chain
Join the ESI-MS collection of illustrative plates of 1 or several DOTA-SCN；
Fig. 6: site-directed coupling DIBO-DOTA Mabthera fixed point labelling radiosiotope
A: paper thin layer chromatography inspection wild type Mabthera (RTX-WT) non-site-directed coupling
Cu64Productivity, left: before crossing PD-10 post；It is right: after PD-10 post is except remaining little molecule excessively；
B: paper thin layer chromatography inspection heavy chain A122 position saltant type Mabthera (RTX-122)
Site-directed coupling Cu64Productivity, left: before crossing PD-10 post；Right: to cross PD-10 post except residue
After little molecule；In diagram, processed by PD-10 post, obtained purer (> 98%)
Radioimmunoassay conjugate；
C: radioactive automatic developing confirms radioisotopic site-directed coupling, and left figure is for examining horse
This light blue dyes, and right figure is radioactivity gamma ray autography, and two figure correspondence positions are from a left side
It is followed successively by the right side
RTX-WT-DTT, RTX-WT+DTT, RTX-122-DTT, RTX-122+DTT, (DTT
For dithiothreitol, DTT, for albumen reducing agent)；Diagram shows, all success couplings of two samples
Radiosiotope Cu64, RTX-WT sample is non-site-directed coupling, and it is at heavy chain and light
Chain position all has radioactive ray to develop, and RTX-122 is site-directed coupling, suddenlys change and even
Connection site is in heavy chain A122 position, therefore only heavy chain has radioactive ray to develop；
Fig. 7: fixed point labelling64The Micro-PET imaging of Cu antibody
Mabthera site-directed coupling Cu64The Micro-PET imaging results of tumor-bearing mice:
A:18 hour Micro-PET imaging results, left for injection 500uCi
RTX-122-Cu64Tumor-bearing mice, right figure is injection 500uCi RTX-122-Cu64And 4
The cold antibody of multiple dose (non-coupling radiosiotope) is as the tumor-bearing mice closed；
B:60 hour Micro-PET imaging results；
Diagram shows to extend in time, and radioimmunoassay thing remains the targeting of tumor,
Higher level be enriched to tumor locus.
In order to be more fully understood that the present invention, concrete test is explained by inventor by embodiment
Stating and illustrate, wherein said embodiment is merely to illustrate, and does not limit the protection of the present invention
Scope.Any variant of equal value with the present invention or embodiment are included in the present invention.
Embodiment 1: comprise the structure of the genophore of the Mabthera of rite-directed mutagenesis
(1) structure of helper plasmid and acquisition
By optimization and the trial of condition, determine that 4 tRNA are by 7sk promoter (its
Sequence is as shown in SEQ ID NO:6) expressing in series is optimum, builds and obtains pXH-N3
Assistant carrier: this carrier is with pUC19 as template, by utilizing BamHI restriction enzyme site,
Introduce 7sk promoter and the tRNA of 4 series connection；It is re-introduced into CMV strong promoter to control
Under tRNA synzyme and poly A termination signal；Utilize EcorI restriction enzyme site,
Introduce eucaryon and replicate original paper f1 ori and SV40；This plasmid can express the non-sky of specific recognition
So tRNA and tRNA synzyme of amino acid N AEK.
(2) acquisition of the plasmid containing Mabthera
Synthesize through full genome, it is thus achieved that the gene (SEQ ID NO:1 and 3) of Mabthera.
Then connect in pEF-1b (Life Technologies) carrier for expression of eukaryon, obtain
Wild type Mabthera expression plasmid (pEF-RTX-HC-WT and
PEF-RTX-LC-WT, its heavy chain expressing Mabthera respectively and chain moiety).
(3) selection in rite-directed mutagenesis site
According to the binding site of the crystal structure of Mabthera, Mabthera and its receptor, and comprehensively
Consider epitope, information [the Ding Jianping et al. such as enzymolysis site
J.Biol.Chem.2007,282:15073-15080], have chosen several applicable site and modify,
It is based primarily upon following factor: 1. aminoacid is exposed to protein surface to facilitate coupling；Do not affect
Antibody combines with the normal of receptor；The most do not affect the character of antibody itself, such as stability etc.；
[Sondermann et is consulted by more detailed documents and materials
al.Nature,2000,406:267-273；Mulkerrin et al.J.Immunol.2000,
164:4178-4184；Sun et al.J.Biol.Chem.2001,276:16469-16477], inventor
Choose the A122 position (HC-A122) of heavy chain, K168 (LC-K168) position of light chain,
P15 (LC-P15) is that specific site carries out point mutation, with this saltant type Metro Huawei raw material also
It is pinpointed the coupling of little molecule.
(4) design of primers and the mutational vector of rite-directed mutagenesis builds
For the A122 position of Mabthera heavy chain, the K168 position of light chain and P15 position, if
Meter can make the described amino acid whose codon mutation of coding be the primer of amber codon, tool
Body primer is as shown in the table.
Table 1: mutant primer list
Utilize site-directed mutagenesis kit ( Lightning Site-Directed
Mutagenesis Kits, Catalog #210518), by specification operates with above-mentioned steps (2)
The wild type Mabthera expression vector pEF-RTX-HC-WT of middle acquisition and
PEF-RTX-LC-WT is the template A122 position by Mabthera heavy chain, the of light chain
The amino acid codes in K168 position and these sites, P15 position sports succinum and terminates close
Numeral, build obtain expression plasmid (pEF-RTX-HC122, pEF-RTX-LC168,
PEF-RTX-LC15), suddenly change successfully through sequence verification.
Embodiment 2: the expression of the Mabthera of rite-directed mutagenesis and purification
The present invention builds pXH-N3Containing the tRNA being derived from ancient methanosarcina in plasmid
(tRNAPyl) and pyrrolysyl-tRNA synthetase (pheRS), at express cell
In, with Amber stop codon (TAG) for there being justice coding, it is possible to make non-natural ammonia
Base acid NAEK is incorporated in albumen, thus causes the rite-directed mutagenesis of Mabthera.Below,
Inventor mixes the production performance of probability and mutein and is examined NAEK
Survey.
1: the synthesis of alpha-non-natural amino acid NAEK and qualification
The chemosynthesis reaction formula of alpha-non-natural amino acid Lys-azido is as follows
As described in above formula, raw material 1 (ethylene bromohyrin) 2.3mL is dissolved in 90mL acetone with
And the mixed solution of 15mL water, adding NaN33.12g, 60 DEG C of oil baths are heated to reflux instead
Answer 20h.Being cooled to room temperature, rotation is evaporated off acetone, and absolute ether extracts (30mL × 8),
Anhydrous Na 2SO4 is dried, and rotation is evaporated off solvent and obtains 2.62g colorless liquid product 2.
Product 2 (500mg, 5.74mmol) is joined triphosgene (1.70g, 5.74mmol)
THF (10ml) solution in.0 DEG C of stirring reaction 8h, solvent is evaporated.Residue is in vacuum
Under be dried 1h, obtain colorless oil as product 3.
Be dissolved in 3 in the THF of 1.5ml and be slowly added to Boc-Lys-OH (1.7g,
In the solution of 1M NaOH (20ml) 6.88mmol)/THF (5ml).0 DEG C of stirring is anti-
Answer 12h and be gradually warmed up to room temperature.Again reactant liquor is cooled to 0 DEG C and 1M with 0 DEG C
Hydrochloric acid solution reacting liquid pH value is adjusted to 2～3.Reactant liquor EtOAc extracts
(30mL × 5), the organic layer saturated aqueous common salt of 2 × 100ml washs.Anhydrous Na 2SO4
It is dried organic layer, filters, revolve and solvent is evaporated off obtains 1.65g colourless viscous liquid product 4
Need not be further purified.
It is dissolved in 4 in 15mL CH2Cl2, under stirring, is slowly added dropwise 15mL TFA, room
Steaming solvent after the lower reaction 30min of temperature, remaining liq product 5mL methanol dissolves, and adds
Entering 100mL ether, separate out a large amount of white solid precipitation, it is white that filtration drying obtains 1.38g
Color solid end product 5.1H NMR (D2O): δ=1.22-1.45 (m, 4H), 1.67-1.73
(m,2H),2.99(m,2H),3.38(m,2H),3.70(m,1H),4.09(m,2H).
13C NMR (D2O): δ=21.4,28.4,29.6,39.5,53.4,56.2,57.8,116.0
(TFA),153.1,162.3(TFA),172.9.HRMS:m/z calcd for C9H17N5O4
[M]+:259.1281；Found:259.1283, it was demonstrated that the Lys-azido structure obtained is correct.
2:pXH-N3Structure
By the investigation of documents and materials, tRNAPylHigh efficient expression be that non-natural amino acid has
One of key factor that effect is inserted, and host cell can be produced bright by too high expression
Aobvious cellulotoxic effect, the most reasonably optimizes tRNAPylExpress, can aid in sudden change
The great expression of type albumen.
(1) first fix the number (1) of tRNA, attempt different startups by changing
Son, including U1, mu6, H1, hu6 and 7sk promoter, with the 39th amino acids
Green fluorescent protein (GFP39TAG) for alpha-non-natural amino acid is reporter protein, checking
Alpha-non-natural amino acid is inserted the impact of efficiency by different promoters, such as Fig. 2-B, shown in C；Knot
Fruit shows that 7sk (SEQ ID NO:6) promoter is in the promoter selected, it is possible to have most
The insertion alpha-non-natural amino acid of effect；
(2) fixing promoter is 7sk promoter, by changing tRNA series connection number, including
1,2,3,4 and 5, with LUC Photinus pyralis LUC Photinus pyralis FL as reporter gene, sea pansy fluorescein
Enzyme is reference gene, includes the linking arm containing TAG termination codon between two genes,
Read over efficiency by TAG to detect different tRNA series connection number alpha-non-natural amino acid is inserted
Enter the impact of efficiency, as shown in Fig. 2-E；Diagram shows when 4 tRNA of series connection when,
The insertion efficiency of alpha-non-natural amino acid is the highest；
(3) by by tRNA synzyme pheRS, 4 promoteres 7sk-tRNApylPass through base
Because of subcloning procedures clone into identical carrier pUC19 (utilizing BamHI restriction enzyme site) as
Shown in Fig. 2-A, it is built into assistant carrier pXH-N3, its sequence such as SEQ ID NO:5
Shown in.
3: the NAEK of sudden change Mabthera mixes and expresses and purification
(1) as a example by the saltant type (RTX-H122) expressing heavy chain A122 position: will implement
(it expresses anti-Rituximab antibodies for the step 2 of example 1 and 4 pEF-RTX-LC-WT obtained
Light chain amino acid) and pEF-RTX-HC122 (the heavy chain ammonia of its expression anti-Rituximab antibodies
Base acid, and contain point mutation in No. 122 site), and the step 3 of embodiment 2
pXH-N3Mix with certain proportion, then mix by a certain percentage with transfection reagent PEI, altogether
With adding mammalian cell, it is simultaneously introduced NAEK to final concentration 1mM, 100rpm,
37 DEG C, 8%CO2Culture supernatant is collected after expressing some skies；
(2) subsequently to express cell system, transfection reagent, each carrier ratio, carrier and transfection examination
Agent ratio, saltant type expression condition is optimized by additive types etc., as shown in Figure 4,
Finally determining that expression condition is Freestyle293 cell line, 40kDa branch chain PEI transfects,
Carrier and PEI ratio are 1:4 (mass ratio), three kinds of carrier ratio pEF-RTX-LC-WT:
pEF-RTX-HC122:pXH-N3=1:1:3, adds VPA, expresses 7 days；
(3) culture supernatant 9000rpm that will collect, 4 DEG C of high speed centrifugation 10min, except cell and
Cell debris, carries out microfiltration and ultrafiltration processes dense further through hollow fibre filtering system
Contracting (more than 40 times), and carry out the exchange of Ni-NTA-Bind buffer, recentrifuge
Remove cell debris, through Ni-NTA metal chelate affinity chromatography, use Ni-NTA-Wash
Buffer fully washs, and finally uses Ni-NTA-Elute buffer solution elution, obtains the purest
The Mabthera sample changed, purity is about 95%.Protein sample after preliminary purification is passed through
Liquid is changed in 4-5 ultrafiltration, shift in little molecule coupling buffer (20mM Tris-HCl pH=8.0,
200mM NaCl, processes three times through Chelex100 5 × 20cm, thoroughly cleans residual metal
Ion).
4: the qualification of sudden change Mabthera
(1) design matched group, using do not add NAEK express cell as comparison, do identical turn
Dye processes；
(2) collect culture supernatant, be directly added into SDS-PAGE sample-loading buffer and boil sample process,
And do SDS-PAGE electrophoresis and Western Blot analysis, comparative purpose position protein bars
Band, can substantially observe that addition NAEK group gives expression to total length Mabthera, as shown in Figure 3.
Embodiment 3: mutant and the site-directed coupling of difunctional linking arm (DIBO-DOTA)
1: the synthesis of difunctional linking arm DIBO-DOTA and qualification:
Compound 1 (2.88g, 14.0mmol) is dissolved in the anhydrous CH2Cl2 of 20mL,
N2 protects, and is slowly added to BF3 OEt2 (2.59mL, 21.0mmol), system is moved afterwards
To-10 DEG C of cold wells, under stirring, it is slowly added dropwise trimethyl silicone hydride Azimethylene.
CH2Cl2 solution (the 10.5mL trimethyl silicone hydride diazonium first of (2.0mol/Lin hexanes)
Alkane is dissolved in the anhydrous CH2Cl2 of 20mL), dropping in 1 hour is complete.Reaction is continued at-10 DEG C
After 2-4 hour, reactant liquor is poured into cancellation reaction in 50mL frozen water, isolates organic facies,
Aqueous phase CH2Cl2 extracts (2 × 50mL).Organic facies is washed with saturated aqueous common salt after merging
(2 × 40mL), the dried filtering and concentrating of anhydrous sodium sulfate, silicagel column separates (oil
Ether: CH2Cl2, v/v, 2/1) obtain faint yellow product 2 (2.22g, 72%) .1H NMR (300
MHz, CDCl3): δ 8.26 (1H, q, J=1.4,6.6Hz), 7.13-7.43 (7H, m), 7.05
(2H, q, J=3.8,12.9Hz), 4.06 (2H, s) .13C NMR (75MHz, CDCl3): δ
196.6,136.9,136.3,135.4,133.8,133.1,132.4,131.4,130.6,129.3,
128.8,128.0,127.3,126.9,48.4.MALDI HRMS:m/z 243.0767
[M+Na+].Calcd for C16H12NaO+:243.0780.
Compound 2 (2.20g, 10mmol) is dissolved in the mixed solvent of EtOH and THF
(1/1, v/v, 80mL), is slowly added to sodium borohydride (0.76g, 20mmol), room under stirring
Temperature reaction is overnight.After TLC detection reaction completely, in system, it is slowly added dropwise 1mL acetic acid
Cancellation is reacted.Rotation adds 80mL CH2Cl2 suspendible, saturated aqueous common salt after solvent is evaporated off
Washing (3 × 80mL), the dried filtering and concentrating of anhydrous sodium sulfate, silicagel column separates (oil
Ether: ethyl acetate, v/v, 8/1；Gradient elution effect is bad) obtain white solid product 3 (2.00
G, 91%) .1H NMR (300MHz, CDCl3): δ 7.50 (1H, m), 7.14-7.30 (7H,
M), 6.90 (2H, q, J=2.7,12.0Hz), 5.31 (1H, q, J=6.3,10.0Hz), 3.41
(2H,m).13C NMR(75MHz,CDCl3):δ141.7,136.7,136.2,134.5,
131.7,131.5,130.1,129.9,129.3,128.7,127.4,127.2,126.9,125.9,
74.4,42.7.MALDI HRMS:m/z 245.0949[M+Na+].Calcd for
C16H14NaO+:245.0937.
Being dissolved in 25mL CHCl3 by compound 3 (1.11g, 5mmol), stirring is lower slowly
Dropping bromine (0.26mL, 5mmol).React 0.5h, TLC under room temperature and detect raw material reaction
After Wan Quan, revolve and solvent is evaporated off, silicagel column separation (petroleum ether: CH2Cl2, v/v, 2/1,
Or gradient elution) obtain yellow viscous liquid product 4 (1.11g, 58%).1H NMR
(300MHz,CDCl3):δ7.54-7.47(2H,aromatics),7.31-6.72(6H,
Aromatics), 5.77 (1H, d, J=5.4Hz, CHBr), 5.22 (1H, dd, J=3.6,15.9
Hz, CHOH), 5.19 (1H, d, J=5.4Hz, CHBr), 3.50 (1H, dd, J=3.6,
15.9Hz, CH2), 2.75 (1H, dd, J=3.6,15.9Hz, CH2) .13C NMR (75
MHz,CDCl3):δ141.3,140.0,137.2,134.0,133.4,131.5,131.3,130.9,
127.8,126.2,123.7,121.3,76.5,70.0,62.3,32.2.MALDI HRMS:m/z
402.9313[M+Na+].Calcd for C16H14Br2NaO+:402.9304.
Being dissolved in the THF that 20mL is dried by compound 4 (0.77g, 2mmol), N2 protects
Protect, under stirring, be slowly added dropwise the THF solution (4mL, 8mmol) of 2.0M LDA.Room
Temperature reaction 0.5h, is slowly added dropwise 0.5mL distilled water cancellation reaction in reaction system.Rotation
Solvent being evaporated off, dissolves with 80mlDCM, wash twice with saturated aqueous common salt, oil phase is done
Dry sample of mixing, silicagel column separates (petroleum ether: ethyl acetate, v/v, 8/1, or gradient elution) and obtains
To white solid product 5 (0.25g, 57%).1H NMR(300MHz,CDCl3):δ7.67
(1H, aromatics), 7.37-7.18 (7H, aromatics), 4.57 (1H, dd, J=2.1,
14.7Hz, CHOH), 3.04 (1H, dd, J=2.1,14.7Hz, CH2), 2.86 (1H, dd,
J=2.1,14.7Hz, CH2) .13C NMR (75MHz, CDCl3): δ 154.5,150.6,
128.6,127.1,1127.0,126.0,125.8,125.1,124.7,123.0,122.7,121.7,
111.9,109.6,74.2,47.7
Compound 5 (0.22g, 1mmol) is dissolved in 30mLCH2Cl2, adds 0.4mL
Pyridine and to nitroxyl chloride phenyl formate (0.4g, 2mmol).React overnight under room temperature, TLC
After detection raw material 5 reacts completely, reactant liquor saturated aqueous common salt washs (2 × 40mL), nothing
The dried filtering and concentrating of aqueous sodium persulfate, silicagel column separates (petroleum ether: CH2Cl2, v/v, 2/1)
Obtain white solid product 6 (0.34g, 72%).1H NMR(300MHz,CDCl3):
δ8.23-8.18(2H,aromatics),7.56-7.54(2H,aromatics),7.46-7.18(8H,
Aromatics), 5.52 (1H, dd, J=3.9,15.3Hz, CHOH), 3.26 (1H, dd, J=
3.9,15.3Hz, CH2), 2.97 (1H, dd, J=3.9,15.3Hz, CH2)；13C NMR
(75MHz,CDCl3):δ154.5,150.7,149.1,148.7,129.0,127.4,127.3,
126.7,126.5,125.5,125.2,124.3,124.0,122.6,122.4,120.8,120.6,
120.2,112.2,108.5,80.6,44.8；MALDI HRMS:m/z 408.0852[M+
Na+].Calcd for C23H15NNaO5+:408.0842.
Ethylenediamine (30mg, 0.5mmol) is dissolved in 10mL (anhydrous) CH2Cl2,
Adding 300 μ L triethylamines, N2 protects, stirring is lower add compound 6 (38mg, 0.1
mmol).Post (30:1-5:1) is rushed by dichloromethane methanol system.Obtain compound 7.1H
NMR(300MHz,CDCl3):δ7.51-7.50(2H,aromatics),7.35-7.28(6H,
aromatics),5.50(1H,s,CHOH),3.26-3.15(3H),2.93-2.85(3H)；13C
NMR(75MHz,CDCl3):δ155.6,152.0,150.9,129.8,127.9,127.8,
126.9,126.1,125.8,123.7,123.6,121.2,112.8,109.9,46.1,43.6,
41.5.
Difunctional linking arm site-directed quantitative coupling (see Fig. 5) of 2:NAEK site directed mutant proteins
Containing the site directed mutant proteins of NAEK, by the azido group on NAEK, with
DIBO-DOTA (containing the compound of cyclooctyne structure) is real by the ring strain of cyclooctyne
Now without the Click coupled reaction of copper catalysis.As follows without copper catalysis coupled reaction system:
RTX-122-NAEK (it is embodiment 2 preparation) 1ug/ul
DIBO-DOTA 1mM
Reaction condition: 4 DEG C, vertical suspendible 12 hours.
Result verification difunctional linking arm DIBO-DOTA site-directed quantitative be coupled to Metro
(see Fig. 5-A, B) in China, can be by 95% in 12 hours through above-mentioned reaction condition
The above difunctional linking arm of Mabthera site-directed coupling, reacted complex is through ultrafiltration
Desalination, solution exchanges to (20mM ammonium acetate-acetic acid in isotope coupling buffer
PH=5.5, Chelex100 process 3 times), the connection product obtained can be used as pinpointing labelling
Isotope is used.
Embodiment 4: the radioisotopic site-directed coupling of mutant and qualification
Positron emission tomography (Positron emission tomography, PET) is the most increasingly
Become the guardian technique in oncotherapy, and Cu64Owing to its half-life is short, (～12 is little
Time), little to harm, it is relatively easily formed stable complex, therefore with DOTA
Applicable research carries out the phenomenon treatment of PET；And Lu177Due to its half-life moderate (～6
My god), main release beta ray, radiation radius is short, thus conveniently carries out therapeutic
The research and development of radioimmunity conjugate.
1: Mabthera-Cu64Site-directed coupling:
One end DIBO of difunctional linking arm (DIBO-DOTA) passes through octatomic ring octyne
Carrying out reacting without copper Click with the azido group of Mutant Antibodies, other end DOTA passes through
Chelation reacts with radioactive metal isotope.Radiosiotope Cu64Big by Beijing
Learn Nuclear Medicine Department of tumour hospital and produce preparation, Mabthera-Cu64Coupled reaction system is as follows:
RTX-122-DIBO-DOTA 100ug(1mg/ml)
Cu64 2mCi
Reaction condition: 42 DEG C stand 1 hour.
Result carries out Radio-TLC successively, and SDS-PAGE analyze (as Fig. 6-A,
Shown in B, C), after confirming that isotope carries out coupling, through the simple desalination of PD-10 post,
Be obtained in that to put and exempt from purity the radioimmunity conjugate of 98%；
2: the Mabthera-Cu of tumor-bearing mice64Micro-PET imaging
CD20 is expressed positive cell Ramos A1 cell with 1 × 107/ oxter, a right side note
Penetrate severe immune deficiency (Severe Combined Immune-deficiency, SCID) little
Mus, after about two weeks, tail vein injection 300-350uCi Mabthera-Cu64Conjugate, separately makees
Closed group, injection 500ug non-coupling radioactive substance Mabthera is as sealer simultaneously.
In latter 18 hours of injection, 60 hours, with 1.5% isoflurane anesthesia mice, carry out
Micro-PET images, as shown in Figure 7.
From image results, elapsing in time, radioactive substance is substantially enriched in tumor
Position, PET visible tumor clearly body image.
Although with above embodiments describing the present invention, it should be appreciated that do not carrying on the back
On the premise of the spirit of the present invention, the present invention can further be modified and be changed,
And within these modifications and variation belong to protection scope of the present invention.Such as, the application
Although with Mabthera coupling Cu64As a example by be illustrated, it is obvious that the present invention is not
Mabthera coupling Cu should be only limited to64, the present invention can be applicable to by people in the art
Any little molecule of any destination protein coupling.
Claims (13)
1. the polypeptide of sudden change, the aminoacid on its at least 1 site is sported non-natural ammonia
Base acid, described alpha-non-natural amino acid is:
Shown Lys-azido (NAEK),
Or other contains the alpha-non-natural amino acid of nitrine structure；
Preferably, described polypeptide is Rituximab；
It is highly preferred that described sudden change is positioned at SEQ ID NO:2 and/or SEQ ID NO:4
In arbitrary one or more sites；
Most preferably, described mutational site is selected from: be shown in SEQ ID NO:2 K168
Position, the A122 position of SEQ ID NO:4, or SEQ ID NO:2 or SEQ ID NO:
One or more in the site that on 4, other are less to activity influence.
2. the polypeptide of sudden change as claimed in claim 1, its mutating acid is N position
Aminoacid, described mutating acid connected mode in described polypeptide is shown below:
Wherein, by R1To R2The N-terminal that direction is aminoacid sequence to C-terminal side
To, R1For the 1st to N-1 amino acids residue in polypeptide,
R2For the amino acid residue of the N+1 position in polypeptide to C-terminal, R4For
3. the polypeptide of sudden change as claimed in claim 1, its comprise be available for coupling nitrine and
The difunctional linking arm of metal isotope, cyclooctyne (DIBO) is contained in described linking arm one end
Group, the other end contains DOTA, and described difunctional linking arm structure is shown below:
The polypeptide of the sudden change in the most aforementioned any one of claim 1-3, its heavy chain amino
Sequence is SEQ ID NO:10 and/or its light-chain amino acid sequence is SEQ ID NO:8.
5. the polypeptide of the sudden change as claimed in claim 4 after site-directed coupling, its knot
Structure is shown below:
Wherein, wherein n-th aminoacid is undergone mutation, and R1 is the 1st to N-1 position
Amino acid residue, R2 is N+1 position to the amino acid residue of C-terminal, and R3 is DOTA
Or other directly or indirectly provide the little molecule of function；
Preferably, it is SEQ ID NO:2 that the polypeptide before wherein suddenling change comprises aminoacid sequence
Heavy chain and/or light chain that sequence is SEQ ID NO:4；
It is highly preferred that R therein3For DOTA, it chelates radiosiotope；
Most preferably, described radiosiotope is selected from Lu177, Cu64Or Y90。
6. the polypeptide of the sudden change after site-directed coupling as claimed in claim 5, described
R in conjugate3Selected from the auspicious statin of amycin (doxorubicin) or monomethyl Australia
(Monomethyl auristatin E, MMAE).
7. the nucleic acid molecules of the polypeptide of the sudden change in coding any one of claim 1-6.
8. comprise the carrier of nucleic acid molecules described in claim 7.
9. a carrier, it is the carrier pXH-N3 as shown in SEQ ID NO:5.
10. a pharmaceutical composition, it contains any one of claim 1-4 of effective dose
The polypeptide of described sudden change, or described in claim 5 or 6 through site-directed coupling
After the polypeptide of sudden change.
11. pharmaceutical compositions as claimed in claim 10, preparation for Radionuclide imaging,
Purposes in the medicine for the treatment of refractory malignancies or tumor recurrence.
12. 1 kinds of microorganisms, it contains the carrier described in claim 8 and/or 9；
Preferably, described microorganism is escherichia coli.
13. 1 kinds of zooblasts, it contains the carrier described in claim 8 and/or 9；
Preferably, described zooblast is Freestyle293 cell line.
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| CN110642934A (en) * | 2019-09-10 | 2020-01-03 | 中国医学科学院北京协和医院 | Long-acting interleukin-2 of target regulatory T cell and application thereof in treating autoimmune disease |
| CN111304187A (en) * | 2020-02-27 | 2020-06-19 | 杭州师范大学 | Method for directly and rapidly preparing cross-linked enzyme aggregate from cell lysate |
| CN111304187B (en) * | 2020-02-27 | 2021-09-21 | 杭州师范大学 | Method for directly and rapidly preparing cross-linked enzyme aggregate from cell lysate |
| CN111747869A (en) * | 2020-06-12 | 2020-10-09 | 北京大学深圳研究生院 | Genetically encoded formaldehyde reactive unnatural amino acid, preparation method and application thereof |
| WO2023004687A1 (en) | 2021-07-29 | 2023-02-02 | 浙江新码生物医药有限公司 | Unnatural amino acid and application thereof, recombinant protein containing same, and recombinant protein conjugate |
| WO2023004686A1 (en) | 2021-07-29 | 2023-02-02 | 浙江新码生物医药有限公司 | Unnatural amino acid, and use thereof, recombinant protein containing same, and recombinant protein conjugate |
| CN113548984A (en) * | 2021-07-29 | 2021-10-26 | 浙江新码生物医药有限公司 | Unnatural amino acid, application thereof, recombinant protein containing unnatural amino acid and recombinant protein conjugate |
| CN113548984B (en) * | 2021-07-29 | 2023-08-11 | 浙江新码生物医药有限公司 | Unnatural amino acid and application thereof, recombinant protein containing unnatural amino acid and recombinant protein conjugate |
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