CN107217042A - A kind of genetically engineered cell system for producing no fucosylation albumen and its method for building up - Google Patents
A kind of genetically engineered cell system for producing no fucosylation albumen and its method for building up Download PDFInfo
- Publication number
- CN107217042A CN107217042A CN201710641279.5A CN201710641279A CN107217042A CN 107217042 A CN107217042 A CN 107217042A CN 201710641279 A CN201710641279 A CN 201710641279A CN 107217042 A CN107217042 A CN 107217042A
- Authority
- CN
- China
- Prior art keywords
- genetically engineered
- cell
- cell system
- antibody
- engineered cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01068—Glycoprotein 6-alpha-L-fucosyltransferase (2.4.1.68), i.e. FUT8
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Produced the invention discloses a kind of without the genetically engineered cell system of fucosylated albumen and its method for building up.The present invention knocks out Slc35c1 genes, and/or Fut8 genes in host cell by using CRISPR/Cas9 technologies, obtains the stable genetically engineered cell system of Slc35c1 gene silencings, and/or Fut8 gene silencings.It can be produced using genetically engineered cell system of the invention and remove fucosylated albumen completely, and still stablized after 30 generations of biography, solve the fucosylated removal two large problems unstable not exclusively with passage in the prior art.Enhanced ADCC activity is shown without fucosylated antibody so as to enhance antibody clinical therapeutic effect by what the method for the present invention was produced.
Description
Technical field
The invention belongs to technical field of bioengineering, being related to a kind of can produce the genetic engineering of no fucosylation albumen
Cell line and its method for building up.
Technical background
Since 1986 approval first antibody drug since, antibody class medicine by more than 30 years flourish, by
There is the clear superiorities such as the targeting, specificity, selectivity of height in antibody drug, therapy field also from traditional cancer, from
Body immunity disease is progressively expanded in terms of the diagnosis and treatment of anti-infective, metabolic disease and angiocardiopathy etc., and is achieved and looked steadily
Purpose curative effect, while it improves life in patients, also creates huge economic benefit.For tumour antibody except
The signal transduction of target spot mediation is blocked so as to which outside inducing cell apoptosis, more therapeutic antibodies are played by following effector function
Therapeutic action：Cytotoxic effect (the antibody-dependent cell-mediated that cell is relied on
Cytotoxicity, ADCC), cell rely on phagocytosis (antibody-dependent cellular
Phagocytosis, ADCP), the cytotoxic effect of Complement Dependent (complement-dependent cytotoxicity,
CDC) etc..
The cytotoxic effect (ADCC) that cell is relied on refers to the NKT for expressing antibody Fc acceptor (such as Fc γ RIIIa)
Cell (NK), macrophage or neutrophil leucocyte etc., pass through Fc acceptors and the Fc sections for the IgG antibody for having been incorporated into target cells
With reference to, and then activate, the effect of the cell toxicant matter direct killing target cell such as release perforin, granzyme.ADCC is therapeutic
One of key mechanism of antibody clinical curative effect, most important for antibody performance curative effect, this point has also obtained clinical research
Support.The combination situation of antibody and Fc acceptors (such as Fc γ RIIIa) directly affects the ADCC effector functions of antibody, can be by changing
The protein structure or Fc sections of oligonucleotide chain for becoming antibody Fc section are modified to strengthen the combination of antibody and Fc acceptors (such as Fc γ RIIIa)
Ability, and then strengthen ADCC effects, strengthen antibody clinical therapeutic effect.The effector function of enhancing therapeutic antibodies can not only be obtained
More preferable therapeutic effect is obtained, while antibody consumption is so as to reduce treatment cost needed for can reducing treatment.
In recent years, research find to remove or reduction fucosylation after, therapeutic antibodies are in animal body or clinical manifestation
Go out more preferable curative effect.Improved and Fc γ RIIIa affinity, Ke Yi mainly due to the antibody for removing or reduce fucosylation
Stronger ADCC effects are shown under lower concentration by the high-affinity with Fc γ RIIIa, are removed or reduction fucosido
It is melted into as one of effective ways of raising therapeutic antibodies efficiency of future generation.Therefore, working out can produce anti-without fucosylation
The engineering cell strain of body is the problem of those skilled in the art is anxious to be resolved.
But current most of monoclonal antibodies are prepared by Chinese hamster ovary cell (CHO) cell, this antibody-like
Core fucosylation level it is very high, how to remove antibody Fc district core fucose improve its ADCC effect be at present it is such
One of main focus of antibody research.Wherein, went through the Gazyva (Obinutuzumab) listed in 2013, is on granted
The Glyco-engineered monoclonal antibody in city, it is the GlycoMab based on Roche that its fucose, which is knocked out,TMTechnology platform, by height expression β-
Isosorbide-5-Nitrae-N acetyl glucosamines amido-transferase I II (GnTIII enzymes), promotes the sugared ends of N- to divide the addition of type galactoside equally, and then
Suppress the modification of antibody core fucose, further enhancing the ADCC activity of antibody.But the glycosylation engineered technology has one
Fixed defect：The technology divides type galactoside equally by introducing so that up to more than 30 kinds of the sugar-type on antibody, its species is more
Many, increasingly complexization, adds the heterogeneity of sugar-type and the quality control difficulty of product；In addition the fucose of this method removes effect
Rate is low, and the sugar-type for still having about 30% contains core fucose.
The application can remove the fucosido of removing protein completely by being knocked out to two genes of Slc35c1 and Fut8 simultaneously
Change, and the albumen of fucosylation can not be still detected after 30 generations of biography, solve fucosylation and remove not exclusively and pass
For unstable two large problems.
The content of the invention
In order to overcome the defect of prior art, the side of removing protein fucosylation can be gone to completely the invention provides a kind of
Method, methods described is to realize to remove the albumen rock algae that cell is produced by knocking out Slc35c1 genes, and/or Fut8 genes
Glycosylated purpose.According to the above method, it can be used for gene of the production without fucosylation albumen present invention also offers a kind of
Engineering cell system, the albumen of no fucosylation can be prepared using the genetically engineered cell system.In the specific embodiment of the invention
In, the anti-CD20 of no fucosylation monoclonal antibody can be prepared using the genetically engineered cell system.Without fucosylation
Anti- CD20 monoclonal antibody compared with the anti-CD20 of fucosylation monoclonal antibody, with enhanced ADCC effects.
According to an aspect of the present invention, the invention provides a kind of genetic engineering for producing no fucosylation albumen is thin
Born of the same parents are that the genetically engineered cell system is the cell line of Slc35c1 gene silencings, and/or Fut8 gene silencings.
Further, the genetically engineered cell system is resistant to following at least one agglutinin：LcA, pea
Beans agglutinin, bean lectin, dried orange peel agaric agglutinin.
Further, the albumen is the albumen containing Fc areas.
Further, the nucleotide sequence containing albumen of the coding containing Fc areas in the genetically engineered cell system.It is described to compile
The nucleotide sequence of albumen of the code containing Fc areas can exist independently of the genome of the genetically engineered cell system, can also be whole
In the genome for closing the genetically engineered cell system.
Further, Cas9 gene orders are contained in the genetically engineered cell system.The Cas9 gene orders can be independent
Exist in the genome of the genetically engineered cell system, in the genome that the genetically engineered cell system can also be incorporated into.
The Cas9 of the present invention includes but is not limited to SpCas9, Cas9-nickase (Cas9n), dCas9-FokI etc..
In the embodiment of the present invention, the present invention uses SpCas9.
Further, in the genetically engineered cell system contain for Slc35c1 genes sgRNA gene order, and/or
Gene order containing the sgRNA for Fut8 genes.The gene order of the sgRNA can be thin independently of the genetic engineering
The genome of born of the same parents system is present, in the genome that can also be incorporated into the genetically engineered cell system.
According to another aspect of the present invention, the invention provides the foundation side of foregoing genetically engineered cell system
Method, the method for building up comprises the following steps：The Slc35c1 genes, and/or Fut8 genes in host cell are knocked out, is obtained
The cell line of Slc35c1 gene silencings, and/or Fut8 gene silencings.
Further, the method for building up comprises the following steps：Knocked out using CRISPR/Cas9 technologies in host cell
Slc35c1 genes, and/or Fut8 genes, obtain the cell line of Slc35c1 gene silencings, and/or Fut8 gene silencings.
Further, the method for building up comprises the following steps：
For individually knockout Slc35c1 genes,
(1) CRISPR/Cas9 technologies are utilized, sgRNA sequences are designed for the Slc35c1 genes in host cell, by it
Gene order is imported in host cell simultaneously with Cas9 gene orders；
(2) screening of resistance pressure obtains genetically engineered cell system of the production without fucosylation albumen；
For individually knockout Fut8 genes,
(1) CRISPR/Cas9 technologies are utilized, sgRNA sequences are designed for the Fut8 genes in host cell, by its gene
Sequence is imported in host cell simultaneously with Cas9 gene orders；
(2) screening of resistance pressure obtains genetically engineered cell system of the production without fucosylation albumen；
For knockout Slc35c1 genes simultaneously and Fut8 genes,
(1) CRISPR/Cas9 technologies are utilized, are separately designed for the Slc35c1 genes and Fut8 genes in host cell
SgRNA sequences, its gene order and Cas9 gene orders are imported in host cell simultaneously；
(2) screening of resistance pressure obtains stable genetically engineered cell system of the production without fucosylation albumen.
In above-mentioned method for building up, the step that sgRNA gene orders and Cas9 gene orders are imported in host cell simultaneously
It can include：SgRNA gene orders and Cas9 gene orders are connected respectively on carrier and build knockout carrier, by two knockouts
Carrier is transfected simultaneously to be entered in host cell.
As alternative mode, the step that sgRNA gene orders and Cas9 gene orders are imported in host cell simultaneously
Suddenly it can include：SgRNA gene orders and Cas9 gene orders are commonly connected to build knockout carrier on a carrier, by structure
The knockout carrier transfection built enters in host cell.
In specific embodiments of the present invention, by sgRNA gene orders, importing host is thin simultaneously with Cas9 gene orders
Step in born of the same parents includes：SgRNA gene orders and Cas9 gene orders are commonly connected to build knockout carrier on a carrier,
The knockout carrier transfection of structure is entered in host cell.
According to a further aspect of the invention, the invention provides a kind of albumen without fucosylation.The albumen is
Produced by foregoing genetically engineered cell system.
According to a further aspect of the invention, the invention provides a kind of foregoing albumen without fucosylation
Preparation method, it is characterised in that the preparation method comprises the following steps：
(1) foregoing genetically engineered cell system is set up according to foregoing method for building up；
(2) in the genetically engineered cell system for obtaining the coded sequence steps for importing (1) of albumen, obtain without fucosylation
Albumen.
According to a further aspect of the invention, preparation is tied up to without rock the invention provides foregoing genetically engineered cell
Application in the glycosylated albumen of algae.
The albumen of the present invention can be recombinant protein or endogenous protein.Recombinant protein is by encoding proteins
Nucleotide sequence is building up in expression vector and is conducted into host cell to express the foreign protein of formation.
In a particular embodiment, albumen of the invention is recombinant protein.
In a particular embodiment, albumen of the invention is the albumen containing Fc.
The albumen containing Fc of the present invention includes antibody, bispecific antibody, immunoadhesin and other comprise at least and exempted from
The associated proteins of the funtion part in epidemic disease globulin CH2 and CH3 regions." funtion part " is to refer to combine Fc acceptors (such as Fc
γ R or FcRN) CH2 and CH3 regions, and/or the region of complement activation can be participated in.If CH2 and CH3 regions include and deleted
Remove, replace, and/or insert or prevent its from reference to any Fc acceptors and can not activating complement other modifications, then the CH2
It is not functional with CH3 regions.
The albumen containing Fc of the present invention also includes antibody derivatives, and antibody derivatives include antibody or the Fc comprising antibody
Domain or Fc areas, are modified by covalent bond heterologous molecule.The example of antibody derivatives melts including binding structural domain-Ig
Zoarium, wherein the binding structural domain can be, for example, the peptide of the ectodomain of part, acceptor, peptide, non-natural formation, etc.
Deng.The example of the exemplary fusion in immunoglobulin or Fc areas includes：Etanercept, it is merging for sTNFRII and Fc areas
Albumen (United States Patent (USP) 5,605,690)；A Laifasai, it is that egg is merged in the LFA-3 that expresses and Fc areas on antigen presenting cell
In vain (United States Patent (USP) 5,914,111)；Cytotoxic T lymphocyte-associated antigen -4 (CTLA-4) and the fusion protein in Fc areas
(J.Exp.Med.181:1869(1995))；Interleukin 15 and the fusion protein (J.Immunol.160 in Fc areas:5742
(1998))；The factor Ⅴ II and fusion protein (Proc.Natl.Acad.Sci.USA98 in Fc areas:12180(2001))；Interleukin
The 10 and fusion protein (J.Immunol.154 in Fc areas:5590(1995))；Interleukin-22 and the fusion protein in Fc areas
J.Immunol.146:915(1991))；The CD40 and fusion protein (Surgery132 in Fc areas:149(2002))；Flt-3
(the fms- samples EGFR-TK) and fusion protein (Acta.Haemato.95 of antibody Fc district:218(1996))；OX40 and antibody
Fusion protein (the J.Leu.Biol.72 in Fc areas:522(2002))；And the fusion protein with following molecules：Other CD molecules
(for example, CD2, CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-I), CD137), adhesion molecule is (for example, ALCAM (swashs
Leukocyte adhesion molecule living), cadherin, ICAM (intracellular adhesion molecule) -1, ICAM-2, ICAM-3) cell factor receptor
Body (for example, interleukin-4 R, IL-5 R, interleukin-6 R, IL-9 R, interleukin-10 R, IL-12 R, interleukin-
13R α 1, interleukin-13 R α 2, interleukin-15 R, interleukin-21 R α), chemotactic factor (CF), cell death inducement signal molecule (example
Such as, B7-H1, DR6 (death receptor 6), PD-1 (programmed cell death -1), TRAIL R1), costimulatory molecules is (for example, B7-1, B7-
2, B7-H2, ICOS (can induce costimulation thing)), growth factor (for example, ErbB2, ErbB3, ErbB4, HGFR), induction
Molecule (for example, B7-H3), activation factor (for example, NKG2D), signal transfer of molecules (for example, gpl30), BCMA, and TACI.
It can be prepared using the foregoing genetically engineered cell system of the present invention any kind of without fucosylation
Antibody or antibody derivatives.Using the foregoing genetically engineered cell system of the present invention prepare without fucosylation
The example of antibody or antibody derivatives includes but is not limited to：Anti-HER2 monoclonal antibody：Herceptin (Trastuzumab, United States Patent (USP)
No 5821337), handkerchief trastuzumab (Pertuzumab, United States Patent (USP) No 7862817)；Anti- EGFR monoclonal antibodies：Cetuximab
(Cetuximab, United States Patent (USP) No 6217866)；Anti-CD20 monoclonal antibody：Rituximab (Rituximab, world patent
WO9411026), Obinutuzumab (Chinese patent Authorization Notice No.：CN1902231B), (Chinese patent is public by Ocrelizumab
The number of opening：CN101151278)；Anti- CD19 monoclonal antibodies：Light and weight chain CDR and HD37 hybridomas (pezzutto (1997),
J.Immunol.138,2793-9) identical IgG1 chimeric antibodies；Anti- CLD18A2 antibody：(Chinese patent mandate is public by IMAB362
Announcement number：CN101687929).
In a particular embodiment of the present invention, the nothing prepared using the foregoing genetically engineered cell system of the present invention
The albumen of fucosylation is the anti-CD20 of no fucosylation antibody.
The heavy chain protein sequence of anti-CD20 antibody is as shown in SEQ ID NO.1, and the DNA sequence dna of encoding heavy chain albumen is such as
SEQ ID NO.29；Light chain protein sequence encodes the DNA sequence dna such as SEQ ID of light chain protein as shown in SEQ ID NO.2
NO.30。
Using the antibody prepared of genetically engineered cell of the present invention include but is not limited to mouse source antibody, humanized antibody,
Embedding and antibody, human antibody.Preferably, the antibody that genetically engineered cell of the invention is prepared is IgG type monoclonal antibodies.
Can be IgG different subtype using the antibody prepared of genetically engineered cell of the present invention, including IgG1,
IgG2、IgG3、IgG4。
Antibody and antibody derivatives prepared by the method according to the present invention can be used for various treatments or non-therapeutic use.Example
Such as, the antibody can be used as treatment antibody.Antibody derivatives (for example, receptor-Fc fusion) can be used as treating molecule.It is described anti-
Body or antibody derivatives can be with other molecule coupling labeleds.The antibody coupling is in suitable medicine (for example, antibody drug conjugates)
Or other activating agents.The antibody and antibody derivatives can also be used for non-treatment purpose, such as diagnostic test, prognostic assay, release
Experiment, etc..
The antibody and antibody derivatives prepared according to the inventive method can be made into treatment articles or non-treatment articles for use.It is described
Antibody and derivative can be made into pharmaceutical composition, wherein including the antibody or derivative and one kind that treat or prevent effective dose
Or (acceptable) composition of a variety of pharmaceutically compatibles.For example, medicine or non-drug composition generally comprise one or more medicines
Thing carrier (such as sterile liquid, Ru Shui and oil, including oil, animal, the oil of plant or synthesis source, such as peanut oil, soybean
Oil, mineral oil, sesame oil etc.).During the intravenous administration pharmaceutical composition, water is more conventional carrier.Saline solution and dextrorotation
Sugar aqueous solution and glycerite also are used as liquid-carrier, are particularly suitable for use in parenteral solution.Suitable excipient includes, for example, ammonia
Base acid, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk (chalk), silica gel, odium stearate, list are hard
Glycerol, talcum powder, sodium chloride, skimmed milk power, glycerine, propylene, glycol, water, ethanol etc..If desired, composition
A small amount of wetting agent or emulsifying agent or pH buffer can be contained.These compositions can take solution, supensoid agent, emulsion, tablet, ball
The forms such as agent, capsule, powder agent, sustained release preparation.E.W.Martin's《Remington pharmaceutical science》(Remington's
Pharmaceutical Sciences) describe the examples of suitable pharmaceutical carriers.This composition usually contains therapeutically effective amount
Protein, usually purified form, and appropriate carrier, in the form of providing and be suitable for administering to patient.Preparation corresponds to administration
Mode.
The composition of intravenous administration is typically the solution of the aqueous buffer of sterile isotonic.If desired, medicine
Solubilizer and local anesthetic can be contained, such as lidocaine mitigates the pain of injection site.Generally, each composition is provided separately
Or offer, such as the sealing container such as ampoule or anther sac in lined out activity content of material in the form of unit dosage forms are provided
In freeze-dried powder or without the form of aqueous concentrate., can be with defeated containing sterile pharmaceutical grade water or salt solution when giving the medicine by transfusion
Liquid bottle distributes the medicine.By injection give the medicine when, it is possible to provide the sterile water for injection or salt solution of an ampoule, so as to
All compositions are mixed before medicine.
The foregoing genetically engineered cell system that the present invention is built can be the cell line that transient transfection/transduction is produced,
Can also be the cell line that stable transfection/transduction is produced.
In a particular embodiment, it is stable cell lines in the genetically engineered cell system of the present invention.
This paper terms " stable cell lines " refer to cell property can stablize heredity, generally referred to as stable transfection/turn
The cell line of artificial delivery life, the i.e. nucleotide sequence of entrance cell is incorporated on the genome of cell stable heredity and gone down.
This paper terms " no fucosylation " be finger protein it is glycosylation modified in be free of fucosylation.
This paper terms " core fucose " refer to：In the core pentasaccharides of N- sugar, on the GlcNAC being connected with asparagine
The fucose of link.
Terms used herein " ADCC " refers to cell-mediated cell-cytotoxic reaction.
Terms used herein " strengthens ADCC " to refer in the presence of effector cell, the phase with contacting defucosylated antibody
Compared with the cell killing of cell, when contacting without defucosylated antibody, the increase of any measurable cell cracking, for example,
Cell cracking increase at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%th, 250%, 300% or 325%.
Terms used herein " carrier " is the nucleic acid molecules for referring to transport connected another nucleic acid.One kind is carried
Body type is " plasmid ", and it refers to circular double-stranded DNA ring, wherein other DNA fragmentation can be connected.Another bearer type is
Viral vector, wherein other DNA fragmentation may be coupled in viral genome.The host that some carriers can be imported at it
Autonomous replication (such as bacteria carrier and episomal mammalian vectors with bacterial origin of replication) in cell.Other carriers
(such as non-add type mammalian vector) import host cell after can be incorporated into the genome of host cell, thus with
Host genome is replicated together.In addition, some carriers can guide the expression for the gene being effectively connected with them.These carriers exist
Herein referred to as " recombinant expression carrier " (or being referred to as " expression vector ").The table generally used in recombinant DNA technology
Up to carrier usually from plasmid form.In this manual, " plasmid " and " carrier " can be with used interchangeably, because plasmid is the most frequently used
Carrier format.But, the present invention also includes the expression vector of other forms, and such as (for example replication defect type is reversed viral vector
Record virus, adenovirus and adeno-associated virus), they play equivalent function.
This paper terms " host cell " include any cell for being suitable for expressing recombinant nucleic acid sequence.It is thin that cell includes eucaryon
(such as saccharomyces cerevisiae, schizosaccharomyces pombe, pichia pastoris phaff, methanol type are finished for born of the same parents' (unicellular or many cells), yeast cells
Red yeast etc.), plant cell, insect cell (such as SF-9, SF-21, the insect cell of baculovirus infection, cabbage looper
(Trichoplusia ni) etc.), inhuman zooblast, human cell or cell fusion thing, such as hybridoma or four sources are miscellaneous
Hand over knurl (quadroma).
Host cell can be people, monkey, ape, hamster, rat or mouse cell.In some embodiments, cell is
Eukaryotic and selected from following cell：CHO (such as CHOK1, DXB-11CHO, Veggie-CHO), COS (such as COS-7),
Syria hamster, rat melanoma, mouse black-in lymphoma (such as SP2/0, NS0), retina cell, Vero, CV1, kidney is (for example
HEK293,293EBNA, MSR293, MDCK, HaK, BHK, BHK21), HeLa, HepG2, WI38, MRC5, Colo205, HB
8065, HL-60, Jurkat, Daudi, A431 (epidermis), CV-1, U937,3T3, L cells, C127 cells, MMT 060562,
Sertoli cells, BRL 3A cells, HT1080 cells, human melanoma cell, tumour cell, human lymphoma cell is (for example
Namalwa cells) and cell line from aforementioned cells.
In a particular embodiment of the present invention, that host cell is selected is CHO.
This paper terms " antibody " are represented：(1) the immunologic competence part of immunoglobulin polypeptides and immunoglobulin polypeptides,
I.e. polypeptide of immunoglobulin class or part thereof, includes the antigen of immunologic opsonin binding domain specific antigen (for example, CD20)
Binding site and the Fc domains for including complicated N- glucosides-connection sugar chain, or (b) this immunoglobulin polypeptides or immune special
The conservative replaces derivative of the fragment of property combination antigen (for example, CD20).The description of antibody is generally see, for example, Harlow＆
Lane,《Antibody：Laboratory manual》(Antibodies：A Laboratory Manual) (CSH Press
(Cold Spring Harbor Laboratory Press), 1988).
Term " bispecific antibody " includes the antibody for being capable of selective binding two or more epitopes.Bispecific
Antibody generally comprises 2 different heavy chains, and each heavy chain specifically binds the different tables on different-two different moleculars of epitope
Different epitopes on position (such as two different epitopes not on synantigen) or same molecule are (for example, the difference on same antigen
Epitope).If bispecific antibody being capable of two different epitopes of selective binding (the first epitope and the second epitope), the first weight
Chain is typically directed to the compatibility low at least 1 to 2 or 3 or 4 or more of the second epitope for the compatibility of the first epitope than the first heavy chain
Many orders of magnitude, vice versa.The epitope specifically bound by bispecific antibody can on identical or different target (example
Such as on identical or different albumen).The heavy chain of the different epitopes on same antigen can be recognized for example, by combination to prepare
Bispecific antibody.For example, can be by the nucleotide sequence of the variable heavy chain sequence of the different epitopes in code identification same antigen
The nucleotide sequence for encoding identical or different heavy chain constant region is fused to, can be expressed in the cell of expression light chain immunoglobulin
Such sequence.Typical bispecific antibody has 2 heavy chains, each to have 3 heavy chain CDR, and heel is (last from N- ends to C-
End) CH1 domains, hinge area, CH2 domains and CH3 domains；Also there is light chain immunoglobulin, it does not assign epitope knot
Close specificity but can be combined with each heavy chain, or can be combined with each heavy chain and the combination of heavy chain epitope can be combined
One or more epitopes that area is combined, or can be combined with each heavy chain and 1 or 2 heavy chains and 1 or 2 tables can be made
Position is combined.
Term " monoclonal antibody " refers to derived from individual cells clone, including any eucaryon or procaryotic cell clone or phagocytosis
The antibody of body clone.
Term " Fc areas " represents the constant region of antibody, for example, CH1- hinge-CH2-CH3 domains, optionally have CH4 knots
Structure domain, or the conservative replaces in this Fc areas derivative.
Term " Fc domains " represents the constant region domain of antibody, for example, CH1, hinge, CH2, CH3 or CH4 structure
Domain, or this Fc domains conservative replaces derivative.
Beneficial effects of the present invention：
The present invention utilizes the CRISPR/Cas9 technique constructions base of Slc35c1 gene silencings, and/or Fut8 gene silencings
Because of engineering cell system, the genetically engineered cell system can prepare the antibody of no fucosylation, and the property of cell line can be with
Heredity steady in a long-term is gone down.
Brief description of the drawings
Fig. 1 plasmid pX330 structures and insertion point schematic diagram；
Fig. 2 shows the EM201 antibody N- sugar-type analysis charts of different cell line expression；
Fig. 3 shows the N- sugar-type analysis charts of different cell line mitotic stabilities；
Fig. 4 shows that anti-CD20 antibody is schemed with Fc γ RIIIa binding activity detection；
Fig. 5 shows anti-CD20 ADCC effect figure of the antibody to Daudi cells；
Fig. 6 shows anti-CD20 influence figure of the Antybody therapy to Daudi cell nude mouse subcutaneous transplantation knurl volumes.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage
Solution, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment
Technology or condition person, (write, Huang according to the technology described by document in the art or condition such as with reference to J. Pehanorm Brookers
What training hall etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Examination used
Agent or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
The structure of the Slc35c1 gene knockout carriers of embodiment 1
The mRNA sequence (NM_001246808.1, as shown in SEQ ID NO.3) for targetting Chinese hamster Slc35c1 genes
Code area devises 4 sections of gRNA (table 1), is synthesized by Nanjing Genscript Biotechnology Co., Ltd., two pairing it is single-stranded (F and
R) after Gradient annealing, (purchase is certainly with the pX330-U6-Chimeric_BB-CBh-hSpCas9 of BbsI digestion with restriction enzyme
Addgene, article No. Plasmid42230；Multiplex Genome Engineering Using CRISPR/Cas
Systems.Cong L,Ran FA,Cox D,Lin S,Barretto R,Habib N,Hsu PD,Wu X,Jiang W,
Marraffini LA,Zhang F.Science.2013Jan 3.10.1126/science.1231143PubMed
23287718) carrier is connected.Carrier structure and insertion point schematic diagram are as shown in Figure 1.
Confirm to obtain correct clone after sequence verification, transfect shaking flask after Top-10 competent cells, picking monoclonal
Interior culture, transfected Chinese hamster cell is obtained with the big extraction reagent kit of endotoxin-free plasmid (Tiangeng biochemical technology Beijing Co., Ltd)
Plasmid used in CHO.
The gRNA designs of the targeting Slc35c1 genes of table 1
Embodiment 2：The structure of FUT8 gene knockout carriers
Target the volume of the mRNA sequence (XM_003501735.2, as shown in SEQ ID NO.14) of Chinese hamster FUT8 genes
Code area devises 5 sections of gRNA (table 2), is synthesized by Nanjing Genscript Biotechnology Co., Ltd., single-stranded (F and the R) of two pairings
After Gradient annealing, (purchase is certainly with the pX330-U6-Chimeric_BB-CBh-hSpCas9 of BbsI digestion with restriction enzyme
Addgene, article No. Plasmid42230；Multiplex Genome Engineering Using CRISPR/Cas
Systems.Cong L,Ran FA,Cox D,Lin S,Barretto R,Habib N,Hsu PD,Wu X,Jiang W,
Marraffini LA,ZhangF.Science.2013Jan 3.10.1126/science.1231143PubMed
23287718) carrier is connected.
The gRNA designs of the targeting FUT8 genes of table 2
Confirm to obtain correct clone after sequence verification, transfect shaking flask after Top-10 competent cells, picking monoclonal
Interior culture, transfected Chinese hamster cell is obtained with the big extraction reagent kit of endotoxin-free plasmid (Tiangeng biochemical technology Beijing Co., Ltd)
Plasmid used in CHO.
The Chinese hamster ovary celI of the Slc35c1 gene knockouts of embodiment 3 is built
30ml density 5 × 10 is inoculated with 125ml cell culture shaking flasks5CHO-K1 (ATCC) cell, Cell viability exists
More than 98%, after cell centrifugation, buffer solution is turned with electricity and is diluted to 1 × 107Cells/ml density, the targeting that 40 μ g are built
The pX330 plasmids of Slc35c1 genes are added in electric shock cup, are added 0.7ml cell suspensions, are finally filled into electricity and turn buffer solution extremely
0.8ml, is gently mixed, and is shocked by electricity under conditions of 300V, 20ms once, electric shock cup is placed in into ice bath 5min in ice chest, after transfection
Culture medium is added, 37 DEG C, 5%CO is placed in2, rotating speed 130rpm cell culture table on cultivate.After 2 days, add lens and coagulate
Collect element LCA (Lensculinaris agglutinin) to 200 μ g/ml, screen stable resistance clone；After dosing 5 days, centrifugation
Cell is collected, using specific affinities of the LcA LCA to albumen fucosido, the cell after cotransfection is used
Biotin-LCA is marked, is carried out negativity sorting with reference to anti-biotin microBeads and MACs LD posts；In 300 μ after sorting
G/ml LcA LCA culture 24hr, limiting dilution assay seeds cells into progress cell monoclonal in 96 orifice plates,
In 300 μ g/ml LcA LCA colonized cultures, 12 holes or 6 holes are further inoculated into afterwards until being transferred to culture
In bottle, monoclonal cell strain is obtained.After the monoclonal cell strain extracting genomic DNA of acquisition, PCR amplification Slc35c1 gene pieces
Section detects the catastrophe of wherein gene, and PCR forward primer sequences are：5'-ATGTGACAATTGAAGGCTGTACC-3'(SEQ
ID NO.12)；Reverse primer sequences are：5'-AGAAGGAAGTGGTCTGTTTGAG-3'(SEQ ID NO.13).PCR primer is passed through
After agarose gel electrophoresis purifying is reclaimed, carrier T pGM-Simple-T Fast Vector (Tiangeng biochemical technology Beijing is cloned into
Co., Ltd), the carrier after clone is used for being sequenced, and filters out the monoclonal that frameshift mutation all occurs for Slc35c1 gene reading frames
Cell line, is denoted as CHO-K1-S-/-。
The Chinese hamster ovary celI of the Fut8 gene knockouts of embodiment 4 is built
30ml density 5 × 10 is inoculated with 125ml cell culture shaking flasks5CHO-K1 (ATCC) cell, Cell viability exists
More than 98%, after cell centrifugation, buffer solution is turned with electricity and is diluted to 1 × 107Cells/ml density, the targeting that 40 μ g are built
The pX330 plasmids of Fut8 genes are added in electric shock cup, are added 0.7ml cell suspensions, are finally filled into electricity and turn buffer solution extremely
0.8ml, is gently mixed, and is shocked by electricity under conditions of 300V, 20ms once, electric shock cup is placed in into ice bath 5min in ice chest, after transfection
Culture medium is added, 37 DEG C, 5%CO is placed in2, rotating speed 130rpm cell culture table on cultivate.After 2 days, add lens and coagulate
Collect element LCA (Lens culinaris agglutinin) to 200 μ g/ml, screen stable resistance clone；After dosing 5 days, from
The heart collects cell, and using specific affinities of the LcA LCA to albumen fucosido, the cell after cotransfection is made
Marked with biotin-LCA, carry out negativity sorting with reference to anti-biotin microBeads and MACs LD posts；After sorting
300 μ g/ml LcA LCA culture 24hr, limiting dilution assay, which is seeded cells into, carries out cell Dan Ke in 96 orifice plates
Longhua, in 300 μ g/ml LcA LCA colonized cultures, is further inoculated into 12 holes or 6 holes until transfer afterwards
Into blake bottle, monoclonal cell strain is obtained.
After the monoclonal cell strain extracting genomic DNA of acquisition, PCR expands the prominent of Fut8 genetic fragments detection wherein gene
Change situation, PCR forward primer sequences are：5'-GATTCCAGGTTCCCATATATTC-3'(SEQ ID NO.25), reverse primer
Sequence is：5'-TGATGACTGCTAGTGATGCTAC-3'(SEQ ID NO.26), or forward primer sequence is：5'-
TGTCTGAAGCATCATGTGTTG-3'(SEQ ID NO.27), reverse primer sequences are：5'-
ACAGTATTTCATCAAATCCTTG-3'(SEQ ID NO.28).After PCR primer is reclaimed through agarose gel electrophoresis purifying, gram
The grand carrier arrived after carrier T pGM-Simple-T Fast Vector (Tiangeng biochemical technology Beijing Co., Ltd), clone is used for
Sequencing, filters out the monoclonal cell strain that frameshift mutation all occurs for Fut8 gene reading frames, is denoted as CHO-K1-F-/-。
The Chinese hamster ovary celI that the Slc35c1 and Fut8 Gene Doubles of embodiment 5 are knocked out is built
By the stable strain CHO-K1-S for having knocked out Slc35c1 genes of structure-/-Cell, Fut8 bases are knocked out according to embodiment 4
Cause, the clone strain filtered out is the Chinese hamster ovary celI that Slc35c1 and Fut8 Gene Doubles are knocked out, and is denoted as CHO-K1-SF-/-。
The expression and purifying of the anti-CD 20 antibodies of embodiment 6
Using pcDNA3.1 as skeleton carrier, by the selection label excision of Neomycin neomycins, GS (glutamy is replaced with
Amine synzyme, glutamine synthetase) expression box, it is used as selection markers；Wherein GS cDNA can be from expression GS's
Obtained in cell line CHO by RT-PCR, it is engineered after carrier be named as GS screening vectors.It is respectively synthesized coding EM201 weights
(DNA sequence dna of encoding heavy chain encodes the DNA sequence dna such as SEQ of light chain to the DNA sequence dna of chain and light chain as shown in SEQ ID NO.29
Shown in ID NO.30), inserted between light chain and heavy chain after CMV promoter, HindIII digestions are introduced at 5 ' ends by Standard PCR
Site, introduces NotI restriction enzyme sites at 3 ' ends, is inserted between the HindIII of GS screening vectors and NotI, build and obtain eucaryon
Expression vector pEM201.
30ml density 5 × 10 is inoculated with 125ml cell culture shaking flasks5CHO-K1, CHO-K1-S-/-、CHO-K1-F-/-
And CHO-K1-SF-/-Cell, Cell viability is more than 98%, and cell is with 1 × 107Cells/ml density is inoculated in electricity and turns buffering
Liquid, 40 μ g pEM201 plasmids are added in electric shock cup, 0.7ml cell suspensions is added, finally fills into electricity and turn buffer solution extremely
0.8ml, is gently mixed, and should not produce bubble.Shocked by electricity under conditions of 300V, 20ms once, electric shock cup is placed in ice in ice chest
5min is bathed, is diluted in 6ml culture mediums in 37 DEG C of CO2Recover 24h in incubator, 75 μM of MSX warps are added into seed culture medium
Cross limiting dilution assay and carry out subclone screening.Divided by limited dilution method in monoclonal cell to 96 orifice plates, 37 DEG C, 5%CO2
Incubator culture, treats that cell is expanded to certain amount, carries out ELISA checkings, select positive colony and be amplified to 24 orifice plates, 6 successively
Orifice plate, T25 square vases.By the culture and screening of 6~8 weeks, the monoclonal cell strain for being capable of high efficient expression anti-CD 20 antibodies is obtained.
The antibody of the anti-CD20 is named as EM201, and the protein sequence of its heavy chain and light chain is shown in SEQ ID NO.1 and SEQ ID NO.2.
Expansion culture of the monoclonal cell strain Jing Guo culture medium multistep, inoculum density is 0.5 × 106Cells/ml, is added
MSX to 75 μM, continue after 2 weeks, cell is collected by centrifugation and is cultivated in 125ml shaking flasks, 37 DEG C, 5%CO is placed in2, rotating speed 130rpm
Shaking table on suspend culture 12 days, during which the 3rd, glucose was added with 4g/L final concentration in 6,9 days, after culture terminates in harvest
Clearly, supernatant is purified.Isolating and purifying for EM201 antibody is carried out using AKTA (GE companies).Protein A were collected first
Affinity column (MabSelect SuRe) pH adjusts pH value extremely in the eluent of 3.4-3.6 scopes (being monitored with 280nm)
8.0, anion exchange chromatography (Q-Sepharose FF) is loaded to, 280nm is monitored and collected sample.By collection liquid pH
Value regulation is loaded to cation-exchange chromatography post (Poros) and collects the antibody that anti-CD20 is obtained after sample, ultrafiltration concentration to 5.5.
The sugar-type analysis of the anti-CD 20 antibodies of embodiment 7 (EM201)
Take and 10 μ L G7 enzyme cutting buffering liquids, 1 μ LPNGase F are added after 200 μ g anti-CD 20 antibodies, super filter tube desalination, is added water
To 100 μ L, in 37 DEG C of constant temperature 10hr.- 20 DEG C of ethanol that the good sample of digestion is added into 300 μ L are mixed, and are stood after 20min
12000rpm centrifuges 3min, takes supernatant to be concentrated in vacuo drying, obtains the sugared dry powder of N-.DMSO and acetic acid are according to 350 μ L:150 μ L ratios
Example is mixed, and is taken 5mg 2-AB, 6mg SodiumCyanoborohydride to be dissolved in 100 μ L DMSO and acetic acid mixture, is taken
The μ L of mixed liquor 10 are added in the sugared dry powder of the N- being concentrated in vacuo, 65 DEG C of derivative 3 hours, the acetonitrile and the μ of water mixed liquid 200 of addition 80%
L, centrifuges 2mins, takes supernatant, liquid phase analysis：Chromatographic column：WATERS Acquity UPLC BEH Amide1.7μm；Column temperature：40
℃；Excitation wavelength：λ ex=330nm；λ em=420nm；Sample size：10μL；Liquid phase gradient is as shown in table 3.
The gradient elution timetable of table 3
| Time (min) | Flow A% (ammonium formate) | Mobile phase B % (acetonitrile) |
| 0 | 20 | 80 |
| 20 | 20 | 80 |
| 25 | 25 | 75 |
| 75 | 40 | 60 |
| 80 | 65 | 35 |
| 85 | 65 | 35 |
| 86 | 20 | 80 |
| 96 | 20 | 80 |
Anti- CD20 antibody EM201 is respectively by CHO-K1, CHO-K1-F-/-、CHO-K1-S-/-And CHO-K1-SF-/-Cell
Expression, its sugar-type collection of illustrative plates are as shown in Fig. 2 from Fig. 2 and following table 4, the EM201 phases expressed with CHO-K1 cell lines
Than CHO-K1-F-/-、CHO-K1-S-/-The content of the antibody fucose of cell expression is significantly reduced, only 0.6% and 1.8%
Sugar-type contains fucose；And CHO-K1-SF-/-The antibody of cell expression does not detect the sugar-type containing fucose, shows with individually striking
Except Slc35c1 with Fut8 genes are compared, it is more complete that dual-gene knockout removes fucose.
The EM201 antibody N- sugar-type ratios of the different cell line expression of table 4
Legend：2-Acetamido-2-deoxy-D-glucose：■；Fucose：；Mannose and galactolipin：●
The fucose of embodiment 8 knocks out the mitotic stability of stable strain
To meet protein drug industrialization production scale to passage and the requirement of cell doubling level, the stabilization to structure is thin
Born of the same parents' strain carries out mitotic stability research.It was passaged to for 30 generations since cell bank, covers the industrialization production scale drafted to passing
Generation and the requirement of cell doubling level, and increase by 15 generations on this basis to ensure stability study to following manufacturing specification request
Spreadability.Cell per 2-4 days biography once, totally 30 generation, by inquiry cell different phase doubling time, surge again
Flat, maximum cell density, production capacity, antibody specific production rate, and assess antibody expression amount and quality, the passage to cell
Stability is analyzed.Cell line mitotic stability result shows, passage to P30, cell density, cell doubling time,
Motility rate, antibody specific production rate, antibody expression amount and antibody mass are basicly stable, meet the requirement as engineering cell strain.But
It is CHO-K1-S-/-And CHO-K1-F-/-Cell line increases with passage number, and the ratio of fucose sugar-type dramatically increases (Fig. 3
With table 5), 1.7% and 4.7% are increased to respectively, it may be possible to caused by other glycosylated bypasses are activated；And dual-gene knockout
CHO-K1-FS-/-Cell line was passaged to for 30 generations, still detected (Fig. 4 and table 5) without the sugar-type containing fucose.Show with individually striking
Except Slc35c1 with Fut8 genes are compared, its CHO-K1-FS of the cell line of dual-gene knockout-/-Mitotic stability is more preferable, and core
Fucose knocks out more thorough, thoroughly to be knocked out preferably as fucose cell line progress application and development.
The N- sugar-type ratios of the different cell line mitotic stabilities of table 5
Legend：2-Acetamido-2-deoxy-D-glucose：■；Fucose：；Mannose and galactolipin：●
The anti-CD 20 antibodies of embodiment 9 are analyzed with Fc γ RIIIa binding activity
Anti-His (R＆D companies) antibody will be diluted to 1 μ g/mL with pH9.6 carbonate buffer solution, add in ELISA Plate
4 DEG C of overnight incubations.After board-washing and closing (phosphate buffer for containing 5% skimmed milk power), 1 μ g/ml Fc γ RIIIa are added
(158V) (R＆D companies), 37 DEG C of incubation 1h.By testing sample：EM201(CHO-K1-FS-/-Expression), EM201 (CHO-K1 tables
Up to), Gazyva (Genentech companies) is respectively with anti-human κ (R＆D companies) with 1:2 ratio is mixed, 37 DEG C of incubation 1h
Antibody is diluted to 50 μ g/ml afterwards, then carries out 3 times of gradient dilution 10 concentration points of acquisition.ELISA Plate adds anti-after dilution
Body and anti-human κ the μ l of mixed liquor 100,37 DEG C are incubated 1 hour.GoatF (ab ') 2anti- for being coupled HRP after board-washing
Human IgG F (ab ') 2 (R＆D companies) are with 1:5000 dilution proportion, is added in ELISA Plate, 37 with the amount in 100 μ l/ holes
DEG C be incubated 1h.The TMB color development at room temperature 30min in after board-washing plus 100 μ l/ holes, add the 2mol/L H in 100 μ l/ holes2SO4Color development stopping
Reaction, puts and measure light absorption value at 450nm wavelength is determined on ELIASA, using sample concentration as abscissa, absorbance values are vertical
Coordinate, quadruplex parameters curve matching.
EM201(CHO-K1-FS-/-Expression) for the sugar-type without core fucose anti-CD20 antibody；EM201(CHO-
K1 express) be with the sugar-type of core fucose (>84% ratio) based on anti-CD20 antibody；Gazyva contains core for part
The antibody of anti-CD20 based on the sugar-type (~50% ratio) of fucose.EM201(CHO-K1-FS-/-Expression), EM201 (CHO-
K1 is expressed) and Gazyva (Genentech companies) and Fc γ RIIIa (158V) binding activity experimental result as shown in figure 4, reality
Test result and show EM201 (CHO-K1-FS-/-Expression) it is about 2 times of Gazyva with Fc γ RIIIa (158V) binding activity, more
It is the binding activity (about 6 times) for being significantly stronger than EM201 (CHO-K1 expression), shows no fucosylation anti-CD 20 antibodies to Fc γ
RIIIa has higher affinity.
The ADCC effects to human B cell lymthoma Daudi cells of the anti-CD 20 antibodies of embodiment 10
People Burkitt's lymphoma cell line Daudi cells are purchased from American type culture collection (ATCC), as
Target cell in ADCC tests；The NK-92MI-CD16a of the high a of expression Fc γ R III using on cell membrane is effector cell's (rich biology of China
Medical science Co., Ltd).5 × 10 are added in 96 orifice plates per hole4Individual NK-92MI-CD16a and 1 × 104Individual 40 μ l Daudi are thin
Born of the same parents；By EM201 (CHO-K1-FS-/-Expression), EM201 (CHO-K1 expression), Gazyva (Genentech companies) be separately added into 96
In orifice plate, to sample final concentration 1 × 10-1~1 × 10-8 μ g/ml (10 times of gradients, totally 8 concentration points), 37 DEG C, 5%CO2Carefully
4h is cultivated in born of the same parents' incubator.OD values are read after being developed the color with lactate dehydrogenase L DH test agents box at ELIASA 492nm, target are calculated thin
Cellular lysate rate, using sample concentration as abscissa, target cell lysis rate is ordinate, quadruplex parameters Fitting Analysis.
EM201(CHO-K1-FS-/-Expression), EM201 (CHO-K1 expression) and Gazyva (Genentech companies) ADCC
Activity experiment result is as shown in figure 5, test result indicates that EM201 (CHO-K1-FS-/-Expression) ADCC activity is about the 4 of Gazyva
Times, the more notable ADCC activity (about 20 times) for being better than EM201 (CHO-K1 expression) shows that the anti-CD20 of no fucosylation resists
Body has higher ADCC efficiency than defucosylated antibody.
Curative effect of the anti-CD 20 antibodies of embodiment 11 to human B cell lymthoma Daudi nude mouse subcutaneous transplantation knurls
To assess the internal antitumor activity of anti-CD 20 antibodies, using BALB/cA-nude nude mouses, (6-7 weeks, ♀ was purchased from
Shanghai Ling Chang bio tech ltd) be host human tumour transplantation model.Nude mouse subcutaneous vaccination human B cell lymthoma
Daudi cells, after after tumour growth to 130-180mm3, animal is grouped at random.By blank control (PBS) and three kinds of anti-CD20
Antibody：EM201(CHO-K1-FS-/-Expression), EM201 (CHO-K1 expression) and Gazyva (Genentech companies) be administered respectively,
Three kinds of anti-CD 20 antibodies dosage regimens are 3mg/kg, 2 times a week, totally 4 times.2-3 knurl volume is surveyed weekly, is claimed mouse weight, is recorded number
According to.
As a result show：As shown in fig. 6, the gross tumor volume of blank control group mouse is significantly increased；Compared with control group, resist
CD20 three groups of antibody can significantly inhibit the growth of tumour；EM201(CHO-K1-FS-/-Expression) it is without core fucose
The anti-CD20 of sugar-type antibody；With EM201 (CHO-K1 expression) antibody based on the sugar-type with core fucose, compare, have
Antibody EM201 (the CHO-K1-FS of relatively low fucose content-/-Expression) and Gazyva (Genentech companies) have preferably
Suppress knurl effect, and tumor-bearing mice can be resistant to very well to the anti-CD 20 antibodies medicine for knocking out fucose, without diseases such as weight loss
Shape occurs.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>Jiangsu east antibiont Pharmaceutical Technology Co., Ltd
<120>A kind of genetically engineered cell system for producing no fucosylation albumen and its method for building up
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 449
<212> PRT
<213>People source
<400> 1
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser
20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 2
<211> 219
<212> PRT
<213>People source
<400> 2
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
85 90 95
Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 3
<211> 1098
<212> DNA
<213>Mouse source
<400> 3
atgaacaggg cgcctctgaa gcggtccagg atcctgcgca tggcgctgac tggaggctcc 60
actgcctctg aggaggcaga tgaggacagc aggaacaagc cgtttctgct gcgggcgctg 120
cagatcgcgc tggtcgtctc tctctactgg gtcacctcca tctccatggt attcctcaac 180
aagtacctgc tggacagccc ctccctgcag ctggataccc ctatcttcgt cactttctac 240
caatgcctgg tgacctctct gctgtgcaag ggcctcagca ctctggccac ctgctgccct 300
ggcaccgttg acttccccac cctgaacctg gaccttaagg tggcccgcag cgtgctgcca 360
ctgtcggtag tcttcattgg catgataagt ttcaataacc tctgcctcaa gtacgtaggg 420
gtggccttct acaacgtggg gcgctcgctc accaccgtgt tcaatgtgct tctgtcctac 480
ctgctgctca aacagaccac ttccttctat gccctgctca catgtggcat catcattggt 540
ggtttctggc tgggtataga ccaagaggga gctgagggca ccctgtccct cataggcacc 600
atcttcgggg tgctggccag cctctgcgtc tccctcaatg ccatctatac caagaaggtg 660
ctcccagcag tggacaacag catctggcgc ctaaccttct ataacaatgt caatgcctgt 720
gtgctcttct tgcccctgat ggttctgctg ggtgagctcc gtgccctcct tgactttgct 780
catctgtaca gtgcccactt ctggctcatg atgacgctgg gtggcctctt cggctttgcc 840
attggctatg tgacaggact gcagatcaaa ttcaccagtc ccctgaccca caatgtatca 900
ggcacagcca aggcctgtgc gcagacagtg ctggccgtgc tctactatga agagactaag 960
agcttcctgt ggtggacaag caacctgatg gtgctgggtg gctcctcagc ctatacctgg 1020
gtcaggggct gggagatgca gaagacccaa gaggacccca gctccaaaga gggtgagaag 1080
agtgctatca gggtgtga 1098
<210> 4
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 4
caccggcggt ccaggatcct gcgcangg 28
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
aaactgcgca ggatcctgga ccgcc 25
<210> 6
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 6
caccgatcct gcgcatggcg ctgacngg 28
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence
<400> 7
aaacgtcagc gccatgcgca ggatc 25
<210> 8
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 8
caccggcggg cgctgcagat cgcgcngg 28
<210> 9
<211> 25
<212> DNA
<213>Artificial sequence
<400> 9
aaacgcgcga tctgcagcgc ccgcc 25
<210> 10
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 10
caccgaacct ctgcctcaag tacgtngg 28
<210> 11
<211> 25
<212> DNA
<213>Artificial sequence
<400> 11
aaacacgtac ttgaggcaga ggttc 25
<210> 12
<211> 23
<212> DNA
<213>Artificial sequence
<400> 12
atgtgacaat tgaaggctgt acc 23
<210> 13
<211> 22
<212> DNA
<213>Artificial sequence
<400> 13
agaaggaagt ggtctgtttg ag 22
<210> 14
<211> 2823
<212> DNA
<213>Mouse source
<400> 14
ttcaggttgc tgctctggct taggccatct atgaccctgg tggtgttttc attcactata 60
agtccttccc atctttatta actgagcaag ttcagctagt aattttagag accgaggttc 120
aagcaataac acctatctct gcaataccgt gtggctttct tcaatgtctt acatcctaag 180
gaaaggaagc atgtagagcc caggaagcac aggacaagaa agctgcctcc ttgtatcacc 240
aggaagatct ttttgtaaga gtcatcacag taaccagaga gactaatttt gtctgaagca 300
tcatgtgttg aaacaacaga aacttatttt cctgtgtggc taactagaac cagagtacaa 360
tgtttccaat tctttgagct ccgagaagac agaagggagt tgaaactctg aaaatgcggg 420
catggactgg ttcctggcgt tggattatgc tcattctttt tgcctggggg accttattgt 480
tttatatagg tggtcatttg gttcgagata atgaccaccc tgaccattct agcagagaac 540
tctccaagat tcttgcaaag ctggagcgct taaaacaaca aaatgaagac ttgaggagaa 600
tggctgagtc tctccgaata ccagaaggcc ctattgatca ggggacagct acaggaagag 660
tccgtgtttt agaagaacag cttgttaagg ccaaagaaca gattgaaaat tacaagaaac 720
aagctaggaa tgatctggga aaggatcatg aaatcttaag gaggaggatt gaaaatggag 780
ctaaagagct ctggtttttt ctacaaagtg aattgaagaa attaaagaaa ttagaaggaa 840
acgaactcca aagacatgca gatgaaattc ttttggattt aggacatcat gaaaggtcta 900
tcatgacaga tctatactac ctcagtcaaa cagatggagc aggtgagtgg cgggaaaaag 960
aagccaaaga tctgacagag ctggtccagc ggagaataac atatctgcag aatcccaagg 1020
actgcagcaa agccagaaag ctggtatgta atatcaacaa aggctgtggc tatggatgtc 1080
aactccatca tgtggtttac tgcttcatga ttgcttatgg cacccagcga acactcatct 1140
tggaatctca gaattggcgc tatgctactg gaggatggga gactgtgttt agacctgtaa 1200
gtgagacatg cacagacagg tctggcctct ccactggaca ctggtcaggt gaagtgaagg 1260
acaaaaatgt tcaagtggtc gagctcccca ttgtagacag cctccatcct cgtcctcctt 1320
acttaccctt ggctgtacca gaagaccttg cagatcgact cctgagagtc catggtgatc 1380
ctgcagtgtg gtgggtatcc cagtttgtca aatacttgat ccgtccacaa ccttggctgg 1440
aaagggaaat agaagaaacc accaagaagc ttggcttcaa acatccagtt attggagtcc 1500
atgtcagacg cactgacaaa gtgggaacag aagcagcctt ccatcccatt gaggaataca 1560
tggtacacgt tgaagaacat tttcagcttc tcgaacgcag aatgaaagtg gataaaaaaa 1620
gagtgtatct ggccactgat gacccttctt tgttaaagga ggcaaagaca aagtactcca 1680
attatgaatt tattagtgat aactctattt cttggtcagc tggactacac aaccgataca 1740
cagaaaattc acttcggggc gtgatcctgg atatacactt tctctcccag gctgacttcc 1800
ttgtgtgtac tttttcatcc caggtctgta gggttgctta tgaaatcatg caaacactgc 1860
atcctgatgc ctctgcaaac ttccattctt tagatgacat ctactatttt ggaggccaaa 1920
atgcccacaa ccagattgca gtttatcctc accaacctcg aactaaagag gaaatcccca 1980
tggaacctgg agatatcatt ggtgtggctg gaaaccattg gaatggttac tctaaaggtg 2040
tcaacagaaa actaggaaaa acaggcctgt acccttccta caaagtccga gagaagatag 2100
aaacagtcaa ataccctaca tatcctgaag ctgaaaaata gagatggagt gtaagagatt 2160
aacaacagaa tttagttcag accatctcag ccaagcagaa gacccagact aacatatggt 2220
tcattgacag acatgctccg caccaagagc aagtgggaac cctcagatgc tgcactggtg 2280
gaacgcctct ttgtgaaggg ctgctgtgcc ctcaagccca tgcacagtaa aataatgtac 2340
tcacacataa catacaaatg gattattttc tactttgccc tttaaatatt ctgtccccat 2400
gaaacaaaca ctgccacatt atgtaattta agtgacacag acgttttgtg tgagacttca 2460
aacatggtgc ctatatctga gagacctctg tgatttactg agaagatgag aacagctccc 2520
ttctgtgggg aagttggttc ttagtcagtg gtggactggc cactgaattc actgcaatca 2580
acagattcag aatgagaatg gatgtttttc ctttatatgg ttgtctggat tttttttaaa 2640
gtaatttcat cagttcagtt catccacctc attaataaat gaaggaatat accaataaaa 2700
tcaaatgaaa tattcactgt ccattaggaa gttttataaa acaatgccat gaacaaaaaa 2760
ttctttagta ctcaatgttt ctggacattc tctttgataa caaaaataaa ttttaaaaag 2820
gaa 2823
<210> 15
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 15
caccggtcag acgcactgac aaagtngg 28
<210> 16
<211> 25
<212> DNA
<213>Artificial sequence
<400> 16
aaacactttg tcagtgcgtc tgacc 25
<210> 17
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 17
caccgggata aaaaaagagt gtatcngg 28
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence
<400> 18
aaacgataca ctcttttttt atccc 25
<210> 19
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 19
caccggtgta ccatgtattc ctcaangg 28
<210> 20
<211> 25
<212> DNA
<213>Artificial sequence
<400> 20
aaacttgagg aatacatggt acacc 25
<210> 21
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 21
caccggggac cttattgttt tatatngg 28
<210> 22
<211> 25
<212> DNA
<213>Artificial sequence
<400> 22
aaacatataa aacaataagg tcccc 25
<210> 23
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (26)..(26)
<223> n is a, c, g, or t
<400> 23
caccggtttt atataggtgg tcattngg 28
<210> 24
<211> 25
<212> DNA
<213>Artificial sequence
<400> 24
aaacaatgac cacctatata aaacc 25
<210> 25
<211> 22
<212> DNA
<213>Artificial sequence
<400> 25
gattccaggt tcccatatat tc 22
<210> 26
<211> 22
<212> DNA
<213>Artificial sequence
<400> 26
tgatgactgc tagtgatgct ac 22
<210> 27
<211> 21
<212> DNA
<213>Artificial sequence
<400> 27
tgtctgaagc atcatgtgtt g 21
<210> 28
<211> 22
<212> DNA
<213>Artificial sequence
<400> 28
acagtatttc atcaaatcct tg 22
<210> 29
<211> 1347
<212> DNA
<213>Artificial sequence
<400> 29
caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60
tcctgcaagg cctccggcta cgccttctcc tactcctgga tcaactgggt gaggcaggcc 120
cccggccagg gcctggagtg gatgggcagg atcttccccg gcgacggcga caccgactac 180
aacggcaagt tcaagggcag ggtgaccatc accgccgaca agtccacctc caccgcctac 240
atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggaacgtg 300
ttcgacggct actggctggt gtactggggc cagggcaccc tggtgaccgt gtcctccgcc 360
tccaccaagg gcccctccgt gttccccctg gccccctcct ccaagtccac ctccggcggc 420
accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480
aactccggcg ccctgacctc cggcgtgcac accttccccg ccgtgctgca gtcctccggc 540
ctgtactccc tgtcctccgt ggtgaccgtg ccctcctcct ccctgggcac ccagacctac 600
atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagaaggt ggagcccaag 660
tcctgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720
tccgtgttcc tgttcccccc caagcccaag gacaccctga tgatctccag gacccccgag 780
gtgacctgcg tggtggtgga cgtgtcccac gaggaccccg aggtgaagtt caactggtac 840
gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gtacaactcc 900
acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960
tacaagtgca aggtgtccaa caaggccctg cccgccccca tcgagaagac catctccaag 1020
gccaagggcc agcccaggga gccccaggtg tacaccctgc ccccctccag ggacgagctg 1080
accaagaacc aggtgtccct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 1140
gtggagtggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200
gactccgacg gctccttctt cctgtactcc aagctgaccg tggacaagtc caggtggcag 1260
cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320
aagtccctgt ccctgtcccc cggcaag 1347
<210> 30
<211> 657
<212> DNA
<213>Artificial sequence
<400> 30
gacatcgtga tgacccagac ccccctgtcc ctgcccgtga cccccggcga gcccgcctcc 60
atctcctgca ggtcctccaa gtccctgctg cactccaacg gcatcaccta cctgtactgg 120
tacctgcaga agcccggcca gtccccccag ctgctgatct accagatgtc caacctggtg 180
tccggcgtgc ccgacaggtt ctccggctcc ggctccggca ccgacttcac cctgaagatc 240
tccagggtgg aggccgagga cgtgggcgtg tactactgcg cccagaacct ggagctgccc 300
tacaccttcg gcggcggcac caaggtggag atcaagagga ccgtggccgc cccctccgtg 360
ttcatcttcc ccccctccga cgagcagctg aagtccggca ccgcctccgt ggtgtgcctg 420
ctgaacaact tctaccccag ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480
tccggcaact cccaggagtc cgtgaccgag caggactcca aggactccac ctactccctg 540
tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600
gtgacccacc agggcctgtc ctcccccgtg accaagtcct tcaacagggg cgagtgc 657
Claims (10)
1. a kind of genetically engineered cell system for producing no fucosylation albumen, it is characterised in that the genetically engineered cell system
It is the cell line of Slc35c1 gene silencings, and/or Fut8 gene silencings.
2. genetically engineered cell system according to claim 1, it is characterised in that the albumen is the albumen containing Fc areas.
3. genetically engineered cell system according to claim 2, it is characterised in that contain volume in the genetically engineered cell system
The nucleotide sequence of albumen of the code containing Fc areas.
4. Cas9 gene orders are contained in genetically engineered cell system according to claim 1, the genetically engineered cell system.
5. genetically engineered cell system according to claim 1, the genetically engineered cell system is contained for Slc35c1 genes
SgRNA gene order, and/or gene order containing the sgRNA for Fut8 genes.
6. the method for building up of the genetically engineered cell system any one of claim 1-5, it is characterised in that the foundation side
Method comprises the following steps：Knock out cell in Slc35c1 genes, and/or Fut8 genes, obtain Slc35c1 gene silencings and/
Or the cell line of Fut8 gene silencings.
7. method according to claim 6, it is characterised in that the method for building up comprises the following steps：Utilize CRISPR/
Cas9 technologies knock out the Slc35c1 genes, and/or Fut8 genes in cell, obtain Slc35c1 gene silencings, and/or Fut8 bases
Because of the cell line of silence.
8. a kind of albumen without fucosylation, it is characterised in that the albumen is as any one of claim 1-5
What genetically engineered cell system produced.
9. the preparation method of the albumen without fucosylation described in claim 8, it is characterised in that the preparation method includes
Following steps：
(1) method for building up according to claim 6 or 7 obtains Slc35c1 gene silencings, and/or Fut8 gene silencings
Genetically engineered cell system；
(2) in the genetically engineered cell system for obtaining the coded sequence steps for importing (1) of albumen.
10. the genetically engineered cell any one of claim 1-5, which is tied up to, prepares answering in the albumen without fucosylation
With.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710641279.5A CN107217042B (en) | 2017-07-31 | 2017-07-31 | Genetic engineering cell line for producing afucosylated protein and establishing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710641279.5A CN107217042B (en) | 2017-07-31 | 2017-07-31 | Genetic engineering cell line for producing afucosylated protein and establishing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107217042A true CN107217042A (en) | 2017-09-29 |
| CN107217042B CN107217042B (en) | 2020-03-06 |
Family
ID=59954978
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710641279.5A Active CN107217042B (en) | 2017-07-31 | 2017-07-31 | Genetic engineering cell line for producing afucosylated protein and establishing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107217042B (en) |
Cited By (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108138186A (en) * | 2016-08-31 | 2018-06-08 | 北京天广实生物技术股份有限公司 | Anti-CD 20 antibodies |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| CN110669770A (en) * | 2018-07-02 | 2020-01-10 | 盛禾(中国)生物制药有限公司 | Humanized monoclonal antibody, preparation method and application thereof |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN114457110A (en) * | 2020-11-10 | 2022-05-10 | 盛禾(中国)生物制药有限公司 | Method for knocking out FUT8 gene |
| CN114934053A (en) * | 2022-06-30 | 2022-08-23 | 澳斯康生物(南通)股份有限公司 | Fucosyltransferase 8-deficient CHO cell line and preparation method and application thereof |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US11999947B2 (en) | 2023-02-24 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104024404A (en) * | 2011-11-30 | 2014-09-03 | 分子生物技术院有限公司 | Haploid cells |
| CN105392885A (en) * | 2013-07-19 | 2016-03-09 | 赖瑞克斯生物科技公司 | Methods and compositions for producing double allele knock outs |
| WO2016196343A1 (en) * | 2015-05-29 | 2016-12-08 | Genentech, Inc. | Humanized anti-ebola virus glycoprotein antibodies and methods of use |
| CN106399360A (en) * | 2015-07-27 | 2017-02-15 | 上海药明生物技术有限公司 | FUT8 gene knockout method based on CRISPR technology |
| CN106701823A (en) * | 2017-01-18 | 2017-05-24 | 上海交通大学 | Establishment and application of CHO cell line for producing fucose-free monoclonal antibody |
-
2017
- 2017-07-31 CN CN201710641279.5A patent/CN107217042B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104024404A (en) * | 2011-11-30 | 2014-09-03 | 分子生物技术院有限公司 | Haploid cells |
| CN105392885A (en) * | 2013-07-19 | 2016-03-09 | 赖瑞克斯生物科技公司 | Methods and compositions for producing double allele knock outs |
| WO2016196343A1 (en) * | 2015-05-29 | 2016-12-08 | Genentech, Inc. | Humanized anti-ebola virus glycoprotein antibodies and methods of use |
| CN106399360A (en) * | 2015-07-27 | 2017-02-15 | 上海药明生物技术有限公司 | FUT8 gene knockout method based on CRISPR technology |
| CN106701823A (en) * | 2017-01-18 | 2017-05-24 | 上海交通大学 | Establishment and application of CHO cell line for producing fucose-free monoclonal antibody |
Non-Patent Citations (1)
| Title |
|---|
| KAH FAI CHAN等: "Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies", 《BIOTECHNOLOGY JOURNAL》 * |
Cited By (48)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11208492B2 (en) | 2016-08-31 | 2021-12-28 | Beijing Mabworks Biotech Co. Ltd. | Anti-CD20 antibody |
| CN108138186A (en) * | 2016-08-31 | 2018-06-08 | 北京天广实生物技术股份有限公司 | Anti-CD 20 antibodies |
| US11970541B2 (en) | 2016-08-31 | 2024-04-30 | Beijing Mabworks Biotech Co. Ltd. | Anti-CD20 antibody |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN110669770B (en) * | 2018-07-02 | 2022-07-12 | 盛禾(中国)生物制药有限公司 | Humanized monoclonal antibody, preparation method and application thereof |
| CN110669770A (en) * | 2018-07-02 | 2020-01-10 | 盛禾(中国)生物制药有限公司 | Humanized monoclonal antibody, preparation method and application thereof |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US12006520B2 (en) | 2019-06-14 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN114457110A (en) * | 2020-11-10 | 2022-05-10 | 盛禾(中国)生物制药有限公司 | Method for knocking out FUT8 gene |
| CN114934053B (en) * | 2022-06-30 | 2024-02-06 | 澳斯康生物(南通)股份有限公司 | Fucosyltransferase 8-defective CHO cell line and preparation method and application thereof |
| CN114934053A (en) * | 2022-06-30 | 2022-08-23 | 澳斯康生物(南通)股份有限公司 | Fucosyltransferase 8-deficient CHO cell line and preparation method and application thereof |
| US11999947B2 (en) | 2023-02-24 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107217042B (en) | 2020-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107217042A (en) | A kind of genetically engineered cell system for producing no fucosylation albumen and its method for building up | |
| US11060097B2 (en) | Immunotherapy methods using anti-PD-L1 antibodies in combination with EGFR1 targeted-TGF-beta immunomodulatory fusion proteins | |
| JP7262597B2 (en) | Bispecific antibodies and methods of making and using the same | |
| Al‐Shamkhani et al. | OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand | |
| CN102850458B (en) | Novel recombined dual-function fusion protein and its preparation method and application | |
| CN102939305B (en) | Antibody to CD122 | |
| US6949360B2 (en) | DNA coding for human cell surface antigen | |
| JP2019519223A (en) | Immune modulatory protein and bispecific binding protein binding to tumor antigens | |
| CN110267677A (en) | Use the Chimeric antigen receptor treating cancer combined with former M2 macrophage molecule inhibitor | |
| WO2018233574A1 (en) | Anti-pd-l1 humanized nanobody and use thereof | |
| CN107814845A (en) | The new nano antibodies of anti-PD 1 and its application | |
| CN102321173B (en) | Humanized macrophage inhibitory factor 1 monoclonal antibody and application thereof | |
| CN106573969A (en) | Drug related transgene expression | |
| CN106574272A (en) | Universal chimeric antigen receptor expressing immune cells for targeting of diverse multiple antigens and method of manufacturing the same and use of the same for treatment of cancer, infections and autoimmune disorders | |
| CN105461808A (en) | Monoclonal antibody and application thereof | |
| CN104788567B (en) | A kind of bispecific antibody preparation method and application of targeted mouse T lymphocyte CD3 and human tumor antigen EpCAM | |
| CN105622753B (en) | A kind of PD-1 monoclonal antibody and its application | |
| US20220348650A1 (en) | Anti-tigit immunosuppressant and application thereof | |
| CN108690138A (en) | It is a kind of can be with people CD19 or CD20 and people the CD3 bispecific antibody combined and its application | |
| JP2022528030A (en) | Multifunctional fusion protein and its use | |
| CN108864307A (en) | The Chimeric antigen receptor of signal peptide optimization targeting CD19, the T cell and preparation method and application for expressing the Chimeric antigen receptor | |
| CN108997497A (en) | The monoclonal antibody and the preparation method and application thereof of specific bond hostage's film film bubble related protein PV-1 | |
| CN104974262B (en) | Recombination double functions fusion protein and its preparation method and purposes | |
| CN106573978A (en) | Method for producing variants having an Fc with improved sialylation | |
| CN107840889A (en) | The anti-CD123 antibody of high-affinity and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wei Huawei Inventor after: Huang Yajie Inventor before: Zhao Jian Inventor before: Wei Huawei Inventor before: Huang Yajie |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |