WO2011136602A2 - Procédé pour sélectionner une lignée de cellules produisant des anticorps, et trousse correspondante - Google Patents

Procédé pour sélectionner une lignée de cellules produisant des anticorps, et trousse correspondante Download PDF

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WO2011136602A2
WO2011136602A2 PCT/KR2011/003189 KR2011003189W WO2011136602A2 WO 2011136602 A2 WO2011136602 A2 WO 2011136602A2 KR 2011003189 W KR2011003189 W KR 2011003189W WO 2011136602 A2 WO2011136602 A2 WO 2011136602A2
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fluorescent protein
antibody
sequence encoding
expression vector
fragment
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PCT/KR2011/003189
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Korean (ko)
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WO2011136602A3 (fr
WO2011136602A9 (fr
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이은교
이홍원
김연구
정준기
안정오
김천석
이혁원
이주환
이혜림
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한국생명공학연구원
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Priority to US13/643,952 priority Critical patent/US20130157262A1/en
Publication of WO2011136602A2 publication Critical patent/WO2011136602A2/fr
Publication of WO2011136602A9 publication Critical patent/WO2011136602A9/fr
Publication of WO2011136602A3 publication Critical patent/WO2011136602A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the present invention relates to a method for selecting antibody producing cell lines using split fluorescent protein and a selection kit for selecting antibody producing cell lines.
  • the selection of highly productive cell lines is an important step in the production of therapeutic antibodies using animal cell lines.
  • Conventionally known methods for screening high productivity animal cell lines include limiting dilution methods, gel microdrop technology, and automated machines.
  • the limiting dilution method is currently used most widely because of the simplicity and low cost of the method itself, but it is inefficient compared to other methods and is not suitable for high throughput screening (HTS).
  • HTS high throughput screening
  • the gel microdrop technique and the method of using an automated device have great advantages in HTS, but they have problems in public use due to the complexity and high cost of the method itself.
  • the gel microdrop technique requires a skilled process and has limitations such as low efficiency gel formation.
  • the relatively high cost of using an automated device is a big problem in use.
  • An object of the present invention is to provide a method for easily selecting antibody-producing cell lines using recombination force of a cleavage fluorescence protein, and a selection kit therefor.
  • the present invention provides a first expression vector comprising a sequence encoding a first fragment of a fluorescent protein and a sequence encoding a heavy chain of an antibody and a second fragment of the fluorescent protein. Transfecting the cell with a second expression vector comprising the sequence and the sequence encoding the light chain of the antibody; And it provides a method for screening antibody-producing cell lines comprising the step of selecting the antibody-producing cell line by identifying the fluorescence shown by the recombination of the first and second fragments of the fluorescent protein.
  • the present invention also provides a first expression vector comprising a sequence encoding a first fragment of a fluorescent protein and an introduction region into which a sequence encoding a heavy chain of an antibody can be introduced; And a second expression vector comprising a sequence encoding a second fragment of the fluorescent protein and an introduction region into which a sequence encoding a light chain of the antibody can be introduced.
  • cleavage fluoroproteins in the selection of antibody-producing cell lines according to the present invention, it is possible to easily detect antibody-producing cell lines only by observing the expression of a single fluorescence color by recombination of cleavage fluorescence proteins.
  • the screening time and screening cost of the production cell line can be significantly reduced.
  • GFP green fluorescent protein
  • Figure 2 is a view showing the results observed by confocal microscopy of the formation of recombination GFP through the co-expression of expression vectors pcDNA-NGFP and pcDNA-CGFP.
  • Figure 3 is a structure for the structure of pCGFP-Heavy, a vector which simultaneously expresses the N-terminal fragment of GFP and the light chain structure of the antibody, pNGFP-Light, and the C-terminal fragment of GFP and the heavy chain structure of the antibody It is a schematic diagram.
  • Figure 4A is a view showing the results observed through the confocal microscope to form the recombination GFP through the co-expression of the expression vectors pNGFP-Light and pCGFP-Heavy according to the present invention.
  • Figure 4B is a view showing the result of measuring the amount of antibody generated through the co-expression of the expression vector pNGFP-Light and pCGFP-Heavy in accordance with the present invention by enzyme immunoassay.
  • FIG. 5 is a primary cell established by isolating individual cells (high 1%) expressing GFP in a CHO cell line (non-separated pool) coexpressing the expression vectors pNGFP-Light and pCGFP-Heavy according to the present invention by flow cytometry. It is a graph showing GFP expressing cell ratio and antibody specific productivity of the separation pool and the secondary separation pool.
  • FIG. 6 is a graph showing the correlation between GFP expression and antibody specific productivity for 30 individual cell lines established according to the present invention.
  • the present invention provides a first expression vector comprising a sequence encoding a first fragment of a fluorescent protein and a sequence encoding a heavy chain of the antibody, and a sequence encoding a second fragment of the fluorescent protein and a light chain of the antibody. transfecting a cell with a second expression vector comprising a sequence encoding a chain); And it provides a method for screening antibody-producing cell lines comprising the step of selecting the antibody-producing cell line by identifying the fluorescence shown by the recombination of the first and second fragments of the fluorescent protein.
  • the fluorescent protein is, for example, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescence Proteins (yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) or enhanced fluorescent protein (EFP)) may be, but not limited to, fluorescent proteins that fluoresce by stimulation of light Anything can be used.
  • the fluorescent protein fragment according to the present invention means split fluorescent protein, and the split fluorescent protein loses the ability to fluoresce when the fluorescent protein is cut into a plurality of fragments, and the plurality of fragments recombine. (reassembly) refers to a fragment of the fluorescent protein that restores its ability to fluoresce again.
  • reassembly means that the fluorescent protein fragments that have lost the ability to generate fluorescence bind to each other to form a fluorescent protein having recovered fluorescence.
  • a first expression vector comprising a sequence encoding a first fragment of a fluorescent protein and a sequence encoding a heavy chain of the antibody and a sequence encoding a second fragment of the fluorescent protein and a light chain of the antibody
  • a second expression vector comprising a sequence encoding a light chain simultaneously expresses the first fragment of the fluorescent protein and the heavy chain of the antibody and simultaneously expresses the second fragment of the fluorescent protein and the light chain of the antibody.
  • the ratio of the heavy and light chains of the antibody to form an antibody protein and the first and second fragments of the fluorescent protein recombine and fluorescence Since the ratio of the ratio is proportional, the amount of fluorescence represented by the recombined fluorescent protein can be used as a marker indicating the amount of antibody produced. That is, by confirming the amount of fluorescence caused by the recombination of the first and second fragments of the fluorescent protein, it is possible to easily select a highly productive antibody-producing cell line with a large amount of fluorescence. At this time, the cell line may be selected using any method known in the art, for example, fluorescence activated cell sorting (FACS) may be used.
  • FACS fluorescence activated cell sorting
  • any one of the first and second fragments of the fluorescent protein may be a C-terminal fragment of the fluorescent protein, the other may be an N-terminal fragment of the fluorescent protein. That is, if the first fragment of the fluorescent protein is a C terminal fragment of the fluorescent protein, the second fragment of the fluorescent protein is the N-terminal fragment of the fluorescent protein, and if the first fragment of the fluorescent protein is the N-terminal fragment of the fluorescent protein, The two fragments are the C terminal fragments of the fluorescent protein.
  • the cleavage site in the full-length fluorescence protein for producing the first and second fragments of the fluorescence protein the fluorophore is preserved in any one fragment of the fluorescence protein, so that the fluorescence by recombination of each fragment It can be appropriately selected by those skilled in the art as long as the ability to regenerate can be restored, and some sequences can be inserted or deleted in the full-length fluorescent protein sequence.
  • the cleavage site for generating the first and second fragments of GFP may be between the 157 and 158 amino acid residues of full length GFP (US Pat. No. 6,780,599). Reference), but is not limited thereto.
  • the sequence of SEQ ID NO: 5 was used as the sequence encoding the first fluorescent protein fragment
  • the sequence of SEQ ID NO: 11 was used as the sequence encoding the second fluorescent protein fragment.
  • the first expression vector further comprises a sequence encoding a first linker peptide linked to a sequence encoding a first fragment of the fluorescent protein, wherein the second expression vector is a vector of the fluorescent protein. And further comprising a sequence encoding a second linker peptide linked to a sequence encoding a two segment, wherein the first linker peptide and the second linker peptide may be configured to bind to each other.
  • an "expression vector” refers to a DNA construct comprising an external DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host.
  • Expression vectors of the invention can typically be constructed as a vector for expression.
  • the expression vector of the present invention is a vector for expressing a recombinant peptide or protein.
  • the expression vector of the present invention can be constructed using prokaryotic or eukaryotic cells as host cells.
  • the recombinant expression vector of the present invention may be, for example, a bacteriophage vector, cosmid vector, YAC (Yeast Artificial Chromosome) vector and the like.
  • plasmid vectors it is preferred to use plasmid vectors.
  • Typical plasmid vectors that can be used for such purposes include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with plasmid vectors to be selected. It has a structure comprising a marker gene and a restriction enzyme cleavage site (c) can be inserted into foreign DNA fragments. Although no suitable restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA. Expression vectors used in the present invention can be constructed through a variety of methods known in the art.
  • a sequence encoding a first segment of a fluorescent protein in a first expression vector according to the present invention A sequence encoding a first linker peptide linked to said sequence; And a sequence encoding a heavy chain of the antibody is operably linked, the sequence encoding a second fragment of a fluorescent protein in the second expression vector; A sequence encoding a second linker peptide linked to said sequence; And sequences encoding the light chain of the antibody are also operably linked.
  • “Operably linked” means that the functional binding between a nucleic acid expression control sequence (eg, an array of promoter, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence (eg, a sequence encoding a fluorescent protein fragment) Wherein the regulatory sequence controls the transcriptional and / or translational process of the other nucleic acid sequence.
  • a nucleic acid expression control sequence eg, an array of promoter, signal sequences, or transcriptional regulator binding sites
  • another nucleic acid sequence eg, a sequence encoding a fluorescent protein fragment
  • the first linker peptide and the second linker peptide are configured to bind to each other.
  • the sequences encoding the first linker peptide and the second linker peptide are directly bound to the sequences encoding the first and second fragments of the fluorescent protein, respectively, so that when the protein is expressed, the first linker peptide and the first fragment of the fluorescent protein and The second linker peptide and the second fragment of the fluorescent protein are each expressed in the form of a fusion protein.
  • the first and second fragments of the fluorescent protein recombine close to each other to restore the fluorescence property.
  • the linker peptide may be a leucine zipper, for example as shown in US Pat. No. 6,780,599, and described in Chen, N. et al. J. Biotechnol., 2009, 142, 205-213 and Lindman, S. et al. Protein Sci., 2009, 18, 1221-1229, but may be an EF1 / EF2 peptide, but is not limited thereto.
  • the EF1 sequence was used as the first linker peptide sequence
  • the EF2 sequence was used as the second linker peptide sequence.
  • a vector for simultaneously expressing a fluorescent protein fragment and a heavy or light chain of an antibody in a vector may be appropriately implemented by those skilled in the art.
  • an expression vector of a bicystronic mode may be used.
  • internal ribosome entry sites (IRS) inside the pIRES expression vector may be used, but are not limited thereto.
  • an internal ribosome entry site (IRES) sequence between the sequence encoding the first fragment of the fluorescent protein of the first expression vector and the introduction region into which the heavy chain sequence of the antibody is introduced; And an internal ribosome entry site (IRES) sequence between the sequence encoding the second fragment of the fluorescent protein of the second expression vector and the introduction region into which the light chain sequence of the antibody is introduced.
  • IRES internal ribosome entry site
  • the antibody producing cell lines usable in the present invention are generally animal cell lines but are not particularly limited and may be E. coli, yeast or plant cell lines.
  • the cell line is an animal cell line
  • the animal cell may be, for example, HEK293, COS7, HeLa, CHO cells, but is not limited thereto.
  • the method for screening an antibody-producing cell line may further comprise the step of confirming whether the antibody-producing cell line has been produced by the method described above.
  • This step is an additional step to verify the correlation between the amount of fluorescence generated by recombination of the first and second fragments of the fluorescent protein and the amount of antibody produced by the binding of the heavy and light chains of the antibody.
  • the antibody-producing cell line can be accurately selected by secondarily verifying the actual antibody-producing ability of the selected antibody-producing cell line.
  • the method for determining whether the antibody-producing cell line is produced in the antibody may be used by any method known in the art, for example, it may be confirmed by an enzyme immunoassay.
  • the present invention also provides a first expression vector comprising a sequence encoding a first fragment of a fluorescent protein and an introduction region into which a sequence encoding a heavy chain of an antibody can be introduced; And a second expression vector comprising a sequence encoding a second fragment of the fluorescent protein and an introduction region into which a sequence encoding a light chain of the antibody can be introduced.
  • the "introduction region into which the sequence encoding the heavy chain of the antibody can be introduced” or the “introduction region into which the sequence encoding the light chain of the antibody can be introduced” in the second expression vector are to be produced, respectively. It refers to a region containing a restriction enzyme cleavage site into which foreign DNA fragments encoding the heavy or light chain of the antibody of interest can be inserted. Alternatively, when no appropriate restriction enzyme cleavage site is present, a vector and a foreign DNA fragment can be easily ligation using a synthetic oligonucleotide adapter or linker according to a conventional method. It means the area consisting of. The specific configuration of the introduction region into which such foreign DNA fragments can be introduced can be appropriately constructed by those skilled in the art.
  • the antibody cell line selection kit of the present invention comprising a first expression vector and a second expression vector, each containing an introduction region into which a sequence encoding a heavy or light chain of an antibody can be introduced, the heavy and light chains of the desired antibody
  • a sequence encoding each into the introduction region an expression vector for selecting a cell line producing the target antibody can be easily and quickly prepared, thereby easily selecting a cell line producing the target antibody.
  • Antibody-producing cell line selection kit is transfection for introducing the expression vector prepared in the restriction enzyme, primer, etc. for inserting the target antibody gene in the expression vector in addition to the first expression vector and the second expression vector It may include all biological or chemical reagents, instructions for use, etc., for the selection of antibody producing cell lines, including preparations, parent cell lines, and the like. Other configurations of such kits may be appropriately selected by those skilled in the art.
  • the fluorescent protein is a green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP; cyan fluorescent protein) Or enhanced fluorescent protein (EFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • BFP blue fluorescent protein
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • EFP enhanced fluorescent protein
  • any one of the first and second fragments of the fluorescent protein may be a C-terminal fragment of the fluorescent protein, and the other may be an N-terminal fragment of the fluorescent protein.
  • the first expression vector further comprises a sequence encoding a first linker peptide linked to a sequence encoding a first fragment of the fluorescent protein, wherein the second expression vector is a fluorescent protein And further comprising a sequence encoding a second linker peptide linked to a sequence encoding a second segment, wherein the first linker peptide and the second linker peptide may be configured to bind to each other.
  • an internal ribosome entry site (IRES) sequence between the sequence encoding the first fragment of the fluorescent protein of the first expression vector and the region encoding the sequence encoding the heavy chain of the antibody may be introduced; And an internal ribosome entry site (IRES) sequence between the sequence encoding the second fragment of the fluorescent protein of the second expression vector and the introduction region into which the sequence encoding the light chain of the antibody can be introduced.
  • IRS internal ribosome entry site
  • the characteristics of the first expression vector and the second expression vector included in the selection kit, the heavy chain of any antibody instead of including a sequence encoding a heavy or light chain of a specific antibody includes all of the characteristics of the first expression vector and the second expression vector previously used in the method for screening an antibody-producing cell line, except that the sequence encoding the light chain includes an introduction region into which it can be introduced. Detailed descriptions are omitted to avoid explanation.
  • Example 1 Preparation of expression vectors pcDNA-NGFP and pcDNA-CGFP
  • pEGFP-C1 green fluorescent protein
  • the linking protein of the terminal fragment was synthesized by DNA from Bioneer Corporation.
  • pEGFP-C1 was used as a template with F1 primer (SEQ ID NO: 1) and R1 primer (SEQ ID NO: 2) for 1 minute 30 seconds at 92 ° C and 1 minute 30 at 55 ° C.
  • Amplification was carried out by performing a total of 25 PCR reactions for 2 minutes at 72 ° C for 2 seconds.
  • the amplified N terminal fragment DNA was named Seq-1 DNA (SEQ ID NO: 5).
  • the portion corresponding to Seq-2 DNA (SEQ ID NO: 6) for synthesizing a linker peptide consisting of an EF1 sequence and a restriction enzyme site was synthesized by Bioneer Corporation.
  • the two DNA conjugates were used as templates, using F2 primer (SEQ ID NO: 3) and R2 primer (SEQ ID NO: 4) for 1 minute and 30 seconds at 92 ° C, 2 minutes at 50 ° C, and Amplification was performed by a total of 35 PCR reactions at 72 ° C. for 2 minutes.
  • the fragments were digested with restriction enzymes HindIII and NotI and then inserted into the expression vector pcDNA3.1 / Zeo vector (Invitrogen), which were digested with the same enzyme, and named as the expression vector pcDNA-NGFP.
  • pEGFP-C1 was used as a template using F3 primer (SEQ ID NO: 7) and R3 primer (SEQ ID NO: 8) for 1 minute 30 seconds at 92 ° C and 1 minute 30 at 55 ° C. Amplification was carried out by a total of 25 PCR reactions for 2 minutes at 72 ° C for 2 seconds.
  • the amplified C terminal fragment DNA was named Seq-3 DNA (SEQ ID NO: 11).
  • the part corresponding to Seq-4 DNA (SEQ ID NO: 12) for synthesizing a linker peptide consisting of an EF2 sequence and a restriction enzyme site was synthesized by Bioneer Corporation.
  • the two DNA conjugates were used as templates, using F4 primer (SEQ ID NO: 9) and R4 primer (SEQ ID NO: 10) for 1 minute and 30 seconds at 92 ° C, 2 minutes at 50 ° C, and Amplification was performed by a total of 35 PCR reactions at 72 ° C. for 2 minutes.
  • the fragments were digested with restriction enzymes HindIII and NotI and then inserted into the expression vector pcDNA3.1 / Zeo vector, which was digested with the same enzyme, and named as expression vector pcDNA-CGFP.
  • Example 2 Formation of Recombinant Fluorescent Protein through Co-Expression of Expression Vector pcDNA-NGFP and pcDNA-CGFP and Fluorescence Observation through Confocal Microscopy
  • PCDNA-NGFP and pcDNA-CGFP were used to determine whether fluorescence was induced by forming recombined GFP in cells using vectors expressing the N- and C-terminal fragments of the green fluorescent protein prepared in Example 1, respectively.
  • HEK293 cells were passaged in animal cell medium DMEM (HyClone) added with 10% FBS (Invitrogen) and 4 mM glutamine (Sigma). Covers attachable to HEK293 cells were attached to the bottom of a 12-well plate (Nunc), and then HEK293 cells were incubated for 12 hours. Next, 0.5 mg vector was transfected into cells using 1.5 mL transfection solution (Stratagene).
  • pEGFP-C1 and pcDNA3.1 / Zeo which are GFP-expressing vectors
  • pcDNA-NGFP and pcDNA-CGFP which are vectors expressing N- and C-terminal fragments of the fluorescent protein
  • pcDNA-NGFP and pcDNA-CGFP were mixed and transfected. Transfected cells were further incubated at 30 ° C. for 24 hours. Cultures were removed from 12-well plates to observe fluorescence of recombined GFP with confocal microcsopy.
  • Transfected HEK293 cells on the coverslip attached to the bottom were washed once with PBS solution and then fixed for 10 minutes with PBS solution containing 2% paraformaldehyde.
  • the immobilized cells were washed twice with PBS solution and then treated with a mounting solution containing 4,6-diimidino-2-phenylindole (DAPI) capable of nuclear staining.
  • the coverslips to which the finally transfected HEK293 cells were attached were observed using a confocal microscope (Zeiss Corporation).
  • the light chain structure of the model antibody provided by Paweng Shinsa Co., Ltd. to insert the light chain structure of the antibody at the multi cloning site (MCS) -A position of pIRES (Clontech), a vector in which two genes are co-expressed.
  • MCS multi cloning site
  • pIRES pIRES
  • F5 primer SEQ ID NO: 13
  • R5 primer SEQ ID NO: 14
  • 25 PCR reactions were performed and amplified.
  • the amplified DNA was digested with restriction enzymes NheI and MluI, and then inserted into pIRES, an expression vector digested with the same enzyme, and named as the expression vector pIRES-Light.
  • N-terminal fragment of the fluorescent protein was prepared by using the pcDNA-NGFP prepared in Example 1 as a template using F6 primer (SEQ ID NO: 15) and R6 primer (SEQ ID NO: 16) for 1 minute 30 seconds at 92 °C, 1 minute at 55 °C Amplification was performed a total of 25 PCR reactions for 30 seconds and at 72 ° C. for 1 minute.
  • the amplified DNA was digested with restriction enzymes XbaI and SalI and then inserted into MCS-B of pIRES-Light, an expression vector digested with the same enzyme, and named as expression vector pNGFP-Light (FIG. 3).
  • F7 primer SEQ ID NO: 17
  • R7 primer SEQ ID NO: 18
  • the amplified DNA was digested with restriction enzyme EcoRI and then inserted into pIRES, an expression vector digested with the same enzyme, and named as the expression vector pIRES-Heavy.
  • pNGFP-Light and pCGFP-Heavy prepared in Example 3 generate two units of an antibody, a heavy chain structure and a light chain structure, an N-terminal fragment and a C-terminal fragment of the fluorescent protein, respectively.
  • pNGFP-Light and pCGFP-Heavy were mixed in equal amounts to transfect HEK293 cells.
  • HEK293 cells were passaged in animal cell medium DMEM supplemented with 10% FBS and 4 mM glutamine. After transfection as described in Example 3, HEK293 cell cultures were centrifuged (21,000 ⁇ g, 20 minutes, 4 ° C.) for enzyme immunoassay and then stored at ⁇ 80 ° C. Coverslips to which the transfected HEK293 cells were attached were subjected to an immobilization step as in Example 2, and fluorescence of recombined GFP was observed by confocal microscopy.
  • PBST solution which is a PBS solution containing 0.05% Tween20
  • the culture medium and the standard antibody were loaded on a plate and reacted at 37 ° C for 1 hour.
  • the plate was washed three times with PBST solution again and reacted with alkaline phosphatase-bound goat anti-human IgG (Alkaline phosphatase-conjugated goat anti-human IgG, Pierce) for 1 hour at 37 ° C.
  • TMB solution (BD biosciences) was added as a substrate. After completion of the reaction using sulfuric acid, the absorbance was measured at 450 nm on a multipurpose plate reader (BioTek).
  • HEK293 cells produced antibodies when the expression vectors pNGFP-Light and pCGFP-Heavy were mixed and transfected.
  • Example 5 Isolation and antibody production verification of individual cells expressing GFP using a flow cytometer
  • the expression vector pNGFP-Light prepared in Example 3 and pCGFP-Heavy was transfected into Chinese hamster ovay-K1 (hereinafter referred to as CHO-K1) cells and passaged three times in animal cell medium IMDM with an additional 10% FBS, 4 mM glutamine and 500 ⁇ g / mL G418. It was named non-separating pool.
  • the cells were cultured at 30 ° C. for 2 days and then verified by flow cytometry.
  • the cell line of each pool was inoculated in a T-25 flask at a concentration of 0.7 10 5 cells / mL, and then the total number of viable cells was measured on days 2 and 4 of the culture at 37 ° C., respectively, and the antibody produced by enzyme immunoassay was measured. Specific antibody productivity was then measured.
  • the ratio of cells including the recombined GFP in the total cell number of each pool increased after separation through flow cytometry.
  • pools with increased proportion of cells containing recombined GFP showed higher specific productivity.
  • the limiting dilution method in the non-separation pool and the secondary separation pool established in Example 5 was used. Dog clones were randomly selected. After inoculation at a concentration of 1 cell / 3 well in a 96-well plate, the culture was expanded to a T-25 flask through subculture, and each clone was inoculated into a T-25 flask at a concentration of 3.0 10 5 cells / mL. After culturing at 37 ° C. for 3 days, the antibody produced by enzyme immunoassay was measured.
  • Non-separating pool Secondary separation pool 0.5 or less 115/116 33/116 More than 0.5 less than 1.0 1/116 1/116 1.0 or more and 5.0 or less 0/116 28/116 5.0 or more and 10.0 or less 0/116 50/116 Greater than 10.0 0/116 4/116
  • GFP expression level was measured by flow cytometry after inoculating each clone into a T-25 flask at a concentration of 0.7 10 5 cells / mL, incubating for 3 days at 37 ° C, and then inducing GFP recombination through additional culture for 2 days at 30 ° C.
  • GFP mean values were measured using a flow cytometer.
  • Determination of antibody specific productivity by enzyme-immunoassay was performed by inoculating each clone into a T-25 flask at a concentration of 0.7 10 5 cells / mL, and then measuring the total number of viable cells on days 2 and 4 of the culture at 37 ° C, respectively. After measuring the antibody produced by the enzyme immunoassay, the specific productivity was measured. The correlation between antibody specific productivity and GFP mean values for each clone was plotted.

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Abstract

La présente invention concerne un procédé de sélection d'une lignée de cellules produisant des anticorps en utilisant une protéine fluorescente clivée, ainsi qu'une trousse pour sélectionner une lignée de cellules produisant des anticorps. Selon l'invention, l'utilisation d'une protéine fluorescente clivée lors de la sélection d'une lignée de cellules produisant des anticorps permet de détecter facilement une lignée de cellules produisant des anticorps en observant simplement l'expression d'une seule couleur fluorescente grâce au réassemblage d'une protéine fluorescente clivée, ce qui a l'avantage de gagner énormément de temps et de réduire considérablement le coût de la sélection d'une lignée de cellules produisant des anticorps, avec en plus une productivité élevée.
PCT/KR2011/003189 2010-04-29 2011-04-29 Procédé pour sélectionner une lignée de cellules produisant des anticorps, et trousse correspondante WO2011136602A2 (fr)

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KR1020110039550A KR101272817B1 (ko) 2010-04-29 2011-04-27 항체 생산 세포주 선별 방법 및 선별 키트
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