WO2018113753A1 - Complexe et procédé d'inactivation du gène fut8 - Google Patents

Complexe et procédé d'inactivation du gène fut8 Download PDF

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WO2018113753A1
WO2018113753A1 PCT/CN2017/117814 CN2017117814W WO2018113753A1 WO 2018113753 A1 WO2018113753 A1 WO 2018113753A1 CN 2017117814 W CN2017117814 W CN 2017117814W WO 2018113753 A1 WO2018113753 A1 WO 2018113753A1
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cell
cells
target gene
sequencing
sequence
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吴海祥
秦民民
林小花
苏艳芳
吕亚楠
崔笑添
周桢昊
宋利平
龙凯
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上海津曼特生物科技有限公司
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Priority to CN201780068144.2A priority Critical patent/CN109963946B/zh
Publication of WO2018113753A1 publication Critical patent/WO2018113753A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of genetic engineering, cell culture, and protein production, and in particular to complexes and methods for inactivating the Fut8 gene.
  • ADCC complement dependent cytotoxicity
  • Fc antibody constant
  • NK cells recognize the antibody constant (Fc) region primarily via interaction with the NK cell Fc ⁇ RIII receptor. Then, NK cells deposit cytotoxic substances such as perforin and granzyme on the surface of target cells, thereby inducing cell lysis and apoptosis.
  • TALEN Three major gene editing technologies, TALEN, ZFN, and CRISPR/Cas, have been reported in the literature to knock out the Fut8 gene in CHO cells using ZFN, CRISPR/Cas, and use this knockout engineered cell line to produce a non-fucoid Glycosylated monoclonal antibody. There are no reports on the use of TALEN technology to knock out the Fut8 gene in CHO cells.
  • TALEN Transcription Activator-Like Effector Nuclease
  • the three core components of TALEN technology are the nuclear localization signal, the TALEN arm responsible for DNA recognition, and the cleavage domain (Fok I) of the artificially engineered endonuclease responsible for genome cleavage. The combination of the three functions to modify the genome of the cell.
  • TALEN's DNA recognition domain consists of a number of very conserved repeat amino acid sequence modules, each consisting of 34 amino acid residues, of which the 12 and 13 amino acid residues are variable residues to identify the target site. .
  • the target gene DNA can be specifically cleaved.
  • a non-homologous end joining that is prone to error is stimulated by inducing a DNA double-strand break or homologous directed repair at the location of a particular gene.
  • the length of specific DNA sequences recognized by different types of TALEN elements is quite different.
  • the specific DNA sequence recognized by the native TALEN element is generally 17-18 bp in length; whereas the specific DNA sequence recognized by the artificial TALEN element is typically 14-20 bp in length.
  • the amino acid residues at positions 12 and 13 of the DNA-specific recognition domain repeat amino acid sequence module constitute a di-amino acid, and the di-amino acid has a one-to-one correspondence with the four nucleotide bases of AGCT: adenine (A) NI (asparagine-isoleucine) recognition, thymine (T) recognized by NG (asparagine-glycine), guanine (G) recognized by NN (asparagine-asparagine), and cytosine (C) is recognized by HD (histidine-aspartic acid) (Katsuyama T et al (2013), An efficient strategy for TALEN-mediated genome engineering in Drosophila, Nucl.
  • a eukaryotic expression vector system dedicated to the construction of TALEN can be used, and a pair of specific TAL target recognition modules can be cloned into the vector, and then introduced into the cell by transfection, thereby realizing the target sequence. Transformation.
  • the efficiency of gene knockout using mammalian cell genome is generally not high, and the successful simultaneous knockout of the biallelic gene of the target gene is less efficient, requiring a convenient, direct, and efficient screening method from the candidate cell pool.
  • a number of candidate cells were screened for cells lacking FUT8 function.
  • the use of TALEN technology to knock out the Fut8 gene of CHO cells also has technical problems such as high difficulty in screening technology, large workload and unpredictable test results.
  • the most commonly used screening method for cell selection is functional screening.
  • the FUT8 protein encoded by the Fut8 gene is ⁇ -1,6 rock. Alginyl transferase.
  • the method can screen for a double knockout Fut8 gene clone within 3 weeks with a screening efficiency of 5%.
  • the inventors used the concentration of 50 ⁇ g/mL LCA reported in the literature to help screen cell clones, and then cloned the cells in a medium containing 50 ⁇ g/mL LCA for gradual amplification and phenotypic screening, after 9 days. After the culture, the cells grew normally, but no mutation was found in the genome sequencing.
  • the LCA lectin-based phenotypic screening method has the following defects: on the one hand, the cost of LCA is too high, about 5 mg takes about 3,000 yuan, and the delivery period is long and the supply is slow; on the other hand, there is no experiment.
  • the selection advantage of the mutant cells was found, and the analysis may need to further increase the LCA screening concentration based on the concentration of 50 ⁇ g/mL, which further highlights the cost disadvantage of the method.
  • the mis-matching assay can also be used for gene knockout assays.
  • Commercial products produced by Integrated DNA Technologies Mutation Detection Kit for Standard Gel Electrophoresis can detect genomic mutations, and after PCR amplification of the mutated region, through the renaturation step, local incomplete pairing is generated in the region of the mutated sequence, and then these regions are found and cleaved by non-completely paired enzymes in DNA.
  • a DNA band of the size of the target fragment and a band to be cleaved are found to find the mutant clone.
  • the cost of the kit is about 100 yuan/reaction, the delivery period is long, and the cost and time cannot be optimized. At the same time, the process of incompletely paired digestion and electrophoresis separation of small fragments of the enzyme is easy to introduce human error and influence the result judgment. .
  • the present invention utilizes the TALEN technology to perform site-directed modification (knockout) of the Fut8 gene to obtain a cell line lacking the function of ⁇ -1,6-fucosyltransferase; sequencing by genomic DNA
  • the method of direct analysis of the sequence of Fut8 gene was used to screen the mutant cell lines to obtain the FUT8 double knockout monoclonal cells.
  • the direct analysis method of genome sequencing was used to investigate the passage stability of monoclonal cells in the process of continuous passage, so as to obtain stable passage. Monoclonal cells.
  • the invention provides a method for producing a biallelic knockout of a target gene in a eukaryotic cell, comprising: providing a TALEN plasmid pair to a cell, the TALEN plasmid pair recognition sequence for identifying a target gene, a target gene
  • the cleavage site is the sequence of the target gene, and the cleavage site is preferably atctggccactgatg (SEQ ID NO. 16); the cell is expressed to express the TALEN protein, the TALEN protein recognizes the target gene and cleaves the sequence of the target gene Sequencing screening of eukaryotic cells in which the target gene undergoes biallelic knockout.
  • the eukaryotic cell is a mammalian cell. In yet another embodiment, the eukaryotic cell is a CHO-K1, CHO-S, CHOK1SV, DG44, DXB11, NS0, SP2/0, PER.C6 or HEK293 cell.
  • the target gene is a fucosyltransferase, optionally a Fut8 gene, preferably the cleavage site is on Exon7 of the Fut8 gene.
  • the invention provides a mammalian cell that is subjected to a biallelic knockout of a target gene using a method of biallelic knockout of the above target gene.
  • the target gene is a fucosyltransferase, optionally a Fut8 gene, preferably the cleavage site is on Exon7 of the Fut8 gene, preferably at atctggccactgatg (SEQ ID NO. 16) .
  • the present invention provides a method of producing a recombinant protein of interest in a host cell, the method comprising: providing a TALEN plasmid pair to a cell, the recognition sequence for identifying a target gene, and the corresponding cleavage site is the target gene a sequence, preferably a cleavage site of atctggccactgatg (SEQ ID NO. 16); causing the cell to express a TALEN protein, the TALEN protein recognizing the target gene and cleaving the sequence of the target gene; sequencing screening
  • the target gene undergoes biallelic knockout of eukaryotic cells; the cells are expressed to express the recombinant protein of interest.
  • the target gene is a fucosyltransferase, optionally a Fut8 gene, preferably the cleavage site is on Exon7 of the Fut8 gene.
  • the present invention provides a double-stranded polynucleotide comprising a nuclease target site comprising: a TALEN plasmid pair, the recognition sequence for identifying a target gene, and the corresponding cleavage site being the sequence of the target gene,
  • the cleavage site is preferably atctggccactgatg (SEQ ID NO. 16).
  • the target gene is a fucosyltransferase, optionally a Fut8 gene, preferably the cleavage site is on Exon7 of the Fut8 gene.
  • the invention provides the use of TALEN technology for the production of a biallelic knockout of a target gene in a eukaryotic cell, wherein the target gene is subjected to a biallelic knockout of a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • the eukaryotic cell is a CHO-K1, CHO-S, CHOK1SV, DG44, DXB11, NS0, SP2/0, PER.C6 or HEK293 cell.
  • the target gene is a fucosyltransferase, optionally a Fut8 gene, preferably the cleavage site is on Exon7 of the Fut8 gene.
  • the sequencing is a direct DNA sequencing method.
  • the screening comprises LCA-based phenotypic screening and/or DNA direct sequencing screening.
  • the choice of target sequence has an impact on the success rate of gene knockout, and different targets will produce different results.
  • the present invention selects the region 403-416 (located in Exon7) as a cleavage site in the three-stage sequence of the reported FUT8 enzyme (primary structure amino acid residue sequence 358-370, 403-416, 451-477), specifically A coding sequence, atctggccactgatg, designed a left and right arm binding site upstream and downstream, with the aim of completely terminating the translational expression of the FUT8 enzyme by cleavage and introduced mutations at this position, rendering it 403-416 and 451-477 Functional regions that cause the Fut8 gene to be inactivated.
  • the maximum gene knockout efficiency of the L2R2 TALEN plasmid combined target CHO-S cells disclosed in the present invention can reach 69.6%.
  • the method for directly analyzing the sequence change of Fut8 gene by using genomic DNA sequencing of the present invention has the following advantages in screening mutant cells: (1) It has strong operability. The experiment requires only a small number of cells (such as 10 5 ), and can carry out small-scale DNA extraction and PCR amplification experiments in a routine laboratory; the cost of sequence analysis is low, and the cost of PCR product sequencing is about 10-20 yuan/reaction. Cell pools or cell clones with target gene mutations can be directly selected by comparing the DNA sequencing color maps and analyzing the peaks in the sequenced color maps. (2) High efficiency.
  • the mutation of genomic DNA in the transfected cell pool can be directly analyzed, according to the target sequence in the sequencing color map.
  • the height of the nearby peaks directly determines the optimal transfection conditions, and the subsequent cloning and further screening are carried out at the fastest speed, without long-term plate, cloning and enzyme activity detection to optimize the transfection conditions.
  • the target gene in most cells is not mutated, or only one allele is mutated, and even some mutations in the cell only have a certain degree of influence on the FUT8 enzyme activity (target function) Only a few amino acid deletions or substitutions occur in the region. Generally, only a small number of cells undergo biallelic knockout, and the FUT8 enzyme function is completely lost. Conventional enzyme activity detection is not very effective in the early cell cloning screening process. Only the clones with the decrease in enzyme activity can be found out from the detection results. The degree of decline is not the standard. If the target clone is not pure, the knockout effect is easy.
  • Genomic DNA sequencing provides a clear cut-off value for screening subsequent cell clones, which can continuously reduce the amount of cell screening in accordance with the progress of the screening process:
  • the genomic DNA was cloned from the cloned genomic PCR product, and the distribution of the mixed genomic template in the clone was sequenced one by one.
  • the clones which only caused the deletion of the amino acid residue without causing the translation of the FUT8 protein to be terminated were excluded by sequence analysis. It is a cell in which the enzyme activity is only partially affected without being completely lost.
  • B) The next step of subcloning the selected clone to obtain a single cell clone can greatly reduce the workload.
  • the genomic sequence analysis during the subcloning screening process can further provide guidance and reference for single cell screening: 1 detection of monoclonal cells with genomic DNA of three or more different mutation types is not truly single Cloning, which does not meet the requirements of industrial production for host cells, can be further subcloned as needed; 2 detection of monoclonal cells with two types of mutant genomic DNA may be double knockout heterozygous monoclonal cells, or two double knocks In addition to the homozygous cells, the clones can be cloned according to the sequence mutations determined in the previous step, and combined with other subcloning sequences in the same batch; 3 detection of monoclonal cells with only one mutant type of genomic DNA is double Knockout, homozygous monoclonal cells, a single genetic background, the potential value of industrial production is high.
  • the present invention uses the DNA sequence encoding the 403-416 segment of the Fut8 gene EXON7 as a target sequence, and successfully completes the knockout of the target sequence biallelic using the genome editing technology TALEN, and forcibly terminates the translation extension of the FUT8 enzyme protein downstream of the target sequence. .
  • the method of genome sequencing designed by the present invention guides the direct screening of mutant clones throughout the entire process: First, the use of genome sequencing to evaluate the cell pool obtained by transfecting TALEN plasmids with CHO-S cells can be performed at the lowest cost (one sequencing reaction) Cost), the fastest speed (one day to complete genomic PCR amplification and PCR product sequencing) to quickly and efficiently assess whether the genome of the cell pool is mutated, and observe the peaks of the peaks near the target sequence in the sequenced color map. To judge the efficiency of gene knockout, to provide a cell pool with high mutation rate for the next step.
  • genome sequencing was performed to exclude all clones still showing wild-type sequences, as well as non-target clones. This is achieved in two steps: 1 sequencing color map shows obvious wild-type genomic sequence, or clones showing residues of wild-type genomic sequence are immediately excluded; 2 for the selected clones and some clones that are difficult to judge mutations, pass TA
  • the cloning method analyzes the mutations occurring in the mixed genomic sequence population one by one: clones in which wild-type genomic sequences are found can be excluded; if a sequence mutation of 3 bases or an integer multiple thereof occurs in the genomic sequence mutation, only the individual Amino acid residues cannot completely delete the target functional region and its downstream amino acid sequence, and are also excluded in this step.
  • sequencing detection has three beneficial effects: on the one hand, it ensures that the subsequent screening samples are biallelic knockout clones; on the other hand, it greatly reduces the probability of genomic back mutation to wild type sequence, for the future.
  • the stability of the cell line provides protection; on the other hand, the direct use of the biallelic gene sequence in the genome, and its translation into the corresponding amino acid sequence mutation and the lack of enzyme function for screening, simple and direct, can avoid the high experimental requirements for enzyme activity detection,
  • the detection time is long, there is no clear threshold value standard, and it is not clear whether the single allele or the double allele is a mutation, and the probability of reversion mutation cannot be judged.
  • the selected clones were subcloned, single cells were isolated and normal growing monoclonal cells were formed.
  • the genomic sequence of the monoclonal cell was detected, and it was confirmed that the genomic mutation contained therein was detected in the previous step, and the wild type sequence no longer appeared (the wild type sequence missed in the previous step, or the single cell cloning process was mutated into the wild The type sequence will be excluded).
  • This step of genome sequencing can also help us determine the monoclonality of the single cells obtained.
  • the beneficial effect of this step sequencing design is to realize the screening function that cannot be realized by other detection methods, that is, the detection of monoclonal cells with genomic DNA of three or more different mutation types is not a true monoclonal, and does not meet the requirements of industrial production for host cells.
  • the monoclonal cells detecting the genomic DNA of the two mutant types may be double knockout heterozygous monoclonal cells, or may be clones formed by mixing two double knockout homozygous cells. According to the sequence mutation determined in the previous step, combined with the detection of other subcloning sequences in the same batch, the monoclonal cells with only one mutant type of genomic DNA are double knockout, homozygous monoclonal cells, and the genetic background is single. Work The potential utilization value of industrial production is high.
  • the selected subclones were serially passaged for 10 to 12 generations, and the cells after the passage were again tested for genomic DNA sequences to understand the stability of the sequence mutations during serial passage, and the detection of sequence mutations after the passage was excluded.
  • Variable clones provide reference data for subsequent production applications. In subsequent production applications, the detection control points are scheduled to be further monitored to further monitor the stability of the sequence of the mutation sites.
  • the stable cell lined with FUT8 function can be obtained by the cell screening method of the present invention. The obtained engineered cells were used to produce proteins, and the protein yield and quality were not significantly different from those of the original cells.
  • the method for inactivating cell endogenous Fut8 gene disclosed in the present invention can be used, for example, but not limited to, mammalian cells for antibody production: CHO-K1, CHO-S, CHOK1SV, DG44, DXB11, NS0, SP2/0, PER.C6 And genetic modification of HEK293; and other uses of mammalian cells.
  • the invention adopts the method of genome sequencing to detect gene knockout mutations: after obtaining the cells, the genomic DNA is extracted by using a kit, and the PCR products are amplified by using primers at both ends of the target sequence, and the PCR products are sequenced, and the vicinity of the target sequence is searched from the sequencing map. A set of peaks is used to determine mutations in the genomic sequence.
  • the DNA sequencing method selected by the invention is faster, cheaper and more operable.
  • the genomic DNA sequencing method adopted by the invention can be selected at different stages of screening, and the knockout result of FUT8 can be analyzed by analyzing the measured sequence binding peak, and finally homozygote and heterozygote can be distinguished.
  • Figure 1 shows the design of a TALEN plasmid pair.
  • Figure 2 shows the results of TALEN plasmid pair activity evaluation.
  • Figure 3 shows the sequencing results of 0129-A3.
  • Figure 4 shows the sequencing results of A1-A4 and B1-B4.
  • Figure 5A shows the sequencing results of wild-type control clone 65
  • Figure 5B shows the sequence alignment of the transformants of clone 40
  • Figure 5C shows the result of sequence alignment of the transformants of clone 37.
  • Figure 6 is a graph showing the growth behavior of subclones in the process of continuous passage.
  • Figure 6A shows the results of growth behavior of clone 40 (top panel of Figure 6A) and clone 18 (lower panel of Figure 6A) during serial passage.
  • Fig. 6B is a graph showing the results of the growth behavior of clone 37 in the process of continuous passage.
  • Figure 7 is a sequence comparison of subclone 6-C10 before and after serial passage.
  • Fig. 8A is a sequence comparison of subclones 18-44 before and after serial passage
  • Fig. 8B is a sequencing result of subclone 37-9
  • Fig. 8C is a sequencing result of subclone 37-30
  • Fig. 8D is a sequencing result of subclone 37-24
  • Figure 8E shows the sequencing results of subcloning 37-41 before passage (left panel of Figure 8E) and after passage (right panel of Figure 8E)
  • Figure 8F is before subcloning 37-4 passage (left panel of Figure 8F) and Sequencing results after passage (right panel of Figure 8F).
  • Figure 9 is a N-glycan profile of antibodies expressed by CHO.
  • Figure 10 shows the results of ADCC activity assay.
  • Figure 11 is a graph showing the effect of the test substance on the survival of the animal in the human B cell lymphoma Raji SCID mouse system xenograft model.
  • Figure 12 is a graph showing the effect of the test substance on the body weight of the animal in the human B cell lymphoma Raji SCID mouse system xenograft model.
  • the genomic sequence database was searched to obtain the Fut8 gene sequence in the CHO cell genome.
  • the DNA sequence encoding the 403-416 segment of the Fut8 gene Exon7 was selected as the gene knockout target sequence, and the TALEN plasmid pair was designed upstream and downstream of the target sequence.
  • the design of the TALEN plasmid pair (shown in Figure 1) is as follows, including two left arm plasmids (DNA binding region sequences, ie, recognition sequences, 15 to 16 bases in length) and three right arm plasmids (recognition sequence length 15 to 17 bases), can be combined to form 6 pairs of TALEN plasmid pairs.
  • G2 FastTALEN TM TALEN kit (Shanghai Stansai Biotechnology Co., Ltd.), from the module base, from the 5' to 3' in units of 1 to 2 bases except the last base of the recognition sequence. Select the corresponding module; then select the corresponding skeleton vector according to the last base of the recognition sequence, mix incubation, ligation, post-treatment, transformation, and transformant screening and sequencing confirmation, then the true expression of the required TALEN protein can be obtained respectively.
  • the nuclear expression plasmid is as follows:
  • the first behavior specifically identifies the sequence
  • the last base determines the type of vector used (selected according to the corresponding number of the G2 FastTALENT TM TALEN kit product specification)
  • the second behavior is the TALEN protein assembly module that is sequentially selected according to the recognition sequence.
  • the recognition sequence of L1 is gtagaaaaaagagtgt
  • the last base t determines that the vector used is L57
  • the corresponding TALEN protein assembly module is G1 ga2 t3 aa4 aa5 aa6 ag7 ag8 tg9.
  • the protein sequence and the corresponding coding gene using the recognition sequences of the plasmids L1-2, L2-1, R1-4, R2-3, R3-3, TALEN are as follows:
  • Possible combinations of plasmid pairs are: L1R1, L1R2, L1R3, L2R1, L2R2, L2R3.
  • Activity assay method The TALEN plasmid was transfected into the cell pool, the genome was sequenced, and the sequencing peak map was compared. The cell pool with high peak height was initially determined to have high gene knockout efficiency and could be used for the next transfection of CHO cells.
  • Figure 2 shows the results of a comparison of the peaks of the genomes of the 6 pairs of TALEN plasmids combined with the transfected cell pool.
  • the L2R2 combination with the high peak height was selected for TA cloning, and the efficiency of gene knockout was evaluated.
  • the higher the peak the higher the knockout efficiency.
  • the L2-1 and R2-3 plasmid pairs are transfected with CHO-S.
  • L2-1 and R2-3 plasmid pairs liposome transfection was carried out, and eGFP and puromycin (PM) carried in the plasmid were used for screening and detection.
  • the CHO cells used were CHO-S suspension cells.
  • the lipofection protocol was as follows (test batch: 0129):
  • Transfected cells 0129-A3 were obtained from the A3 protocol for amplification and cryopreservation.
  • the genomic DNA of the transfected cell pool 0129-A3 was extracted, and the target region fragment was amplified by PCR according to a conventional method, and the obtained PCR product was subjected to TA cloning to calculate the gene knockout efficiency.
  • PCR Cell culture was centrifuged to remove the supernatant, genomic DNA was extracted using a genomic DNA extraction kit, a pair of primers were designed upstream and downstream of the target sequence, and PCR amplification of the target region fragment was performed in a conventional manner.
  • the transfected cell pool 0129-A3 was selected to screen for Fut8 double knockout mutant cells.
  • OBJECTIVE To change the conditions of partial transfection and screening based on the previous CHO-S genome transformation to obtain a transfected cell pool with improved mutation efficiency.
  • the lipofection protocol is as follows:
  • A1 0.25 ⁇ 10 6 , 8P, 3D A2: 0.25 ⁇ 10 6 , 8P, 4D A3: 0.25 ⁇ 10 6 , 12.5P, 3D A4: 0.25 ⁇ 10 6 , 12.5P, 4D
  • B1 0.5 ⁇ 10 6 , 8P, 3D B2: 0.5 ⁇ 10 6 , 8P, 4D B3: 0.5 ⁇ 10 6 , 12.5P, 3D B4: 0.5 ⁇ 10 6 , 12.5P, 4D
  • the knockout efficiency of the gene obtained under the condition of 150611-A4 was 70%, and the knockout efficiency was high, which satisfies the experimental needs.
  • the transfected cell pool 150611-A4 obtained under these conditions was selected to screen for Fut8 double knockout mutant cells.
  • Transfected cell pools 0129-A3, 150611-A4 were screened for Fut8 double knockout mutant cells.
  • a certain volume of globular clones was formed in the semi-solid medium (up to the culturable state), and these clones were selected under microscope to 96-well plates, and according to the growth state of the cells in each well after the culture, The clones were amplified in 24 well plates in batches, and a certain number of cells (10 5 cells) were obtained and subjected to genome sequencing analysis.
  • Genomic sequencing analysis results 0311 batches of semi-solid medium were screened for single-cell clones to obtain two clones with clones (clone 40, 49), and 5,192 batches of semi-solid medium were screened for single-cell clones to obtain four clones with clusters (clone 28, 45, 94, 103). Further TA clone analysis of the template distribution of the above six clones. Clone 65 was used as a wild type control, and its genomic PCR amplification product sequencing map was used as a normal control map for comparison (Fig. 5A).
  • the clone 40 genomic PCR product was subjected to TA cloning, and 17 transformants were successfully obtained and sequenced, and the sequence comparison in the target sequence region is shown in Fig. 5B.
  • Clone 49 also carried out genomic PCR product TA cloning and sequencing analysis, but found the wild type sequence, and the found mutant sequence was 6 bp deletion, which did not meet the screening criteria and was directly excluded.
  • clone 28, 45, 94, 103 genomic PCR products were used for TA, and after transformants were obtained, the template distribution in the clones was analyzed by sequencing, and it was found that the above four clones showed wild-type sequence, in-frame deletion, and in-frame. Insertion, amino acid residue substitution, etc., do not meet the screening criteria and are excluded.
  • the 150611-A4 transfected cell pool was plated by limiting dilution method, 3 cells/200 ⁇ l/well*96 well*3 plates (a total of 0619, 0629 two batches of limiting dilution method were used to screen monoclonal cells). After culturing for 10 days in a 96-well plate, the cells were observed under a microscope, and cloned into a 24-well plate, 1 ml/well, and a certain number of cells (10 5 cells) were obtained, and the genomic DNA of the cells was extracted and extracted according to a conventional method. The target region fragment was PCR amplified, and the obtained PCR product was subjected to genome sequencing analysis. The sequencing results of each cloned genomic PCR product were analyzed in the same manner as in Batch 0129-A3.
  • Genomic sequencing analysis 619 batches of limited-dilution method were used to screen single-cell clones to obtain 6 sets of clones (clone 18, 24, 37, 51, 57, 58), and 0629 batches of limiting dilution method were used to screen single-cell clones to obtain peaks. 6 clones (clone 36, 54, 56, 58, 75, 77). Genomic PCR of the above 12 clones The material was further subjected to TA cloning and analyzed for template distribution.
  • the clone 37 genomic PCR product was cloned by TA to obtain a transformant, and the result of sequencing analysis is shown in Fig. 5C. After analysis, it was found that there were two templates in clone 37, which were sequence inserts of larger fragments:
  • the cryopreserved clone 40 cells were resuscitated to normal viability.
  • Five 96-well plates were plated at 0.5 cells/well, and 5 96-well plates were plated at 1 cell/well. After 11 days of culture, the wells that grew very slowly were excluded. 72 wells with normal growth were transferred to 3 24-well plates and cultured for 4-8 days. After obtaining a certain number of cells (10 5 samples), genomic DNA PCR amplification and sequencing analysis were performed. Eight subclones with failed cultures were excluded and a total of 64 subcloned genome sequencing sequences were analyzed. According to the results of sequencing analysis, 15 subcloned cells were selected and gradually expanded into 6-well plates and shake flask cultures, and finally the selected subcloned cells were preserved.
  • Clones 56 and 77 screened by limiting dilution method were plated by limiting dilution method, 1.5 cells/200 ⁇ l/well, two 96-well plates, and cultured for 10 days, then clones with normal growth were transferred to 24-well plates, 1 ml/ After obtaining a certain number of cells (10 5 ), the amplified genomic DNA of the cells is extracted, and the target region fragment is amplified by PCR according to a conventional method, and the obtained PCR product is subjected to genome sequencing analysis. After the culture, 48 samples were selected from the subclones of clone 56 for genomic sequence analysis, and 24 samples were selected from the subclones of clone 77 for genomic sequence sequencing analysis.
  • the clones 18, 37, and 51 screened by the limiting dilution method were plated by limiting dilution method, and subcloned and cultured in one cell/well and 1.5 cells/well in two 96 plates. After 11 days, according to the growth, from a plate 96 cells / well respectively picked subclones were transferred to 24-well plates to obtain a certain number of cells (10 5), the genomic DNA sequence analysis. The cloned genome sequencing results were analyzed according to the foregoing principles, and after the relevant subclones were excluded, the subclones that met the screening criteria were expanded and then stored.
  • Sequencing analysis method The analysis of the subcloned sequences is based on the template sequence and the template type analyzed by the parent clone, and the subcloned sequence should be included in the parental clone sequence, for example, the subcloning sequence analysis results show the position of the peak. Both are bimodal and conform to the sequence of the two templates superimposed by the sequence analysis of the mother cloned genome, which proves that no new mutant sequence appears and no back mutation is generated; at the same time, the same set of peaks appears in combination with other multiple subclones. It can be judged that these subclones have been produced by single cells. Subclones with high baseline of sequencing, weak amplification of PCR products, disordered sequencing signals, and failed sequencing were excluded.
  • Cryopreservation 37_14 Sequencing attenuation Signal clutter, stop processing 37_18 After the sequencing, the double peak appears, and the third template appears.
  • Cryopreservation 37_25 Double knockout homozygotes The baseline is slightly higher and stops processing 37_26 Double knockout homozygotes Baseline is high, stop processing 37_29 Double knockout homozygotes The baseline is slightly higher and stops processing 37_30 Double knockout homozygotes save 37_31 Double knockout homozygotes - 37_33 Double knockout homozygotes save 37_37 Sequencing without signal Stop processing 37_42 Sequencing without signal Stop processing 37_43 Sequencing without signal Stop processing
  • Subclone cells selected and saved by clone 40 2,4-E3,6-C10,9-F10,10-H1,8-D1,7-E5,5-C5,5-H10,3-D11,2- E10, 1-D9, 2-H6, 1-E7, 2-B5.
  • Subclone cells selected and maintained by clone 56 56-5, 56-6, 56-9, 56-10, 56-13, 56-15, 56-17, 56-28, 56-30, 56-46, 56-2, 56-3, 56-4, 56-8, 56-11, 56-14, 56-16, 56-26, 56-45, 56-48.
  • the sequencing results showed that the subcloned cells above 18 were cloned as double knockout homozygotes.
  • Clone 37 selected and preserved subclone cells: 37-1, 37-4, 37-19, 37-39, 37-40, 37-41, 37-9, 37-11, 37-24, 37-30, 37-33, 37-46.
  • the sequencing results showed that the cloned subclonal cells 37-9, 37-11, 37-24, 37-30, 37-33, and 37-46 were double knockout homozygotes.
  • these subclones were subjected to passage stability studies in batches, serially passaged for 10 to 12 generations, and the growth data of subclones during the passage was analyzed.
  • the genomic DNA sequence was sequenced and confirmed again, and subclones with stable growth characteristics and stable genomic mutations were selected for subsequent project research.
  • FIG. 6A The wild type CHO-S cells were used in parallel control for continuous passage, and the variation of doubling time (DT) of each generation was analyzed to understand whether the growth behavior of these subclones during continuous passage was different.
  • Figure 6A The results in Figure 6A show that the DT of the clone 40 was mostly close to the CHO-S cells in the growth process (top panel of Figure 6A); the homozygous subclones in clone 18 were subjected to serial passage studies, and the growth characteristics of each subclones Stable, consistent with wild-type cell CHO-S growth behavior (lower panel of Figure 6A); selected subclones of clone 37 were serially passaged, and from the growth characteristics, each subclone was associated with wild-type CHO-S cells. The cell doubling time was consistent and stable (Fig. 6B).
  • the subclones with the closest growth characteristics to wild-type CHO-S were selected and analyzed for genomic sequences.
  • the results of subcloning 6-C10 are shown in Figure 7.
  • the left panel shows the genomic sequence of the subcloned pre-passage, and the right panel shows the genomic sequence after passage.
  • the results for subclone 18-44 are shown in Figure 8A, and subcloning 18-44 was identical in sequencing results before and after passage compared to the wild-type genomic DNA sequence.
  • Genomic sequencing analysis was performed on 37-4 and 37-41 subclones, and the results were found to be genomic sequences before and after passage.
  • the middle set of peaks has changed, and the ratio of the two peak types at the same position has changed.
  • the left panel shows the genomic sequence of the pre-subcloning 37-41
  • the right panel shows the 37-41 subcloned genomic sequence after passage.
  • the phenomenon of imbalance of the peaks also appeared (see Figure 8F).
  • the genomic DNA sequence mutations in these clones are all sequence deletions of small fragments, and the deletion length of the sequences varies from 7 bp to 32 bp.
  • the loss of the terminal sequence is easy to occur in the process of non-homologous end joining (NHEJ) initiated by the genomic cleavage. This is also the case in our Fut8 knockout assay, where most of the mutations in the genomic DNA sequence are detected, and several single-base insertion mutations occur.
  • NHEJ non-homologous end joining
  • Mutation template analysis of clone 37 revealed that the insertion of the DNA sequence carrying the large fragment was 184 bp and 202 bp in length, respectively, similar to the results obtained by gene knock-in by specific means, and the genes carried out in this experiment. Knockout usually occurs in different mutations, and among the many clones analyzed, only one case was found, indicating that the mutations in the clone may be individual phenomena, which may be caused by impurities mixed in the TALEN plasmid, individual cells. The special mutation that occurs, the instability of its subcloning is believed to be related to this.
  • the sequencing screening method of the present invention can surprisingly quickly and efficiently identify the stability of different cloned mutant sequences.
  • Transient transfection of anti-CD20 antibodies was performed using subclones 6-C10, 18-44. Reconstitution of wild-type CHO-S cells, 6-C10 Fut8 double knockout heterozygous cells, 18-44 Fut8 double knockout homozygous cells, transient transfection of anti-CD20 antibody, preparation of anti-CD20 antibody by protein A one-step purification .
  • the anti-CD20 antibody for transient transfection.
  • the transfection conditions are as follows:
  • the amount of plasmid is 3 ⁇ g (light chain 1.5 ⁇ g + heavy chain 1.5 ⁇ g) plasmid per 3 ⁇ 10 6 cells, that is, the concentration of the plasmid after adding the cell suspension should be 1 ⁇ g / ml (Note: The concentration of the plasmid obtained by extraction is controlled as much as possible at about 1 mg/ml.
  • the amount of the transfection reagent PEI was 6 ⁇ g of PEI (1 mg/ml) per 3 ⁇ 10 6 cells, that is, the ratio of plasmid to PEI was 1:2.
  • the harvested samples were centrifuged at 10,000 rpm for 30 min at 4 ° C, and the supernatant was taken, and the anti-CD20 antibody was purified by Protein A affinity chromatography column.
  • the purification conditions are as follows:
  • Buffer 1 50 mM Tris-HCl + 150 mM NaCl, pH 7.2
  • Buffer 2 50 mM HAc-NaAc, pH 3.5
  • Chromatography operation steps buffer 3 punch column, 30 min; buffer 1 punch column, 5 CV; take the supernatant sample; buffer 1 punch column, to UV280 baseline stable; buffer 2 punch column, UV280 display 50-max
  • the elution peak was collected at -50 mAU, and the eluted sample was added to buffer 4 to adjust the pH to neutral (that is, the purified anti-CD20 antibody was obtained); the buffer was washed at 3 C, not less than 3 CV; the buffer was 5 punched, 3 CV.
  • CV in the above steps column volume, column volume.
  • 6-C10 was co-transfected with 2.1 L, and the antibody was purified to obtain 37.3 mg (4.78 mg/ml, 7.8 mL); 18-44 was co-transfected with 2.8 L, and protein 51.58 mg (4.96 mg/ml, 10.4 mL) was harvested.
  • the N-glycoside on the glycoprotein was separated from the protein by the PNG F enzyme, and the separated N-glycoside was dried and labeled with the fluorescent reagent 2-AB.
  • the labeled sample was separated by hydrophilic chromatography, and a peak was detected by a fluorescence detector.
  • Experimental equipment and consumables used Waters Acquity UPLC-FLR (fluorescence detector), PNG F enzyme (NEB), column Waters Acquity UPLC Glycan Amide column 1.7 ⁇ m, 2.1 mm ⁇ 150 mm.
  • ADCC activity of the anti-CD20 antibody expressed by the present invention is ADCC activity of the anti-CD20 antibody expressed by the present invention
  • Antibody-dependent cell-mediated cytotoxicity refers to the binding of an IgG antibody to the Fc cell surface Fc ⁇ RIII by specific binding of the Fab fragment to the target cell surface antigenic determinant.
  • NK cells produce a non-specific killing effect on target cells, namely ADCC.
  • a series of diluted anti-CD20 antibody drugs and reference products were added to the target cell Ramos cell line, and the ADCC effect of the antibody drug on the target cells was detected, and the EC 50 value was calculated.
  • 06 anti-CD20 antibody expressing the 06 molecule sequence without host modification 07 anti-CD20 antibody expressing the 07 molecule sequence without host modification (06 and 07 are parallel example data, amino acid sequence is different);
  • 06-6C10, 06-6C10' are anti-CD20 antibodies expressing the 06-molecular sequence modified by host cell line 6C10, "'" represents the second batch of transfection preparation, and the expression of 06-18-44 modified by host cell line 18-44 06
  • the molecular sequence of the anti-CD20 antibody, 07-6C10, 07-6C10' is an anti-CD20 antibody expressing the 07 molecule sequence engineered by the host cell line 6C10, and "'" represents a second batch of transfection preparation.
  • the effector cell density of NK92/FcyRIII cells was adjusted to 2.4 ⁇ 10 6 cells/ml, and 25 ⁇ l was added to each well. Incubate for 5 hours at 37 ° C, 5% CO 2 . At the same time, a blank medium control group, a single target cell spontaneous release control group, a single effector cell spontaneous release control group, a single target cell and an effector cell spontaneous release group, and a single target cell after the end of the incubation period were added to the maximum release control group. .
  • the 96-well plate was equilibrated to 22 ° C for about 30 minutes, mixed with 100 ⁇ l of the test solution, and allowed to stand for 10 minutes, and then 50 ⁇ l of the stop solution was added.
  • Spectra Max M2 detects excitation/emission wavelengths of 560 nm/590 nm fluorescence signal values.
  • the EC 50 ratio of 06, 07 and reference rituximab was about 3 times, indicating that the difference in ADCC between the two samples was not significant at the same level compared with the reference.
  • the EC 50 ratios of 06-6C10, 07-6C10, 06-6C10', 06-18-44 and reference rituximab were between 25-40 times, and the difference in ADCC effect was significantly improved.
  • the four-parameter fit curve is shown in Figure 10.
  • the anti-CD20 antibody expressed by the present invention inhibits tumor growth
  • the human model B cell lymphoma Raji SCID mouse transplantation model was used to test the rituximab, 03 (CHO-S), 03 (6C10), 06 (CHO-S), 06-6C10', 07-6C10'
  • the in vivo antitumor activity of the drug alone was evaluated.
  • each test drug showed significant life extension activity, including rituximab, 03 (CHO-S), 03 (6C10), 06 (CHO-S), 06-6C10', 07-
  • the median survival days of 6C10' were 40 days (p ⁇ 0.0001), 35 days (p ⁇ 0.0001), 38 days (p ⁇ 0.0001), 37.5 days (p ⁇ 0.0001), 40.5 days (p ⁇ 0.0001), and 39 Day (p ⁇ 0.0001), the median survival days of the blank control group was 21.5 days;
  • Percentage of life extension rate relative life extension rate of rituximab, 03 (CHO-S), 03 (6C10), 06 (CHO-S), 06-6C10', 07-6C10' at 1 mg/kg dose
  • the percentages of T/C were 186.05%, 162.79%, 176.74%, 174.42%, 188.37%, and 181.39%, respectively.
  • Fig. 11 The results of the effect of the test substance on the survival of the animal in the human B cell lymphoma RajiSCID mouse system xenograft model are shown in Fig. 11.
  • Fig. 12 The results of the effect of the test substance on animal body weight (Mean ⁇ SEM) in the human B cell lymphoma RajiSCID mouse system xenograft model are shown in Fig. 12.

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Abstract

L'invention concerne un procédé de génération et de séquençage d'une inactivation biallélique d'un gène cible criblé dans une cellule eucaryote au moyen d'une technique TALEN, ainsi qu'un polynucléotide bicaténaire comprenant une paire de plasmides TALEN avec un site de clivage ATCTGGCCACTGATG.
PCT/CN2017/117814 2016-12-21 2017-12-21 Complexe et procédé d'inactivation du gène fut8 WO2018113753A1 (fr)

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