WO2020190031A1 - Transgenic plant with suppressed expression of cgl1 and cgl2 and method for producing target protein using same - Google Patents

Abstract

The present invention relates to a transgenic plant in which the expression of CGL1 and CGL2 is suppressed, and a method for producing a target protein having an increased content of mannose linked to an N-sugar chain using the same. The transgenic plant having suppressed expression of CGL1 and CGL2 according to the present invention, when used to produce the target protein, can increase the content of mannose linked to the N-sugar chain and decrease the content of fucose and/or xylose. Therefore, the transgenic plant according to the present invention can be usefully used for the production of medicinal proteins such as antibody drugs.

Description

Transgenic plants in which the expression of CGL1 and CGL2 is suppressed, and production method of target protein using the same

The present invention relates to a transgenic plant in which the expression of CGL1 and CGL2 is suppressed, and a method for producing a protein of interest in which the content of mannose bound to the N-sugar chain is increased using the same.

The Fc region of an antibody is a form in which various sugar chains are bound, and it is known to affect ADCC (antibody dependent cellular cytotoxicity) efficacy, immunogenicity, stability, etc. depending on the type and content. On the other hand, antibody drugs currently being produced are mostly produced in animal cells, but this is a complicated production process, which takes a long time and is expensive. In addition, there is a disadvantage that infection from pathogens may occur when antibody drugs are produced from animal cells.

In order to improve these shortcomings, studies for producing antibodies using plants have recently been conducted. Production of antibodies using plants can reduce production cost and time, as well as prevent infection from pathogens.

In particular, studies to control the sugar content of the antibody are being conducted as a study to improve the ADCC efficacy of the antibody. This is because the components and structures of sugar chains have a great influence on therapeutic efficacy, residence time in the human body, pharmacological activity, and immune response. In this regard, Abbott's adalimumab patent (US 2012/0276631 and WO 2012/149197) discloses that the sugar chain of an antibody using manganese and galactose can be regulated. However, it is still difficult to prepare antibodies whose sugar chains are regulated so that the target content and specific components are increased or decreased.

Therefore, there is a need for a transgenic plant capable of controlling the component and/or content of the sugar chain and a study for producing an antibody drug using such a plant.

Accordingly, the present inventors produced a transgenic plant in which the expression of CGL1 and CGL2 was suppressed using CRISPR technology in order to develop a transgenic plant capable of controlling the components and/or content of the sugar chain, and the purpose of the transgenic plant In the case of producing a target protein by introducing a specific gene encoding a protein, the present invention was completed by confirming that the target protein with an increased content of mannose bound to the N-sugar chain can be produced.

In order to achieve the above object, an aspect of the present invention provides a transgenic plant in which the expression of CGL1 and CGL2 is suppressed.

Another aspect of the present invention provides a transgenic plant for producing a target protein by introducing a gene encoding a target protein into the transgenic plant.

Another aspect of the invention, i) a recombinant expression vector to guide RNA, CGL1 gene and CGL2 gene coupled to CGL1 gene complementary to include a gene encoding a guide RNA and Chris flops associated proteins that bind complementarily Manufacturing steps; ii) It provides a method for producing a transgenic plant in which the expression of CGL1 and CGL2 is suppressed, comprising the step of transforming plant cells or plant tissues with the recombinant expression vector.

Another aspect of the invention, i) cultivating the transgenic plant; And ii) provides a method for producing a protein of interest comprising the step of recovering the protein of interest from the plant.

Another aspect of the invention, the guide for coupling complementarily to CGL1 gene RNA, CGL1 gene and a guide that bind complementarily to CGL2 gene RNA and Chris flops associated protein transgenic expression of CGL1 and CGL2 suppressed containing It provides a kit for manufacturing plants.

Another aspect of the present invention provides a target protein produced through a transgenic plant for producing the target protein.

When producing a target protein using a transgenic plant in which the expression of CGL1 and CGL2 according to the present invention is suppressed, the content of mannose bound to the N-sugar chain can be increased, and the content of fucose and/or xylose can be increased. Can be reduced. Therefore, the transgenic plant according to the present invention can be usefully used in the production of medical proteins such as antibody pharmaceuticals.

1 is a diagram showing a schematic diagram of the pNGJP0014-NbCGL1sgRNAs recombinant expression vector used in an embodiment of the present invention.

2A is a diagram showing cDNA information of the CGL1 gene and the CGL2 gene.

2B is a diagram showing a target region of a guide RNA for knocking out a CGL1 gene.

2C is a diagram showing a target region of a guide RNA for knocking out a CGL2 gene.

Figures 3a and 3b that the CGL1 and CGL2 gene target region gene target area of the tobacco (Nicotiana benthamiana) knockout diagram confirmed by sequencing the genes.

4A is a diagram showing sequence information of the CGL1 gene target region of cigarettes in which the CGL1 gene is knocked out.

4B is a diagram showing sequence information of the CGL2 gene target region of cigarettes in which the CGL2 gene is knocked out.

5A is a diagram showing the relative amount of N-sugar chains per line of total water-soluble protein (TSP) produced using the transgenic tobacco prepared in an embodiment of the present invention.

5B is a diagram showing the relative amounts of N-sugar chains per line of antibody (GF003) produced using the transgenic tobacco prepared in an embodiment of the present invention.

6 is a diagram showing the results of analyzing the ADCC efficacy of the antibody produced using the transgenic tobacco prepared in an embodiment of the present invention.

One aspect of the present invention provides a transgenic plant in which the expression of CGL1 and CGL2 is suppressed. In this case, the plant may be a plant cell, a plant tissue, or a plant body.

The term "CGL1", as used herein, refers to an enzyme related to the N-glycosylation process, and is also referred to as N-acetylglucosaminyltransferase I (GnTI). The CGL1 may consist of an amino acid sequence represented by SEQ ID NO: 1. The amino acid sequence represented by SEQ ID NO: 1 may be encoded by the nucleotide sequence represented by SEQ ID NO: 2.

The term "CGL2", as used herein, is a major enzyme involved in the process of N-glycosylation, similarly to CGL1, and is also called N-acetylglucosaminyltransferase II (GnTII). The CGL2 may consist of an amino acid sequence represented by SEQ ID NO: 3. The amino acid sequence represented by SEQ ID NO: 3 may be encoded by the nucleotide sequence represented by SEQ ID NO: 4.

The expression of CGL1 and CGL2 can be suppressed by knocking out the genes encoding them, respectively. Specifically, expression may be inhibited by any one method selected from the group consisting of total deletion, partial deletion, partial sequence insertion, and nucleic acid substitution of genes encoding the CGL1 and CGL2.

In one embodiment of the present invention, any one of the CGL1 and CGL2 may be homozygous knockout, and the other may be heterozygous knockout. Specifically, when the CGL1 is homozygous knockout, the CGL2 may be heterozygous knockout. In this case, CGL1 is not expressed and CGL2 can be expressed because one gene encoding it is normal. In addition, when the CGL1 is heterozygous knockout, the CGL2 may be homozygous knockout. In this case, CGL1 is partially expressed and CGL2 is not. At this time, in the transgenic plant, both CGL1 and CGL2 are not homozygous knockout. Plants cannot survive if all are homozygous knockouts.

In one embodiment of the present invention, the plant may have an increased content of mannose bound to the N-sugar chain, and the content of fucose, xylose, or fucose and xylose bound to the N-sugar chain It may be reduced.

The plant may be used without limitation, as long as it is a plant that can be transformed, but specifically tobacco, Arabidopsis, corn, rice, soybean, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper It may be any one selected from the group consisting of. More specifically, the plant may be tobacco ( Nicotiana benthamiana ), and the present invention may be practiced by selecting an appropriate variety according to the purpose of the transformation method and mass production of protein.

In addition, another aspect of the present invention provides a transgenic plant for producing a target protein by introducing a gene encoding a target protein into the transgenic plant.

In one embodiment of the present invention, the protein of interest may be an antibody or an enzyme. Specifically, the antibody may be trastuzumab, bevacizumab, and rituximab, and the enzyme may be β-glucocerebrosidase. In this case, the trastuzumab may include an amino acid sequence represented by SEQ ID NO: 9 and/or an amino acid sequence represented by SEQ ID NO: 10.

In one embodiment of the present invention, the target protein may have an increased content of mannose bound to the N-sugar chain. For example, the protein of interest has a ratio of high mannose-type HexNAc2Hex5 (M5) sugar pattern in the N-sugar chain about 5 times or more, 15 times or more, 25 times or more, 35 times or more, 45 times or more, or 55 times as compared to wild type. It may be more than increased.

In addition, the target protein may have an increase in the content of mannose bound to the N-sugar chain by 5% to 50%.

In one embodiment of the present invention, the target protein may have a reduced content of fucose, xylose, or fucose and xylose bound to the N-sugar chain. For example, the protein of interest has a ratio of plant-specific HexNac4Pent1Hex3 sugar pattern in the N-sugar chain of about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60^ or more, 70% or more, It may be reduced by 80% or more or 90% or more.

The target protein has a ratio of plant-specific Deoxyhex1HexNAc4Pent1Hex3 sugar pattern in the N-sugar chain of about 5% or more, 15% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% compared to wild type. It may be more or less than 90%.

In addition, the target protein may have a content of fucose, xylose, or fucose and xylose bound to the N-sugar chain is reduced by 10% to 50%.

In one embodiment of the present invention, trastzumab, an active ingredient of Herceptin, was produced using a transgenic tobacco in which the expression of CGL1 and CGL2 was suppressed as a specific example of the antibody. At this time, the heavy and light chain amino acid sequences may be composed of amino acid sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. At this time, when one of CGL1 and CGL2 was completely knocked out and the other was produced in a heterozygous knockout plant, it was confirmed that the content of mannose in trastzumab was increased. In the case of the antibody thus produced, it was confirmed that the activity was increased compared to the trastzumab produced in normal plants (Table 5). Further, it was confirmed that the sugar pattern of the target protein produced using the transgenic plant in which the expression of CGL1 and CGL2 was suppressed according to the present invention was effectively changed, and the activity of the target protein produced in the plant was changed. Therefore, the transgenic plant according to the present invention can be usefully used in the production of a protein of interest that requires a change in a sugar pattern.

Another aspect of the present invention is to prepare a recombinant expression vector comprising i) a guide RNA complementary to the CGL1 gene, a guide RNA complementary to the CGL1 gene and a CGL2 gene, and a gene encoding a CRISPR-associated protein. step; ii) It provides a method of producing a transgenic plant in which the expression of CGL1 and CGL2 is suppressed, comprising the step of transforming plant cells or plant tissues with the recombinant expression vector.

The guide RNA includes a sequence specific to the target sequence, and binds all or part of the complementary to the target sequence, so that the CRISPR-associated protein can cleave the target sequence.

Typically, the guide RNA comprises two RNAs, that is, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components of dual RNA (dual RNA); Or a first site comprising a sequence complementary to the target sequence in whole or in part and a second site comprising a sequence interacting with the CRISPR-associated protein, but the CRISPR-associated protein has activity in the target sequence. Any form that can be included in the scope of the present invention without limitation.

Specifically, the guide RNA may include a sequence capable of complementary binding to the CGL1 gene and/or the CGL2 gene. In particular, the guide RNA may include a sequence capable of complementarily binding to a target sequence of the CGL1 gene and/or the CGL2 gene. The guide RNA may be an oligonucleotide comprising 15 to 35 nucleotides capable of complementarily binding to DNA encoding CGL1 and/or CGL2. The guide RNA may be an oligonucleotide comprising 15 to 35, 20 to 25, or 23 nucleotides capable of complementarily binding to the target sequence of the CGL1 gene and/or the CGL2 gene.

As an embodiment of the present invention, the guide RNA complementarily binding to the CGL1 gene may include a nucleotide sequence represented by SEQ ID NO: 5. Furthermore, the gene in CGL1 guide RNA that binds complementarily may be represented by "NbCGL1-1", which binds complementarily to the DNA encoding the gene CGL1 to the target gene only CGL1.

As an embodiment of the present invention, the guide RNA complementarily binding to the CGL1 gene and the CGL2 gene may include a nucleotide sequence represented by SEQ ID NO: 6. Further, the guide RNA that binds complementarily to the gene CGL1 and CGL2 gene may be expressed as "NbCGL1-2", which may be coupled to a complementary gene at the same time in CGL1 and CGL2 gene.

The guide RNA may include a scaffold sequence that helps CRISPR-associated protein bind.

At this time, the sequence information required for the guide RNA can be obtained from a known database such as Addgene and GenBank of NCBI (National Center for Biotechnology Information).

As an embodiment of the present invention, the CRISPR-associated protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb3, Csb2, Csb17 It may be any one selected from the group consisting of Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4, and specifically the CRISPR-related protein may be Cas9.

At this time, the sequence information of the CRISPR-associated protein or gene can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI).

The transgenic plant can be produced through a plant transformation method known in the art. Those skilled in the art can select and carry out a known transformation method suitable for a specific plant in consideration of the characteristics of the plant selected as the host. Specifically, a transformation method using Agrobacteria may be used as a transformation method of plants.

The "transformation method using Agrobacteria" is a method of transferring foreign genes to plant cells by using Agrobacteria, which is a Gram-negative bacterium of soil that causes tumors in the roots and stems of plants. This Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens), Agrobacterium separation tank to Ness (Agrobacterium rhizogenes) including a dielectric (genome of the T-DNA (transfer DNA) is a plant tumor inducing plasmid (Ti plasmid) it found in Agrobacterium bacteria It is a method using the phenomenon inserted in ).

Specifically, a recombinant expression vector in an embodiment of the present invention comprises a sequence encoding a guide RNA and Chris flops associated proteins that bind complementarily to the guide RNA, CGL1 gene and CGL2 gene binding complementarily to CGL1 gene A method of transforming the plant by infecting the plant with Agrobacteria was used.

Another aspect of the present invention provides a method of producing a transformed plant for producing a protein of interest, comprising introducing a gene encoding a target protein into a transformed plant in which the expression of CGL1 and CGL2 is suppressed.

The transformation method is as described above, and the target protein may be various proteins. In particular, it may be an antibody and an enzyme, and specifically, may be trastzumab whose activity changes due to a change in sugar pattern.

Another aspect of the present invention, i) cultivating a transgenic plant for producing the target protein; And ii) provides a method for producing a protein of interest comprising the step of recovering the protein of interest from the plant.

Further, another aspect of the invention, the two in CGL1 gene guide expression of CGL1 and CGL2 to the RNA, CGL1 gene and CGL2 gene comprising a guide RNA and Chris flops associated proteins that bind complementarily binding to a complementary inhibitory It provides a kit for producing a transgenic plant. At this time, the guide and the guide RNA RNA binding to complementary to the gene CGL1 and CGL2 gene binding complementarily to the CGL1 gene is the same as described above.

Another aspect of the present invention provides a target protein produced through a transgenic plant for producing the target protein.

Transgenic plants for producing the target protein are the same as described above.

The protein of interest may be an antibody or an enzyme. Specifically, the antibody may be trastuzumab, bevacizumab, and rituximab, and the enzyme may be β-glucocerebrosidase. In this case, the trastuzumab may include an amino acid sequence represented by SEQ ID NO: 9 and/or an amino acid sequence represented by SEQ ID NO: 10.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1. Preparation of transgenic tobacco with suppressed expression of CGL1 and CGL2

Transgenic cigarettes in which the CGL1 gene and the CGL2 gene were knocked out were constructed using a recombinant expression vector containing guide RNA and CRISPR-associated protein. The specific manufacturing method is as follows.

Example 1.1. Construction of recombinant expression vector

The recombinant expression vector has an antibiotic cassette to be selected by kanamycin and hygromycin antibiotics, and was constructed to have a polycistronic tRNA-gRNA cassette synthesized with Cas9. The Cas9 was constructed to be expressed by the Arabidopsis ubiquitin 10 promoter, and the tRNA-gRNA was constructed to be expressed by the Arabidopsis ubiquitin 6 promoter. In order to deliver the Cas9 protein to the nucleus, the SV40 (PKKKRKV (SEQ ID NO: 13)) nuclear transfer signal sequence and the C-terminal Bipartite (KEPAATKKAGQAKKKK (SEQ ID NO: 14)) nuclear transfer signal sequence were added to the N-terminus of the Cas9 open reading frame. I did. The two guide RNA was designed to target the gene CGL1 and CGL2 gene of Nikko tiahna Ventana Mia or (N. benthamiana). A schematic diagram of the pNGJP0014-NbCGL1sgRNAs recombinant expression vector constructed as described above is shown in FIG. 1.

At this time, a guide RNA target region was designed to knock out each of the CGL1 gene and CGL2 gene in N. benthamiana . The target regions of the CGL1 gene and CGL2 gene to which the guide RNA binds are as shown in FIGS. 2A to 2C. In addition, the sequences complementarily binding to the target region of the guide RNA for knocking out the CGL1 gene and the CGL2 gene are shown in Table 1 below.

In Table 1, NbCGL1-1 shall only CGL1 gene targeting, NbCGL1-2 will target the gene CGL1 and CGL2 genes (Fig. 2b and Fig. 2c).

Example 1.2. Production of transgenic tobacco using Agrobacteria

In order to produce a transgenic tobacco in which the CGL1 gene and the CGL2 gene were knocked out, Agrobacteria containing the recombinant expression vector of Example 1.1 was used. Specifically, seeds of tobacco ( N. benthamiana ) were used, and the tobacco seeds were sterilized with 75% ethanol for 1 minute, and sterilized by immersing in 50% Clorox for 10 minutes. Thereafter, the tobacco seeds were washed 4 times with sterile water, and covered with sterile filter paper to dry the water on the leaf surface. After disinfecting the tobacco seeds, they were placed on a seed sowing medium and grown in a culture chamber at 25°C for 4 weeks. Tobacco leaves grown for a week were cut into 1 cm squares with a knife and placed on a solid medium for transformation (1 MS, 3% sucrose, 2 mg/L 6-BA, 0.2 mg/L NAA, and 1% agar).

Agrobacteria containing the recombinant expression vector prepared in Example 1.1 above were used as a transformation medium at pH 5.8 (1 MS, 3% sucrose, 2 mg/L 6-BA (6-benzyl adenine) and 0.2 mg/L NAA ( Naphthaleneacetic acid)) was used to adjust the OD ₆₀₀ to 0.6 to prepare an Agrobacteria transformation culture.

Subsequently, the cut tobacco leaves were immersed in the Agrobacteria transformation culture solution for 10 minutes. The soaked tobacco leaves were washed once with sterile water and then drained with sterile filter paper. The tobacco main leaf was transferred to a solid medium for transformation (1 MS, 3% sucrose, 2 mg/L 6-BA, 0.2 mg/L NAA and 1% agar) with a pH of 5.8 so that the back side of the tobacco leaf was facing up. . Thereafter, the tobacco leaves were mixed with an antibiotic medium of pH 5.8 (1 MS, 3% sucrose, 2 mg/L 6-BA, 0.2 mg/L NAA, 25 mg/L hygromycin, 200 mg/L thymentin, and 1% Baby). In order to maintain the effectiveness of the antibiotic, the process of transferring to a new solid medium was repeated once every two weeks as described above.

After 1 to 2 months, when callus is formed and leaves and stems come out, a medium of pH 5.8 that induces roots (1 MS, 3% sucrose, 25 mg/L hygromycin, 200 mg/L tea Mentin and 1% agar). When the roots were induced after 2 to 3 weeks, they were transferred to the soil in a large container to obtain new individuals (seeds) after 4 weeks.

Example 2. Analysis of transgenic tobacco

Example 2.1. Next-Generation Sequencing (NGS) analysis

After the above-described embodiment and the first knockout the gene CGL1 and CGL2 gene of tobacco in the same way, by introducing the gene encoding the target protein transgenic tobacco was performed NGS analyzed by the selected object on the antibiotic medium. Specifically, the target region was subjected to primary PCR using an amplicon primer, and then a lean-up was performed on the produced PCR product. A library was constructed by indexing the target region with DNA containing an amplicon. Subsequently, after performing the second PCR, QC was performed with a Bioanalyzer and sequenced. At this time, showed the CGL1 and CGL2 gene target gene target area region of the knockout was cigarettes in Figures 3a and 3b, analysis conditions were as follows.

Analysis equipment: Illumina product, MiniSeq (SY-420-1001), Miniseq mid output kit (300 cycles, FC-420-1004)

Amplicon Primer:

Forward: 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[locus specific sequence] (SEQ ID NO: 7)

Reverse: 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[locus specific sequence] (SEQ ID NO: 8)

The results of the NGS analysis are shown in Table 2 below, and individual selection was performed with gene editing efficiency (%).

As shown in Table 2, among 25 individuals, the gene editing efficiency was found to be 25% or more in #3-1, #3-2, and #3-5 individuals. Among them, the T2 generation genotyping and N-sugar chain structure analysis results of #3-2 individuals showing 50% or more gene editing efficiency are shown in Example 2.2. And shown in Example 2.3.

Example 2.2. Sanger Sequencing

Sanger sequencing was performed on the #3-2 individual selected in Example 2.1. Specifically, the guide RNA target region was amplified from genomic DNA extract using Q5 High-Fidelity DNA Polymerase (NewEngland Biolabs) in a 20 μl reaction. Thereafter, the PCR product was cloned using the All in One Cloning Kit (Biofact, South Korea), and 15 to 20 cloned clones were individually sequenced for each sample. Showed the confirming the knockout CGL1 GCL2 gene and gene sequence information results in Fig. 4a and 4b, to a line-by-line mutation information of the T2 generation are shown in Table 3 below.

In Table 3 below, it means complete knockout (homozygous knockout) of CGL1 gene or CGL2 gene indicated by lowercase letters, and capital letters mean hetero (heterozygous knockout). In addition, the CGL1 gene or the CGL2 gene alone in lowercase letters means a single knockout. Here, it was confirmed that the insertion (insertion) appeared as +1 and the deletion (deletion) appeared in various forms of -4 to -29.

Example 2.3. Analysis of sugar chain structure

The N-sugar chain structure analysis was performed on #3, #5, #10, #13, and #48 individuals selected in Example 2.2. The specific method is as follows.

Example 2.3.1. Protein separation

After freezing the transformed N. benthamiana obtained in Example 1 with liquid nitrogen, the leaves were crushed in a mortar bowl. A phosphate buffer solution (pH 7.2) of 2 times the volume was added to the powder obtained by grinding and mixed. Thereafter, the mixture was allowed to stand on ice for 10 minutes and centrifuged under conditions of 15,000× g , 20 minutes and 4°C to collect the first transparent supernatant. 2 times the volume of phosphate buffer solution (pH 7.2) was additionally added to the remaining powder, and the same procedure as described above was repeated to recover the supernatant, and this was mixed with the first recovered supernatant. Through this, a total soluble protein (TSP) of N. benthamiana was obtained.

The obtained TSP was filtered through 0.45 μm to remove large insoluble particles, and then concentrated under the conditions of 30 kDa, 15,000× g , 30 minutes and 4°C. The concentrated TSP was repeatedly treated three times with ultrapure water, and the phosphate buffer solution was replaced with ultrapure water under the conditions of 30 kDa, 15,000× g , 30 minutes and 4°C. Then, the amount of protein was measured using the Bradford method.

Example 2.3.2. N-sugar chain separation and analysis

A denaturing solution (0.1% RapiGest SF and 10 mM DTT) was added to the TSP sample (50 μg) obtained in Example 2.3.1 and reacted at 56° C. for 45 minutes. Thereafter, 20 mM of iodoacetamide was additionally added and reacted for 1 hour in dark conditions (room temperature). Sugar cleavage enzyme (2 µl, 5 U/µl, PNGase A) was added to the reaction solution, followed by reaction at 37°C overnight. Thereafter, the N-sugar chain was extracted using a PGC cartridge (Porous graphitize carbon SPE cartridge) and dried by a vacuum centrifugation method.

10 μl of a fluorescent labeling solution (5 mg 2-aminobenzoic acid, 6 mg sodium cyanoborohydride/100 μl acetic acid and DMSO) was added to the dried N-sugar chain, followed by reaction at 65° C. for 3 hours. The fluorescently labeled N-sugar chain was extracted using a Cyano SPE cartridge and dried by vacuum centrifugation. The dried anthanilamide (Anthanilamide; A89804-5G, Sigma-Aldrich)) labeled N-sugar chain was dissolved by adding 20 μL of ultrapure water and analyzed using a mass spectrometer (Ultraflex III TOF/TOF, Bruker Daltonics). At this time, the analysis conditions are as follows.

-Instrument Control: Flex Control 3.0 (Bruker Daltonics)

-Analysis mode: reflection mode

-Polarity: Bipolar

-Detection: m/z 100 to 4,000

-Laser repetition rate: 100 Hz

-Number of shots: 500 shots

-Refraction: On, 500 Da

-Voltage (see Table 4 below)

The results of N-sugar chain analysis of the total water-soluble protein are shown in FIG. 5A.

Example 2.4. Production of the protein of interest

Example 2.4.1. Preparation of a recombinant vector containing a recombinant gene

The heavy chain gene of trastuzumab was added by adding Bsa I restriction enzyme from the plasmid containing the heavy chain gene (SEQ ID NO: 11) and light chain gene (SEQ ID NO: 12) synthesized by ordering and requesting from mBiotech. Only SEQ ID NO: 11) and light chain gene (SEQ ID NO: 12) were expressed in plants.

Example 2.4.2. Production of Agrobacteria transformed with a recombinant vector

Agrobacterium cells (GV3101) were transformed by introducing each recombinant expression vector having the heavy chain gene (SEQ ID NO: 11) and light chain gene (SEQ ID NO: 12) of trastuzumab, and then medium (YEP agar plate) Smeared on and incubated for about 2 days at 28°C. Thereafter, the resulting single colony was inoculated into a liquid medium (YEP broth) and pre-cultured for about 2 days under conditions of 200 rpm at 28°C. The pre-culture was inoculated at a ratio of 0.5% of the amount of the new liquid medium, and cultured at a temperature of 28° C. at 200 rpm until an OD ₆₀₀ value of 1.2 to 1.8.

Example 2.4.3. Recombinant protein production using transformed plant cells

The transformed Agrobacteria cultured with shaking in Example 2.4.2 was diluted to an OD ₆₀₀ value of 0.02 by adding a buffer solution for impregnation (10 mM MES, pH 5.6, 10 mM MgSO ₄ ). Diluted transgenic Agrobacteria containing heavy and light chains were mixed in a ratio of 1:1, and the tobacco leaves of the individual obtained in Example 1.2 were immersed in this mixture in a vacuum chamber. A vacuum was applied to the vacuum chamber, and after reaching the target pressure, the pressure was released to take out and dry tobacco leaves to obtain transformed plant cells expressing trastzumab.

Example 2.5. Isolation and analysis of N-sugar chain from GF003 antibody protein

Protein was isolated from the plant cells obtained in Example 2.4.3 in the same manner as in Example 2.3.1, and the N-sugar chain was isolated and analyzed in the same manner as in Example 2.3.2, and the results are shown in FIG. 5B. Done.

As shown in Figure 5b, the structural change of the N- linked sugar chains was remarkably confirmed that the gene appears when sikyeoteul the CGL1 and CGL2 gene expression GF003 the antibody using the knock-out object. Specifically, the ratio of the high mannose-type HexNAc2Hex5 (M5) sugar pattern in the N-sugar chain of the GF003 antibody was approximately 15 to 56 times higher than that of the wild type. On the other hand, the ratio of plant-specific HexNac4Pent1Hex3 and Deoxyhex1HexNAc4Pent1Hex3 sugar patterns in the N-sugar chain of the GF003 antibody was approximately 40% to 80% and 18% to 90% lower than that of the wild type, respectively. This pattern was clearly confirmed in individuals with genotypes in which either the CGL1 gene or the CGL2 gene showed complete knockout and the other was hetero. In particular, the ratio of high mannose-type HexNAc2Hex5(M5) sugar pattern in the N-sugar chain of GF003 antibody produced in #13 individual was approximately 56 times higher than that of wild type, and the ratio of plant-specific HexNac4Pent1Hex3 and Deoxyhex1HexNAc4Pent1Hex3 sugar patterns was wild type The contrast was approximately 80% and 90% lower, respectively.

In addition, when the gene CGL1 and CGL2 sikyeoteul gene expressing GF003 antibody using the object knockout was confirmed that the content of the mannose sugar chain bonded to the N- increase 5% to 50%, coupled to the N- linked sugar chains of the antibody GF003 It was confirmed that the content of fucose and xylose decreased by 10% to 50%.

Example 3. ADCC analysis of trastzumab produced in transgenic tobacco

The following experiment was conducted to analyze the efficacy of ADCC (antibody dependent cellular cytotoxicity) using animal cells. One day before the analysis, SKBR3 cells (target cells, target cells, T, ATCC, Cat#. HTB-30) were dispensed into a 96-well-plate at 10,000 cells/100 µl/well, at this time, a white 96-well-plate Was used to prevent interference from other wells that may occur during fluorescence measurement. Thereafter, Jurkat cells (effector cells, E) were harvested and resuspended in RPMI1640 medium containing 10% low IgG FBS. At this time, since FBS may affect ADCC analysis, low IgG FBS (Gibco, Cat#. 16250-078) was used, and hygromycin and G418 (Geneticin) were not added because they could kill SKBR3. . Subsequently, Jurkat cells were resuspended in RPMI1640 medium containing 10% low IgG FBS at 1.5×10 ⁶ cells/ml so that the ratio of effect cells (E) and target cells (T) was E:T=15:1. . In addition, the maximum concentration of each antibody was 1 ㎍/㎖, and serially diluted by 1/3 to prepare antibodies having a total of 10 different concentrations.

Thereafter, the medium present in the 96-well-plate was removed, and the total volume of antibodies produced from Jurkat cells and #3, #5, #10, #13 and #48 individuals selected in Example 2.2 was 100 μl. It was added to SKBR3 cells so that At this time, 1 mg of antibody was added to 1 ml of Jurkat cells having a density of 1.5×10 ⁶ cells/ml in an e-tube, and 200 μl of each well was added to a 96-well plate. Starting with this, 120 µl of Jurkat cells were added to the next nine wells, and then sequentially diluted by 60 µl each in the first 1 µg/ml well to complete a sample containing 10 different concentrations of antibodies.

The next day, the luciferase (Luciferase) substrate solution was put at 60 μl/well using a multi-channel pipette and incubated for 2 minutes in a CO ₂ incubator. Thereafter, the degree of fluorescence was measured using a loose ferase assay protocol with a FLUO STAR OMEGA microplate reader. Then, the concentration value of IC ₅₀ was obtained using the GraphPAD PRISM program (Table 5 and FIG. 6).

As a result, it was confirmed that the CGL1 gene and the CGL2 gene have high ADCC efficacy in antibodies produced from knock-out individuals. In particular, it was confirmed that the antibody extracted from the #13 individual through the IC ₅₀ value of each antibody exhibited about 10 times higher efficacy. Through this, it was confirmed that the efficacy of the antibodies produced in individuals in which the CGL1 gene and the CGL2 gene were knocked out was improved.

Claims (23)

1. Transgenic plants in which the expression of CGL1 and CGL2 is suppressed.
2. The method of claim 1,

   One of the CGL1 and CGL2 is homozygous knockout, and the other is heterozygous knockout, transgenic plants.
3. The method of claim 1,

   The CGL1 is a transgenic plant consisting of the amino acid sequence represented by SEQ ID NO: 1.
4. The method of claim 3,

   The amino acid sequence represented by SEQ ID NO: 1 is encoded by the nucleotide sequence represented by SEQ ID NO: 2, transgenic plants.
5. The method of claim 1,

   The CGL2 is a transgenic plant consisting of the amino acid sequence represented by SEQ ID NO: 3.
6. The method of claim 5,

   The amino acid sequence represented by SEQ ID NO: 3 is encoded by the nucleotide sequence represented by SEQ ID NO: 4, transgenic plants.
7. The method of claim 1,

   The plant is a transgenic plant with an increased content of mannose bound to the N-sugar chain.
8. The method of claim 1,

   The plant is a transgenic plant in which the content of fucose, xylose, or fucose and xylose bound to the N-sugar chain is reduced.
9. The method of claim 1,

   The plant is any one selected from the group consisting of tobacco, Arabidopsis, corn, rice, soybean, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper.
10. A transgenic plant for producing a target protein to produce a target protein by introducing a gene encoding a target protein into the transgenic plant of claim 1.
11. The method of claim 10,

    The protein of interest is an antibody or an enzyme, a transgenic plant for producing a protein of interest.
12. The method of claim 10,

    The target protein is any one selected from the group consisting of trastuzumab, bevacizumab, rituximab, and β-glucocerebrosidase, producing a target protein For transgenic plants.
13. The method of claim 10,

    The target protein is an increase in the content of mannose bound to the N-sugar chain, transgenic plants for producing the target protein.
14. The method of claim 13,

    The target protein is the content of mannose bound to the N-sugar chain is increased by 5% to 50%, transgenic plants for producing the target protein.
15. The method of claim 10,

    The target protein is the content of fucose, xylose, or fucose and xylose bound to the N-sugar chain is reduced. Transgenic plants for producing a target protein.
16. The method of claim 15,

    The target protein is the content of fucose, xylose, or fucose and xylose bound to the N-sugar chain is reduced by 10% to 50%, transgenic plants for producing the target protein.
17. i) preparing a recombinant expression vector comprising a sequence encoding an RNA guide and Chris flops associated protein that binds to the guide RNA, gene CGL1 and CGL2 genes coupled to a gene CGL1 complementarily complementarily; And

    ii) A method for producing a transgenic plant in which the expression of CGL1 and CGL2 is suppressed, comprising transforming plant cells or plant tissues with the recombinant expression vector.
18. The method of claim 17,

    The guide RNA complementarily binding to the CGL1 gene comprises a nucleotide sequence represented by SEQ ID NO: 5, a method for producing a transgenic plant.
19. The method of claim 17,

    The guide RNA complementarily binding to the CGL1 gene and the CGL2 gene includes the nucleotide sequence represented by SEQ ID NO: 6. A method for producing a transgenic plant.
20. The method of claim 17,

    The CRISPR related proteins are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx10, Csx16, Csx10 Any one selected from the group consisting of Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4, the method of producing a transgenic plant.
21. i) cultivating the transgenic plant of claim 10; And

    ii) A method for producing a protein of interest comprising the step of recovering the protein of interest from the plant.
22. A kit for producing a transgenic plant in which the expression of CGL1 and CGL2 is suppressed, including a guide RNA complementary to the CGL1 gene, a guide RNA complementary to the CGL1 gene and a CGL2 gene, and a CRISPR -associated protein.
23. As the target protein produced by the transgenic plant for producing the target protein of claim 10, the content of mannose bound to the N-sugar chain is increased, and fucose, xylose, or fucose and xylose bound to the N-sugar chain A target protein with a reduced content of os.

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