Recombinant antibodies having a unique glycan spectrum, produced from genome-edited CHO host cells, and methods for producing the same.

Abstract

To provide a method for producing a recombinant antibody having a unique sugar chain spectrum from a genome-edited CHO host cell.SOLUTION: The method of the present invention is a method for editing fut8 genes in CHO cells suitable for suspension culturing in a serum free medium by TALEN technology, so that the edited CHO host cells can produce recombinant antibodies with unique glycan profiles. The unique glycan profile is characterized by having mainly non-fucosylated N-linked oligosaccharide chains, a low degree of heterogeneity of N-glycosylation, and uniform glycan chains. The antibodies prepared by the methods of the invention have increased ADCC activity and antibody stability.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] This invention belongs to the field of biotechnology and relates to the unique process of extracting genome-edited CHO host cells from a unique genome. Recombinant antibodies having a distinctive sugar chain spectrum, and a method for producing the host cells and antibodies. It is. [Background technology]

[0002] CHO cells were developed in 1957 by Dr. Theodore T. Puc at the University of Colorado, USA. k isolated Chinese hamster ovary cells (Chi) from the ovaries of adult female hamsters. Nesse Hamster Ovary (CHO) refers to a type of epithelial adherent culture cell. It is a conventionally common cell system used in biotechnology. Among them, CHO-K1 cells are widely used industrially. It is used. CHO-K1 cells are a transformed cell line, and their chromosome distribution frequency It has 2n=22 cells and belongs to the sub-diploid cell group. It is stored in ATCC under the number: CCL-61. The CHO-K1 cell line used is widely adopted for the expression of recombinant DNA proteins. These cells are initially adherent culture cells, but after several generations of subculturing and screening, they become suspended. They can also grow in a specific way. The genes in CHO cells are prone to mutation, and gene transfer The process is also relatively easy. Early studies have shown that, compared to other process cell systems, CHO This study demonstrated that antibodies produced in cells have the closest glycan type to human serum antibodies. Therefore, CHO cells are good mammalian host cells for gene expression. The mechanism of action of therapeutic antibodies is to neutralize the target antigen by forming a complex with the target molecule. This is triggered, or the antibody Fc segment eliminates the antigen or pathogen through an immune response. The specific binding of antibody drugs to target molecules and the execution of their active effects are due to their complex higher-order structure and translation. It depends on post-translational modifications. Among these, glycosylation is the most important post-translational modification for antibodies. It plays an important role in the biological activity, in vivo metabolism, and immunogenicity of antibodies. The primary form of glycosylation of drugs is N-glycosylation, and the monosaccharide involved is mainly glucose. Galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine These include fucose and cyanates (NANA, NGNA). Depending on the number of galactose molecules at the end... Therefore, the antibody molecule Fc segment Asn297 is linked to a bifurcated or multi-branched double strand (bian (Tennary) Complex oligosaccharides include G0, G1(1,3), G1(1,6) and G2 complex They can be divided into several types. Among them, each type is either fucose (F) or bisecting glass. They can be divided into 16 species depending on whether they contain ctose (B) (Glycobiology, August 2015,1-10.DOI:10.1093 / glycob / cwv0 65). Therefore, the oligosaccharide type of the antibody heavy chain has sialylated and high-mannose types at the terminals. Even without considering whether they exist or not, there are at least 36 types. Also, both heavy chains of an antibody Because random combinations result in over 400 possible types of glycans. The antibodies exhibit a high degree of heterogeneity.

[0003] Different glycan types also have different effects on the pharmaceutical character of therapeutic antibodies. The content of glycans (Man5) allows for rapid elimination of antibodies from the blood and shortens their half-life. This leads to (MAbs, 2012, 4(4):509-520). G0F is involved in the complement pathway. This contributes to accelerating the elimination rate. G2F levels increase in the umbilical cords of pregnant women and newborns. It is being done. Sialic acid modification has a significant effect on the inflammatory effect of intravenous immunoglobulin administration. A decrease in fucose leads to a significant increase in ADCC activity (JBC (2003) Chemi). (Stry 278, 3466-3473). Therefore, the main mechanism of action of therapeutic antibodies and medicine Depending on the pharmaceutical application, it is necessary to design and improve the glycans of antibodies.

[0004] Unlike protein synthesis, antibody glycosylation does not have a template to follow. The lycosylated type and the proportion of each oligosaccharide component are influenced by the host cell type and culture requirements. It resonated with me. By engineering host cells, the oligosaccharide components of monoclonal antibodies were improved. The methods by which the activity mediated by Fc is enhanced are scattered across different documents and patent reports. For example, β(1,4)-N-acetylglucosamine transferase III ( Antibodies prepared in CHO cells overexpressing GnTIII were compared to antibodies expressed in parental cells. All showed higher ADCC activity, with the difference in activity being approximately 10 to 20 times (Biotechn ol Bioeng. (2001) Aug 20;74(4):288-94). However, the overexpression of GnTIII itself is toxic to CHO cells, and its expression Since it is exogenous, the expression of GnTIII increases with the number of passages during the culture process. The amount decreases, so when used as a host cell, the fucose content of the produced antibody Modification affects the uniformity of antibody drugs. Examples of cell lines that produce defucosed antibodies include Therefore, protein fucose-deficient Lec13 CHO cells (Ripka et al. A rch.Biochem.Biophys.249:533-545(1986) is cited. However, since its protein yield is extremely low, it is not suitable as a host cell for producing therapeutic antibodies. (Yutaka Kanda et al., Biotechnol Bioeng. (20 06) Jul 5;94(4):680-8). The α-1,6-fucosyltransferase gene FUT8 was knocked out in CHO cells (Yamane-Ohnuk i et al. (2004), Biotechnol. Bioeng. 87:614), which also results in a reduction in the fucose content of the produced antibodies. For example, in the FUT8 knockout cell line described in the patents of Yamane-Ohnuki and Kyow a Hakko, a method for controlling the degree of antibody fucosylation and enhancing the ADCC (antibody-dependent cell-mediated cytotoxicity) activity was disclosed. The above method suppresses the expression of the fut8 gene in host cells using specific siRNA, thereby reducing the level of fucose in the antibodies produced by the host cells. This method has the same drawbacks as the CHO cell line overexpressing GnTIII described above. First, it is necessary to introduce foreign sequences into the host cells. Second, the suppression efficiency of the target gene by siRNA is at most about 70%. Finally, the stability of siRNA expression may affect the quality attributes of antibody drugs. Recently, different literatures and patents have reported methods of using emerging genome editing technologies to edit target genes in host cells to inactivate the intracellular FUT8 enzyme and reduce the level of fucose in antibodies. For example, Malphettes et al. (2010) adopted the zinc finger enzyme (ZFN) technology and knocked out from the parental cell DG44 to obtain a homozygous

[0005] type fut8 gene knockout DG44-derived clone, and the antibodies produced from this cell line showed a reduced fucose level. For example, Malphettes et al. (2010) adopted the zinc finger enzyme (ZFN) technology and knocked out from the parental cell DG44 to obtain a homozygous type fut8 gene knockout DG44-derived clone, and the antibodies produced from this cell line It has been reported that the antibody contains no fucose at all. Beurdeley et al. (2012) ) employs TALEN technology to edit the fut8 gene in CHO-K1 cells, thereby modifying the host cell structure. It has been reported that the FUT8 enzyme activity in cells is lost. Also, Sun et al. (2015) ) edited the 10th exon of the fut8 gene using CRISPR / Cas9 technology It has been reported that this method causes the deletion of FUT8 enzyme activity in CHO-K1 cells. [Overview of the project]

[0006] Based on this, existing antibody drugs almost exclusively have a single N-glycosylation modification to Fc. Although it is limited, the sugar chain type components and their content do not match and are prone to fluctuation, and Regarding issues such as the impact on manufacturing stability, genome-edited CHO Antibodies produced from host cells that have a unique sugar chain spectrum, and the production of said antibodies A method needs to be provided. The above-mentioned objectives of the present invention are achieved by the following means: The first aspect of this invention is the A shown in SEQ ID NO.10 and SEQ ID NO.11. It has a mino acid sequence, or is shown in SEQ ID NO.10 and SEQ ID NO.11. The sequence that is performed, and at least 90%, at least 91%, at least 92%, at least 9 3%, at least 94%, at least 95%, at least 96%, at least 97%, small A pair of polypeptides having sequences with at least 98% or 99% identity Regarding the code. In some embodiments, SEQ ID NO.10 and SEQ ID NO.1 The pair of polypeptides shown in 1 are the upstream and downstream DNA-binding domains of the TALEN. These are amino acid sequences, each capable of binding to a specific base region of a particular gene.

[0007] The second aspect of this invention is shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively. Provides a pair of polynucleotides that encode a pair of polypeptides. In this, the pair of polynucleotides are SEQ ID NO.12 and SEQ ID It has the nucleic acid sequence shown in NO.13, or SEQ ID NO.12 and SEQ ID The sequence shown in NO.13 and at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, less Both have 97%, at least 98%, or at least 99% identity.

[0008] The third aspect of the present invention is that the pair of polypeptides are transcription activator-like effectors (Fok I) Provides a pair of fusion proteins fused to the amino acid sequence of the DNA cleavage domain. In some embodiments, the DNA cleavage of transcription activator-like effectors (FokI) The amino acid sequence of the amino acid is either natural or artificially modified. In some aspects The pair of fusion proteins are SEQ ID NO.14 and SEQ ID NO.16 It has the amino acid sequence shown, or SEQ ID NO.14 and SEQ ID NO.1 The sequence shown in 6 and at least 90%, at least 91%, at least 92%, and at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , having at least 98% or at least 99% identity. The pair of fusion proteins are derived from the two nucleotide sequences of the Fut8 gene of CHO. The fragment can be recognized specifically. In some embodiments, the two nucleos of the Fut8 gene The cide sequence fragment is located in exon 1 (SEQ ID NO. 7) of the Fut8 gene. These are two nucleotide sequence fragments. In some embodiments, the two Fut8 genes The nucleotide sequence fragments are assigned to SEQ ID NO.3 and SEQ ID NO.4 respectively. It has the nucleotide sequence shown. In some embodiments, SEQ ID N Between O.3 and the nucleotide sequence shown in SEQ ID NO.4, There is one spacer having the arrangement shown in NO.5.

[0009] The fourth aspect of the present invention is also the nucleotide encoding the pair of fusion proteins described above. Provides a pair of nucleotides. In some preferred embodiments, the above nucleotide pair is SEQ It has the nucleic acid sequence shown in ID NO. 15 and SEQ ID NO. 17, or SEQ The sequences shown in ID NO. 15 and SEQ ID NO. 17 are at least 90% and less than 90%. At least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, or at least 99% It possesses the same identity.

[0010] The fifth aspect of the present invention is also that at least one of the pair of polynucleotides The present invention provides a vector containing one of the polynucleotides. In some embodiments, The vector is a plasmid.

[0011] The sixth aspect of the present invention also provides host cells transformed by the vector.

[0012] In some embodiments of this, the cells transformed by these vectors are geno These are edited CHO host cells, and their parent cells are derived from the CHO-K1 cell line.

[0013] In some aspects of this, the CHO host cells whose genomes have been edited are, without parental cells Suitable for serum suspension culture, the parent cells were named CHO-BAT.

[0014] In some aspects of this, the parent CHO host cell of the CHO host cell whose genome has been edited is a CHO host cell. -BAT is a CHO-K1 selected to satisfy one or more of the following characteristics: The above cells have high transfection efficiency, The above cells have a short logarithmic growth time. The cells described above have the ability to achieve high cell density in CD-CHO culture.

[0015] In some embodiments, the CHO host cells in which the genome has been edited are compared to the parent cells. In all cases, the region of the FUT8 gene has base deletions, insertions, or nonsense mutations, so the cells in question The endogenous α1,6-fucosyltransferase (Fut8) loses its enzymatic activity. .

[0016] The cells described above do not contain exogenous DNA sequences. Recombinant antibodies expressed in the above cells as host cells exhibit unique glycan spectral characteristics. Hold.

[0017] In some aspects, CHO host cells in which the above genome has been edited are FUT8 genes Editing of the gene's exon 1 genome causes the endogenous Fut8 in the affected cell to lose its enzymatic activity. In other words, the above cells produce base deletions and nonsense mutations in the FUT8 gene. It is characterized by not containing the DNA sequence of the expression vector incorporated during the process.

[0018] The above cells exhibit unique glycosylation spectral characteristics when recombinant antibodies are expressed as host cells. It possesses. Its characteristics mainly include having non-fucosylated N-linked oligosaccharide chains, and This includes other glycosylation spectral features of the antibody.

[0019] In some aspects, the CHO host cells in which the above genome has been edited contain the FUT8 gene. The gene was knocked out. The cells showed negative results when bound to agglutinin LCA. The cells described above were named CHO-BAT-KF.

[0020] The seventh aspect of the present invention is to use at least one of the aforementioned pair of polypeptides, or the aforementioned pair At least one of the polynucleotides, or a small portion of the aforementioned pair of fusion proteins. We will provide at least one of the above, or the aforementioned vector, or a kit containing the aforementioned host cells. do.

[0021] The eighth aspect of this invention relates to the aforementioned pair of polypeptides / pair of polynucleotides / pair of fusion tannins. This provides the use of a protein / vector for editing the Fut8 gene in CHO cells.

[0022] The ninth aspect of the present invention relates to the aforementioned pair of polypeptides / pair of polynucleotides / pair of fusion tannins. Preparation of antibodies from proteins / vectors / host cells, particularly antibodies with unique glycan spectra. It provides use for the aforementioned pair of polypeptides / pair of polynucleotides / We provide antibodies prepared using a pair of fusion proteins / vectors / host cells.

[0023] The tenth of the present invention relates to the aforementioned pair of fusion proteins, or a pair of polynucleotides, or The chemist was introduced into CHO cells, cultured at 37°C for 14 days, and then pressure screening was performed. This includes obtaining CHO cells in which the Fut8 gene is knocked out by the limiting dilution method. This provides a method for editing the Fut8 gene of CHO. An example of this method is Wood e See t al., J Immunol. 145:3011 (1990).

[0024] One aspect of the present invention is a process comprising the following steps to unify genome-edited CHO host cells from a single host cell. A method for producing recombinant antibodies having a distinctive sugar chain spectrum, or a method for producing antibodies using the same. Provides antibodies:

[0025] (1) The aforementioned pair of fusion proteins, or pair of polynucleotides, or vectors C Transfection into HO cells (e.g., wild-type CHO cells) and pressure screen The process involves obtaining CHO cells in which the Fut8 gene has been knocked out by limiting dilution. , (2) The Fut8 gene knocks the plasmid encoding the antibody gene expression cassette. Electroporation was performed on the CHO cells that were removed, followed by pressure screening and limiting dilution. This process yields a stable CHO cell line that secretes antibodies.

[0026] In a preferred embodiment, step (1) involves transting the vector into wild-type CHO cells. Infection is performed, and the plasmid is comfortably transferred to stable wild-type CHO cells. To perform an action. In a preferred embodiment, the aforementioned CHO cells are CHO-K1, and more preferably... As mentioned above, CHO-K1 is suitable for serum-free culture. In a preferred embodiment, the aforementioned antibody is an anti-CD20 antibody, and more preferably, The antibodies described are humanized or entirely human-derived anti-CD20 antibodies, and more preferably, as described above. The antibody is BAT4306F, and more preferably the aforementioned BAT4306F antibody is SEQ The light chains shown in ID NO. 20 and the heavy chain shown in SEQ ID NO. 21 It possesses both properties. The inventor has used the method, cells, polypeptides, etc. of the present invention to create multiple types It is used in the preparation of various antibodies, and after further investigation, it was found that the multiple types of antibodies prepared are all All of them express highly consistent glycan types, and the degree of heterogeneity of those glycan types is low. They discovered that the method, cells, etc. of the present invention are suitable for the preparation of all types of antibodies. This was shown. In one embodiment, the antibody binds to CD20. In one embodiment, CD The 20-conjugated antibody is a humanized antibody. In a preferred embodiment, the BAT4306F humanized antibody The antibody is the B-Ly1 antibody sequence from patent WO2005044859, specifically the B- of the heavy chain variable region. This is the HH6 amino acid sequence and the B-KV1 amino acid sequence of the light chain variable region. BAT4306F The antibody is the sequence shown in SEQ ID NO.20 and SEQ ID NO.21. It contains a pair of light and heavy chains. In one embodiment, the CD20-conjugated antibody is all of human origin BAT44 This is an 06F antibody, and the sequence shown in SEQ ID NO.22 and SEQ ID NO.23 It comprises a pair of light and heavy chains in a row. In one embodiment, the antibody is BAT1206F. The BAT1206F antibody was used on both light chains indicated by SEQ ID NO. 18, and SEQ I It has both heavy chains as shown in D NO.19. In one embodiment, the antibody is BAT020 It is 6F, BAT0206F binds to EGFR, and this antibody has SEQ ID NO. 24 It has two light chains as shown in [image] and two heavy chains as shown in SEQ ID NO. 25. One embodiment In this case, the antibody is BAT0808, and this antibody binds to Trop2, SEQ I Both light chains shown in D NO.26, and both heavy chains shown in SEQ ID NO.27. It possesses. In some embodiments, the modified glycoprotein is secreted from the host cell. In some embodiments, the modified glycoprotein is an antibody.

[0027] As an example of a specific embodiment, from a CHO host cell in which the genome of the present invention has been edited, A method for producing recombinant antibodies having a unique sugar chain spectrum, or a method for producing recombinant antibodies using the same. The antibody production process includes the following specific steps: The aforementioned pair of fusion proteins, or a pair of polynucleotides, or vectors are wild-type C After introducing it into HO cells and transfecting them, the cells are then treated with C, which contains plant agglutinin (LCA). Add D CHO (Sigma) + 10% FBS (fetal bovine serum) and perform pressure screening. After 14 days, the viable cells were inoculated into 96-well cell culture plates at a rate of 0.5 cells / well. The serum concentration was reduced to 5%, and after 7 days, the cells were introduced into 24-well cell culture plates. Then, return the cells to the CO2 incubator, and after 7 days, remove some of the cells and heat them at 1000 rpm. After 5 minutes, centrifuge, change to PBS once, and label with 2 μl of fluorescein. Take the LCA, mix it with the cells, incubate on ice for 30 minutes, and wash once with PBS. First, read the fluorescence value using a flow cytometer (BD, C6) and use it as a negative control. Using wild-type CHO cells that were not transfected, positive cells were used for cell culture. Transferred to a 6-well plate, serum concentration reduced to 1%, and after 7 days, the cells were removed using a small roller. The cells were transferred to bottles, and the culture medium at this time was serum-free CD CHO, completing the acclimatization process. Cells were taken from the area, and the CHO genome was extracted using a plasmid extraction kit (Omega). Using this as a template, the primers L130for (SEQ ID NO.1) and L130r Polymerase chain reaction (PCR) using ev (SEQ ID NO.2) and taq enzyme. The PCR product is then linked to a T vector (Promega), transformed, and played. The sample was spread on a substrate, and the following day, a single colony was sequenced with T7 primer to obtain DNA. The sequence was analyzed using STAR analysis software, and base deletions appeared when compared to the wild-type CHO genome sequence. CHO cells were scaled up and named CHO-BAT-KF. A cell library was established from CHO-BAT-KF, and CD CH4 containing 7.5% DMSO was used. Cells were cryopreserved in a cryopreservation solution, then introduced into a liquid nitrogen tank for long-term storage. Antibody genes were then processed. The plasmid to be digested is taken, its OD260 is measured, and electroporation is performed. Place a 50μg plasmid in the top 10 7 Mix with CHO-BAT-KF and electroporate Transfection is performed using a Biorad cell culture system, and the cells are then placed in a 96-well cell culture tray. Inoculate the body and 48 hours later, methionine iminosulfone (methionine sulfon Oximine (MSX) was added, and after 14 days, it was coated with an anti-FC polyclonal antibody. After blocking the ELISA plate with 3% BSA, add the supernatant to the plate and bake at 37°C. The cells were incubated for 2 hours. They were washed 5 times with PBST and labeled with anti-horseradish peroxidase. Adding sheep anti-human kappa / lambda light chains and 2M H2SO4, microparticles are used. The OD450 value was read using a rate reader. Those with high titers were cloned and cultured on a larger scale. The supernatant of the collected cells was then gathered, and an antibody protein in which fucose was knocked out was obtained.

[0028] The present invention also provides cells that are genome-edited CHO host cells. In the CHO host cells whose genomes have been edited, the edited Fut8 gene is SEQ ID The sequence shown in NO.28, or the sequence shown in SEQ ID NO.28, and less 90%, at least 91%, at least 92%, at least 93%, at least 94% %, at least 95%, at least 96%, at least 97%, at least 98%, less Sequences with at least 99%, at least 99.5%, or at least 99.8% identity To possess.

[0029] The present invention also relates to nucleic acids containing the sequence shown in SEQ ID NO. 28, or SEQ The sequence shown in ID NO.28 and at least 90%, at least 91%, at 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, at least 99.5%, or This provides nucleic acids containing sequences with at least 99.8% identity.

[0030] This invention has also been deposited with the China Center for Typical Cultures Depositary, deposit number: CCTCC N O:C2017127, Entrustment date: August 10, 2017, Address: Wuhan University, Wuhan, China, Min. Classification and naming: Chinese hamster ovary cells CHO-BAT-KF fut8(- / -) This provides CHO host cells. In some embodiments, the host cells are maintained in serum-free medium. In some embodiments, The host cells are maintained in a suspension culture. The present invention further relates to a culture medium containing the above host cells. The present invention relates to a culture fermentation vessel containing multiple host cells in the culture medium. In some embodiments, The culture medium described above is serum-free.

[0031] The twelfth aspect of this invention is a unique sugar produced from genome-edited CHO host cells. Recombinant antibodies possessing a chain spectrum, the above antibodies are humanized or entirely of human origin. It has a unique glycosylation mode and a low degree of N-glycosylation heterogeneity. The present invention provides an antibody that exhibits significantly increased ADCC activity.

[0032] In some aspects, the above genome is produced from CHO host cells that have been edited. Recombinant antibodies with unique sugar chain spectra bind to CD20 on the cell membrane surface. It is a t-induced antibody.

[0033] In some aspects, the above genome is produced from CHO host cells that have been edited. Recombinant antibodies with unique sugar chain spectra possess unique glycosylation patterns. The above glycosylation pattern is one or more of the N-linked polysaccharides in the above antibody. Glucose (Glc), Fucose (Fuc), Galactose (Gal), Mannose (Man) ), high mannose, glucosamine, G0 and acetoglucosamine It is characterized by a fluctuating level of the sugar moiety selected from the group consisting of (GlcNAc). The characteristics of the glycosylation mode described below satisfy one or more of the following preferred requirements: The above antibodies have a low fucose content (0-5%). The level of galactose contained in the above antibody was relatively low (≤30%). In the above antibodies, the level of mannose was relatively low (≤5%). The levels of high mannose in the above antibodies were relatively low (≤5%). The G0 level of the above antibody is relatively high (≧60%).

[0034] In some embodiments, the level of galactose contained in the above antibody is lower. The percentage is ≤5%. In some embodiments, the level of G0 contained in the above antibody is relatively high, ≥80 It is a percentage.

[0035] In some aspects, the above genome is produced from CHO host cells that have been edited. Recombinant antibodies with unique sugar chain spectra have a fucose content of 0. It meets the desirable requirements. In some aspects, the above genome is produced from CHO host cells that have been edited. Recombinant antibodies with unique sugar chain spectra exhibit extremely low levels of N-polysaccharide heterogeneity. It has low, uniform sugar chains. In some aspects, the above genome is produced from CHO host cells that have been edited. Recombinant antibodies with unique sugar chain spectra exhibit relatively strong ADCC activity of Fc. . In some embodiments, the antibody is BAT4306F, located above Figure 10. It has the sugar chain spectrum shown.

[0036] In some embodiments, the BAT4306F is indicated as SEQ ID NO.20. In cases where it has two light chains and two heavy chains as shown in SEQ ID NO. 21 This means that mutations in the sequence are not ruled out, and these mutations It is acceptable as long as it does not affect the action of the antibody.

[0037] The thirteenth aspect of this invention is that the first exon of the Fut8 gene in CHO host cells contains one This provides CHO host cells in which the Fut8 gene is knocked out, including a deactivating mutation. A mutation is a substitution or deletion of one or more amino acids, or a frameshift mutation, for example. The mutations shown in Figure 6 may also be present.

[0038] The present invention further provides a pharmaceutical composition comprising the aforementioned antibody. A preferred embodiment is: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0039] The present invention further provides an effective amount of the antibody / antibody fragment disclosed in the present invention to the target that requires it. The present invention provides a method for preventing or treating a disease, which includes administering to [a specific substance]. In some embodiments, E-diseases include cancer, allergic diseases, cardiovascular diseases, inflammatory diseases, metabolic diseases, neurological diseases, and This group consists of viral infections and / or bacterial infections. For example, the above disease is cancer. Alternatively, it may be an allergic disease. In some embodiments, the subject is a mammal, for example They are human.

[0040] Compared to conventional antibody drugs, the present invention has the following advantages. Conventionally, commercially available antibody drugs have been almost exclusively limited to single N-glycosylation modifications of Fc. However, the sugar chain type components and their content do not always match and are prone to fluctuations. Complexity exists, particularly in terms of production stability. The genome provided by the present invention Recombinant cells with a unique glycosylation spectrum produced from edited CHO host cells. Antibodies, on the one hand, have a low degree of heterogeneity in N-glycosylation and good homogeneity of sugar chains, ADCC activity is enhanced, and the quality attributes and pharmaceutical characteristics of antibody drugs are greatly improved. Ta. The aforementioned antibody is the corresponding unmodified CHO-K1 (ATCC#CCL-61) or Compared to antibodies produced from parent cells CHO-BAT suitable for suspension, FcγRIIIA receptor The binding affinity to the substance has increased.

[0041] Antibodies produced from the above-described modified host cells are produced from the corresponding unmodified host cells. Compared to the corresponding antibody produced from the cell, it shows increased affinity for FcγRIIIA. [Brief explanation of the drawing]

[0042] [Figure 1] Figure 1 shows CHO-K1 (ATCC#CCL-61) growing on the wall and the CHO-BAT cell line adapted for suspension growth in serum-free medium. [Figure 2] Figure 2 is a schematic diagram of the TALEN expression plasmid pCS2-Fok1. [Figure 3] Figure 3 shows the validation of TLEN protein function. The left side of the electrophoresis graph represents the wild type. PCR products of gene-edited cell genomes showed both bands at 500 bp and 750 bp, while the wild type showed only one band at 750 bp. This is consistent with the predicted results and demonstrates that the Talen protein pair is functional. Here, Lane 1: 100 bp marker, Lane 2: wt, Lane 3: pooled. [Figure 4] Figure 4 shows the results of FACS analysis of cells grown in a 24-well plate. Genetically edited cell clones are cells that are negative for FITC-labeled LCA binding, while wild-type cells are cells that are positive for FITC-labeled LCA binding. [Figure 5] Figure 5 shows that analysis using a glycan chip revealed that the fucose content of clones 41 and 43, which were edited from the tested genes, was reduced to 0-10%, while the fucose content of wild-type antibody 1206 was 80%. [Figure 6]Figure 6 shows the results of TALEN protein targeting sequences being amplified by PCR, sequenced, and then aligned using lasergeneMegAlign sequence analysis software. Cells 191-1, 191-2, 217-1, and 217-3 are cell clones with four selected genomes prepared. Genomes were extracted from each of these clones and used as DNA templates. PCR reactions were performed using primers L130 for and L130 rev, and the amplified products were subjected to CEL-1 base mismatch analysis. The results showed that cell clones 191-1 and 191-2 were heterozygous, while cell clones 217-1 and 217-3 were homozygous. Based on the alignment results, the genome-edited homozygous cells 217-1 and 217-3 were selected and designated as CHO-2G8 and CHO-1D6, respectively. Finally, CHO-2G8 was selected as the host cell and subjected to subsequent tests, and this host cell was named CHO-BAT-KF. [Figure 7] Figure 7 shows a comparison of the proliferation density of CHO-BAT-KF with that of the parent cell CHO-BAT. [Figure 8] Figure 8 shows a comparison of the proliferative capacity of CHO-BAT-KF with that of the parent cell CHO-BAT. [Figure 9] Figure 9 shows the results of MALDI-TOF MS analysis of the N-polysaccharides of BAT4306F and 4306 antibody molecules using a MALDI-TOF MS mass spectrometer. The N-polysaccharides derived from BAT4306F both had one less fucose molecule compared to those derived from 4306. The left figure shows the 4306 antibody molecule produced from parent cells, and the right figure shows the BAT4306F antibody molecule. [Figure 10] Figure 10 shows that BAT4306F has a lower fucose content, a lower degree of glycan heterogeneity, and a product with better uniformity compared to GAZYVA (obinutuzumab). [Figure 11] Figure 11 compares the ADCC activity of BAT4306 wild-type, glycosylated BAT4306F, and anti-CD20 antibodies such as obinutuzumab and rituximab when Raji is used as the target cell and PBMCs are the effector cells. [Figure 12]Figure 12 compares the ability of three antibodies—glycosylated BAT4306F, obinutuzumab, and rituximab—to eliminate B cells in whole blood in vitro at concentrations of 50, 25, and 10 ng / mL. [Figure 13] Figure 13 shows a comparison of the glycosylation spectra of anti-CD20 antibodies BAT4306F and BAT4406F, anti-EGFR antibody BAT0206F, and anti-Trop2 antibody BAT0808 produced in CHO-BAT-KF cells. The genome-edited CHO host cells of the present invention were deposited at the China Center for Typical Cultures Depositary (CCTCC), with deposit number: CCTCC NO: C2017127, deposit date: 2017.8.10, depositary institution: Wuhan University, Wuhan, China, classification and naming: Chinese hamster ovary cells CHO-BAT-KF fut8(- / -). [Modes for carrying out the invention]

[0043] The technical configuration of the present invention will be further described below based on specific embodiments, but the protection of the present invention is limited. The scope of what is required is not limited to these. Based on the spirit of the present invention, Non-essential changes and adjustments are also included within the scope of protection sought by this invention.

[0044] In this invention, expressions such as "level" or "content" of the sugar portion of the antibody are used interchangeably. These terms express the idea that a certain type of sugar portion accounts for the mass ratio of the total sugar portion of the antibody.

[0045] For example, in the present invention, "amino acid" means that it is encoded either directly from nucleic acids or as a precursor. It is a carboxyl α-amino acid. A single amino acid consists of three nucleotides (so-called Encoded by nucleic acids consisting of codons (or base triple bodies). One amino acid is , coded by at least one codon. The same amino acid is coded by different codons. This phenomenon is called "codon degeneracy." The term "amino acid" used in this application refers to: A naturally occurring carboxy-α-amino acid, alanine (three-letter code: ala, single letter) Parent code: A), arginine (arg, R), asparagine (asn, N), asparagine Glutamic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid Isoleucine (glu, E), glycine (gly, G), histidine (his, H), isoleucine ¹(ile, I), leucine (leu, L), lysine (lys, K), methionine (me t, M), phenylalanine (phe, F), proline (pro, P), serine (ser) , S), threonine (thr, T), tryptosawyer (trp, W), tryptophan ( Includes tyr (Y) and valine (val, V).

[0046] In this invention, interchangeable terms include "polynucleotide," "nucleic acid," or "nucleic acid sequence." This refers to a, c, g and t, which are nucleotides (also called bases) in RNA. A multimeric molecule composed of u), such as DNA, RNA, or a variant thereof. The polynucleotide molecule may be a naturally occurring polynucleotide molecule, or it may be synthesized. Polynucleotide molecules, or one or more naturally occurring polynucleotide molecules and This may be a combination of one or more synthesized polynucleotide molecules. So, if one or more of these nucleotides are changed (for example, by inducing mutagenesis), then... This also includes naturally occurring polynucleotide molecules that are lost or added. The nucleic acids are single It may be used to stain other nucleic acids, such as expression cassettes, plasmids, or host cells. It can be something incorporated into the body. Nucleic acids are nucleic acid sequences composed of single nucleotides. Characterized by the amino acid sequence of a polypeptide, which encodes the amino acid sequence. The procedures and methods for transforming with the corresponding nucleic acid sequence are well known to those skilled in the art. Therefore, nucleic acids are characterized by nucleic acid sequences composed of corresponding single nucleotides. It is also acceptable to characterize the polypeptide encoded by the amino acid sequence. It can also be done this way.

[0047] Furthermore, the aforementioned "polynucleotide," "nucleic acid," or "nucleic acid sequence" is present in nucleic acid molecules. The percentage of modified nucleotides relative to the total number of nucleotides is, for example, small. At least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or It is 100%, including modified nucleotides.

[0048] In the present invention, "polypeptide" is made up of amino acids linked by peptide bonds. It is a polymer, which may be natural or synthetic. Approximately 20 or fewer. A polypeptide having two or more amino acid residues is called a "peptide." A molecule composed of peptides, or a single peptide having 100 or more amino acid residues. Molecules containing peptides are called "proteins." Polypeptides include, for example, glycosphing. It may contain non-amino acid components such as metal ions or carboxylates. The component can be added to cells expressing the polypeptide, and in response to variations in cell type. It can also change. Here, polypeptides are defined as having a structure of their amino acid backbone, or Characterized by nucleic acids that encode it. For example, the addition of glycosyl is usually not specified. However, they may also be present. Furthermore, the aforementioned "polypeptides" contain amino acid molecules. The percentage of modified amino acids in relation to the total number of amino acids is, for example, at least Approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% It also includes modified amino acids, which make up %.

[0049] In the present invention, "host cell" refers to a mononuclear cultured microorganism, eukaryotic cell, or cell line. This means that even if it is used as a recombinant vector, or if it is a different transposed polynucleus Recipients of rheotide, or passaged cells of transfected primordial cells This is also acceptable. In some embodiments, the host cell is a non-lymphocyte, and the host cell is the same It exhibits a unique glycan spectrum. In some embodiments, the host cell is, for example, NS These include O cells, monkey COS cells, Chinese hamster ovary (CHO) cells, etc. In this embodiment, the host cells are selected from the group consisting of Chinese hamster ovary (CHO) cells. It will be discovered. In some embodiments, the host cell is CHO-K1, CHO- S, DUXB11, CHO-1E5, CHO3F, CHO / DG44, CHO-BAT It is selected from a group consisting of CHO-2.6 cells. In some embodiments, the above host The cells produce antibodies that exhibit a unique sugar chain spectrum. CHO host cells edited with this gene, for example CHO-BAT-KF fut8(- / -), are cultured. The cells can grow in a device (including a fermentation tank) that enables the growth of nutrients and cultures. It may grow into a single layer, or it may adhere to the surface. Alternatively, the host cell may be a suspension They may be grown inside. These cells may also be grown in a serum-free culture medium. The culture medium may be a commercially available medium, such as DMEM / F12, but is not limited to that. No. Edited CHO host cells, even after many passages in culture medium, will not have their specific characteristics. It can maintain a unique glycan spectrum. For example, edited CHO host cells can maintain at least After approximately 20, 30, 40, or 50 generations, it also maintains its specific and unique sugar chain spectrum. In some embodiments, modified CHO host cells remain after at least approximately 60 generations. It also maintains its specific and unique sugar chain spectrum. In another aspect, it is modified The CHO host cells number at least approximately 100, 150, 200, 500, or 1000 or more. Over subsequent generations, it also maintains its specific and unique sugar chain spectrum.

[0050] Regarding the glycosylation pattern of host cells, N- or O-glycosylation occurs at any protein portion. Cosylation may occur. Note that the position of the nitrogen in the asparagine amide, or This refers to the position of one or more oxygen atoms in the hydroxyl group of hydroxylysine, serine, or threonine. A number of sugar molecules may be added. A characteristic of the glycosylation mode is that at least two or more sugars The molecules or sugars (e.g., monosaccharides, disaccharides, polysaccharides, or oligosaccharides) have been modified. For example, sugar molecules include trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, xunasaccharides, or derivatives thereof. Deoxy sugars (e.g., deoxyhexasaccharides), N- or O-substituted derivatives, such as shealycin. Alternatively, it may be a sugar having an amino group. Sugar molecules include galactose (Gal), budol Glc, mannose (Man), N-acetylneuraminic acid (NeuAc), Coagulant (Fuc), N-acetylgalactoseamine (GalNAc), N-acetylg It may also include lucosamine (GlcNAc) and xylose (Xyl), however these This is not limited to this. Sugar molecules may be linked to other sugar molecules by α or β bonds.

[0051] The "antibody" of this invention refers to any form of antibody, such as recombinant antibodies, humanized antibodies, chimeric antibodies, etc. This includes antibodies, single-chain antibodies, fusion antibodies, monoclonal antibodies, polyclonal antibodies, etc. The antibody may also be a fragment. The antibody may also be a drug, toxin, or therapeutic agent. It may be bound to a radioactive isotope. Bispecific antibodies from the host cells of the present invention Fusion proteins can also be produced, including heterogeneous antibodies that bind to one or more antigens. Therefore, antibodies include naked antibodies, conjugated antibodies, and These may include antibody fragments, and they may be monospecific or multispecific. stomach.

[0052] Possible embodiments include those in which the antibody or antibody fragment is not particularly limited, but are anti-HER 2, anti-CD20, anti-EGF, anti-VEGF, anti-PDGF, anti-EpCam, anti-CD3, anti-CD 4, anti-CD19, anti-CD30, anti-CD33, anti-CD40, anti-CD51, anti-CD55, anti-C D80, anti-CD95, anti-CCR2, anti-CCR3, anti-CCR4, anti-CCR5, anti-folate, anti-C Examples include XCR4, anti-EGFR, or Trop2 antibodies. Preferred embodiments include: The aforementioned antibodies are either humanized or entirely of human origin. In the pharmaceutical composition of the present invention, an antibody of a required purity and a physiologically acceptable substance that can be selected Obtaining carriers, excipients, or stabilizers (Remington's Pharmaceuticals) al Sciences 16th edition,Osol,A.Ed.(1980 By mixing )), the antibody of the present invention is prepared as a lyophilized preparation or aqueous solution for storage. Obtain the product formulation. Acceptable carriers, excipients, or stabilizers are used in the recipe in the amount and concentration specified. It is non-toxic to, for example, phosphites, citrates, and other organic acids. Buffers, antioxidants of ascorbic acid and methionine, (e.g., stearyldimethylbenzoate) Benzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, Benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens For example, methylparaben or propylparaben, catechol, resorcinol, cyclophenate Preservatives (xanol, 3-propanol, and m-cresol), low molecular weight (approximately 10%) Polypeptides (less than 1 / 2 units), such as serum albumin, gelatin, or immunoglobulins. Proteins, such as hydrophilic polymers of polyethylenepyrrolidone, such as glycine and glutamic acid , asparagine, histidine, arginine or lysine amino acids, glucose, mannol Monosaccharides, disaccharides, and other carbohydrates consisting of sucrose or dextrin, such as EDTA. Chelating agents, such as sugars like sucrose, mannitol, trehalose, or sorbitol, e.g. For example, sodium salt-forming counterions, metal complexes (e.g., Zn-protein complexes), and / or For example, Tween, Pluronics TM or polyethylene glycol (MacroGol Examples include nonionic surfactants (e.g., 100%).

[0053] The antibody, pharmaceutical composition, and pharmaceutical preparation of the present invention shall be administered by any suitable means. This can also be done via parenteral, subcutaneous, intraperitoneal, intrapulmonary, or nasal methods, but if necessary... Therefore, when considering local immunosuppressive therapy, methods of administration within the lesion can also be employed. Oral irrigation means include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration methods. The antibody is preferably perfused using pulsed perfusion (especially when the dosage gradient of the antibody of the present invention changes). It can be administered. Depending on the duration of administration, injection is preferred. It is most preferably administered by intravenous or subcutaneous injection. ADCC (Antibody-Dependent Cell-Induced Criticality) Cellular activity is a cell-mediated reaction, and among these, the effector that expresses FcR... Cells (for example, natural killer (NK) cells, neutrophils, and macrophages) target cells The cells recognize the bound antibody and subsequently cleave the target cells. Primary cells mediated by ADCC. This includes NK cells, mononuclear cells, and macrophages. NK cells are usually primarily FcγRI The organism expresses II, while mononuclear cells express FcγRI, FcγRII, and FcγRIII. (This invention) So the parent CHO cell line produces a CHO cell line with a unique sugar chain spectrum. Edited. Subsequently, the edited CHO cell line produced antibodies from the parent CHO cells. It is possible to produce antibodies with higher ADCC activity than the ADCC activity of the target organism.

[0054] Example 1: Screening of parent cells suitable for serum-free suspension culture. CHO-K1 cultured in DMEM / F12 medium containing 10% FBS showed that the cells were conjugated. When the Luens reaches 80-90%, wash with PBS, digest with trypsin, and then 5% FB. The culture was terminated in DMEM / F12 medium containing S, counted, and centrifuged. 5% FBS was used. Re-turbidify the cells with DMEM / F12 medium containing 1 × 10⁶ 5 Cells are attached at a density of cells / ml Seeds were introduced. When the cell confluence reached 80-90%, the cells were washed with PBS and triple-grained. Digestion was performed, and the culture was terminated in DMEM / F12 medium containing 2% FBS. The cells were counted and centrifuged. They were separated. The cells were re-turbidified in DMEM / F12 medium containing 2% FBS, and 1 × 10⁶ 5 Cells / Cells were inoculated at a density of ml. When the confluence of the cells reached 80%-90%, The cells described above are digested with trypsin according to the process described above, and cultured in DMEM / F12 containing 1% FBS. The culture was terminated in soil and subcultured for 3-4 generations. CD CHO medium and DMEM / F12 were mixed in a 1:1 ratio. Mix in a 1(V / V) ratio and adjust to a final concentration of 6 mM glutamine, then use in serum. The concentration was adjusted to 1%. The CHO-K1 cells suitable for the low serum obtained were 3 × 10⁶ 5 Inoculate cells / mL into T25 bottles and culture at 37°C in a 5% CO2 incubator. The cells were digested with trypsin when the confluence reached 80-90%, and 1% FBS. A culture medium containing DMEM / F12 and CD CHO medium (volume ratio: 1:2) The culture was terminated, the cells were counted and centrifuged, and 3 × 10⁻⁶ cells were obtained. 5 T25 bottle at cell / mL density The samples were inoculated and cultured in a 37°C, 5% CO2 incubator. DMEM / F in the mixed medium. The ratio of 12 is gradually reduced to (1:8), and when the cell viability rate exceeds 90%, this Components from DMEM / F12 in the cell culture medium can be completely eliminated, and chemical composition CHO-K1 cells suitable for culture in 1% serum in a limited-time CD CHO medium were established. CHO-K1 cultured in CD CHO medium with a limited chemical composition including 1% FBS was found to be cell When the confluence reaches 80-90%, wash with PBS, digest with trypsin, and 0 The culture was terminated in CD CHO medium containing 0.5% FBS, counted, and centrifuged. 0.5 Re-turbidify the cells in CD CHO medium containing %FBS, and 1 × 10⁶ 5 T Inoculate into 25 bottles, and when the cell proliferation rate reaches 80%-90%, wash with PBS and trim. Digested with psin, cultured in CD CHO medium containing 0.25% FBS, and counted. The cells were centrifuged. The cells were re-turbidified in CD CHO medium containing 0.25% FBS, and 1 × 10⁶ of cells were collected. 5 Cells were inoculated at a density of cells / ml. It takes time for cells to grow healthily at this stage: The serum concentration in the stage began to decrease. CHO-K1 cells suitable for serum-free CD CHO culture. The cells were limitably diluted and inoculated into 30 96-well plates, with a cell density of 1 cell / well. The cells were prepared. After two weeks, they were observed under a microscope, and the cells that produced monoclonal were labeled. Clones with a larger cell area were transferred to a 24-well plate and observed under a microscope after one cycle. Cell clones with relatively high growth density and uniform cell size were labeled, and 6 wells were used. The cells were transferred to a phosphate group and cultured. After one week, they were observed under a microscope and found to be completely suspended and aggregated into clumps. The lighter the sample, the higher the cell density, the more the clones were labeled, and the sample was transferred to a 100 mL triangular bottle. The culture volume was 10 mL. Cell density and proliferation rate were recorded for each cell line. CHO-K1 cells suitable for serum-free culture after acclimatization were newly named CHO-BAT. .

[0055] Example 2: Construction of a FUT8 TALEN recombinant plasmid

[0056] Complete genome sequence of Chinese hamster ovarian cancer cell CHO-K1 (NW.003613) By analyzing 860), the Fut8 genome sequence (Gene ID: 100751648) ) and its cDNA sequence (shown in Table 1, SEQ ID NO. 8) were obtained. Fut8 NM consists of 9 exons and 11 introns. Fut8 enzyme activity center - is the amino acid coded in exon 1 (SEQ ID NO. 7) (SEQ ID N Because it is composed of O.9 (the amino acid sequence shown underlined), Talen's target The TA was designed using the left and right wings of exon 1 of the Fut8 gene as the wing sequence. Based on the LEN design guidelines and gene editing mechanisms, Fut8 TALEN Protein L130P (SEQ ID NO. 10), R184P (SEQ ID NO. 10) .11) has been designed. A fusion protein of L130P and FokI endonuclease. It is formed at L130-FokI (SEQ ID NO.14), and the fusion protein is exo It recognizes the base L130PTN (SEQ ID NO.3) on the left wing of n1. Fusion protein The nucleic acid sequence L130-FokIN, which corresponds to the L130-FokI sequence, has a SEQ ID NO. It is shown in .15. Its length is 19 bp. R184P and FokI endonuclease The fusion protein R184P-FokI (SEQ ID NO.16) is formed, and the fusion The protein recognizes the base R184PTN (SEQ ID NO. 4) on the right wing of exon 1. The nucleic acid sequence R184P-FokIN corresponds to the fusion protein R184P-FokI. This is shown in SEQ ID NO. 17. Its length is 17 bp. TALEN protein Plasmid vector encoding the left wing L130PTN and right wing R184PTN of exon 1. (See Figure 2) is described in Tomas Cermak et al. (2011) It was constructed as follows: Restrictions on both ends of L130-FokIN and R184P-FokIN. The enzymatic digestion sites of crease NcoI and XbaI were added, and the two fragment sequences were synthesized. The pCS2-peas-T vector was cloned using NcoI and XbaI (Figure 2). ). Between the left wing and right wing conjugate sequences, there is a 19bp spacer. (SEQ ID NO.5) possesses both plasmids of Fut8 TALEN: L1 The DNA sequencing results for 30N and R184N are shown in Table 1, SEQ ID NO. 12. As shown in SEQNO.13, the L130N and R184N nucleic acid sequences are translated into amino acids. Therefore, the corresponding amino acid sequences L130P and R184P are shown in Table 1, SEQ ID NO. This is shown in .10, SEQ ID NO.11. Table 1 Sequence Listing

[0057] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

Table 1-9

Table 1-10

Table 1-11

[0058] Example 3 Analysis of the Functional Effectiveness of FUT8 TALEN Protein

[0059] The CEL-I enzyme is a nuclease that can recognize mismatched bases in double-stranded DNA and cleave the double-stranded DNA from the position of the mismatch. When the targeting sequence is edited to Fut 8 TALEN, the region containing the targeting sequence amplified from the parental genome is mixed with the region containing the targeting sequence amplified from the genome of the transformed cells and denatured, and after annealing, there are base mismatches in the annealed double-stranded DNA Then, the CEL-I enzyme cleaves the annealed double-stranded DNA, and its manifestation is the discovery of two bands in the agarose electrophoresis result. 5×10 5 CHO- BAT cells were taken and seeded in a 6-well plate the day before transfection. The medium was DMEM / F12 containing 1 0% fetal bovine serum. Plasmids L130N and R184N were transiently transfected into cells by the method provided in the reagent description. Trans Three days after transfection, the cells were harvested by centrifugation, and the genome was extracted using a genomic extraction kit Using it as a template, primer L130 for (SEQ ID NO.1) and primer p PCR reaction was performed using Rymer L130 rev (SEQ ID NO.2). PCR amplification of the targeting sequence region contained in the cell was performed similarly. Take 20 μl of each CR product, mix them, raise the temperature to 94°C, and then allow to cool to room temperature. 0.5 μl of CEL-I enzyme was added to 200 ng of annealed DNA, and 42 The cells were incubated at °C for 30 minutes. The PCR reaction product was subjected to agarose gel electrophoresis. The reaction product was The results of the analysis by agarose electrophoresis are shown in Figure 3.

[0060] The results showed that, compared to the wild type, the gene-edited PCR products were 500 bp and 750 bp. Both bands were observed at bp, while the wild type showed one band at 750 bp, and the predicted result This proved that the Talen protein pair can function.

[0061] Example 4: Effect of FUT8 TALEN protein on the fucose content of antibodies

[0062] The designed FUT8 TALEN protein modulates the host genome, and the preparation is The purpose is to determine whether it affects the sugar chain of the antibody (whether the fucose content changes), L The 130N and R184 N plasmids are used to stably express the already established anti-CD20 antibody. Transient transfection is performed on the cell line. The method is lipofectamine 2000 ( The method of providing the reagent (in vitrogen) was referred to from the instructions. A simple explanation is that 10 cm In cell culture dish inner, 1 x 10 6 Each of the cells received L130 24 μL of liposomes containing 4 μg of plasmid DNA of N and R184N was added. After two days of lancetfection, 10% (v / v) FBS (GBICO) and 400 μg / m² were administered. The medium was replaced with DMEM / F12 medium containing L LCA(Vector). After one week, the cells Most of the cells curled up and suspended in the culture medium, while some other cells adhered and grew normally. Discard the supernatant. Cells resistant to LCA were digested with 0.25% (v / v) trypsin, and after centrifugation, 10 The cells were re-turbidified in DMEM / F12 medium containing %(v / v)FBS. 0.5 cells were placed in each well. The cells were inoculated into 96-well plates at the specified density. After 2 weeks of culture, monoclonal cells were selected. The cells were then introduced into a 24-well plate using FACS. Cells that were analyzed and showed negative LCA binding with FITC (Figure 4) were scaled up. The cells were cultured to produce antibodies. The oligosaccharide content of the antibodies produced from the two cell types was... The measurements were taken by Biodonor, and the results are shown in Figure 5.

[0063] As a result, transient transfer of L130 and R184 plasmids to cells preparing antibodies The treatment involved reducing the fucose content of the antibody.

[0064] Example 5: Establishment of genome-modified host cells

[0065] CHO-K1 genome modified for fucose-free protein and antibody production. To establish cells, L130 and R184 plasmids were transiently introduced into the CHO-K1 cell line. Lancefection was performed. Screening of anti-LCA monoclonal cells was conducted in Example 3. The same procedure was followed. For each candidate cell clone, the genome was extracted, and primers were used. L130 for (Table 1, SEQ ID NO.1) and primer L130 rev ( Table 1, SEQ ID NO.2) PCR reaction was performed and the candidate cell clones were included CEL-1 base mismatches are separated for PCR amplification products of the target sequence region. The analysis was performed. If the candidate clone is heterozygous, the agarose ion after enzymatic digestion with CEL-1 is analyzed. If both bands are present in pneumophoresis and the type is homozygous, then even if enzyme digestion with CEL-1 is performed, the tissue will not break down. Because it cannot be separated, the agarose electrophoresis shows a single band. The result is then cloned directly into a T vector (pGEM-T Easy Vector). Subsequently, sequencing was performed. The sequencing results and the results of the region in the parent cell were compared. The pair ratio of the lagment sequences is shown in Figure 6. Based on the alignment results, the two genomes are edited together. Selected homozygous types are designated as CHO-2G8, CHO-1D6, etc.

[0066] Example 6 Evaluation of host cell growth characteristics

[0067] We selected clone CHO-2G8, in which the fut8 gene was knocked out, as the host cell. It was newly named CHO-BAT-KF. 30 mL final concentration contains CD containing 6 mM Gln. CHO AGT TM We took samples from each of the three CHO-BA strains with a cell density of 300,000 / mL. T-KF and one strain of CHO-BAT were inoculated into small 125 mL roller bottles. Take 0.5 mL of cells on days 0, 3, 6, and 7, count them, and calculate the cell density. We measured cell viability and observed changes in the growth characteristics of cells after the Fut8 gene was knocked out. The dynamics were evaluated. Cell growth density is shown in Figure 7, and cell growth and proliferation capacity is shown in Figure 8. From Figures 7 and 8, As such, CHO-BAT-KF, in which the fut8 gene is knocked out, Compared to CHO-BAT cells in which offspring are not knocked out, there was no significant difference in growth density or activity. stomach.

[0068] Example 7: Analysis of glycosylation spectra of antibodies produced from host cells

[0069] Glycans of antibodies produced from the genome-modified CHO-2G8 cell line described in this invention However, in order to confirm that it has a mutated N-polysaccharide modification, protein A affinity BAT4306F produced from CHO-2G8 cells and CHO-K1 cells from the culture medium. 4306 produced from was purified and quantified using ultraviolet UV280. Salt-removed monochromator The 1mg antibody was cultured overnight with PNGaseF at 37°C, and N was extracted from the antibody. -Glycan was released. The released N-polysaccharide was analyzed by 30K Amicon ultrafiltration. The antibodies were separated. The flow-through was freeze-dried and re-turbidified in 200 μl of deionized water. Using a MALDI-TOF MS mass spectrometer, the N-polysaccharides from both antibody molecules mentioned above were analyzed. MALDI-TOF MS was then analyzed. BAT430 produced from CHO-2G8 The oligosaccharides in the 6F antibody are present as a single peak and are essentially the same population. It belonged to the group. This population was the oligosaccharide of antibody 4306 produced from parental host cells. This spectrum is different from that of (Figure 9).

[0070] As a result, the three peaks of N-polysaccharide 4306 were G0F, G1F, and G2F, respectively. Yes. Based on the time at which the N-polysaccharide peak appeared in BAT4306F and its molecular weight, the three N-polysaccharides are... The three peaks are estimated to be G0, G1, and G2, respectively, i.e., N-polysaccharides from 4306. It has one less fucose molecule per N-polysaccharide derived from BAT4306F.

[0071] At the same time, the heterogeneity of the sugar chains in commercially available Gazyva compared to BAT4306F The results of analyzing the degree of heterogeneity of the N-polysaccharide in BAT4306F are shown in Figure 10. The degree is lower, and it has more uniform sugar chains. Differentially produced from CHO-BAT-KF cells. The glycan types of the antibodies were analyzed. The results are shown in Table 4.

[0072] Example 8: Analysis of ADCC activity of antibodies produced from host cells

[0073] The modification of antibodies having N-polysaccharides according to the present invention enhances the biofunction of the antibody (e.g., ADCC activity). To confirm whether it increases, purified antibodies targeted to CD20 are used. Therefore, we measured the ADCC activity (LDH method, promega) in vitro. The antibody was produced by CHO-2G8 cells from the culture medium using a protein A affinity column. BAT4306F was purified and quantified using UV280. The parent antibody was not modified. The compound 4306 was expressed in wild-type CHO cells and purified using a similar method. Detection of ADCC Therefore, Wil2-s cells were cultured in RPMI-1640 culture medium containing 10% FBS. The condition is good (4-7 days). Take cells in the logarithmic growth phase and centrifuge at 1000 times for 10 minutes. Then the supernatant was discarded. Solution A (RPMI-164, phenol red free, containing 10% FBS) Add the culture medium (0) and mix well. Centrifuge as before, wash twice, count, and pulverize with solution A. Cells 3 × 10 5 Prepare the solution to cells / mL and add 100 μl to each well of U-96 well cell culture solution. Added to the rate. The final antibody concentrations were 1.2, 0.24, 0.048, and 0.0 for each well, in that order. 0096, 0.00192, 0.000384, 0.0000768, 0.000015 The concentration was adjusted to 36 μg / mL. It was then placed in a 37°C, 5% CO2 incubator for 30 minutes. In culture. Effector cells (PBMCs) were collected and soluble B (phenolic solution without serum). Add the RPMI-1640 culture medium (free of dead skin cells). Centrifuge and wash twice as described above. Count the cells and fill them with solution B in a 3x10 5 Adjust to pcs / ml, mix well, and add 50u per well. The solution was added to the aforementioned U-96 well cell culture plate at 1. Incubate at 37°C in 5% CO2. The cells were placed in a container and cultured for 3 hours. With 45 minutes remaining in the 3-hour culture time, the target cells were most... Add 20 μl of the dissolution solution to the large release well and incubate for 45 minutes in a 37°C, 5% CO2 incubator. The culture was continued. The U-96 well cell culture plate was placed in a centrifuge and 250g was used for 4 It was centrifuged for a minimum of 1 minute. 50 μl / well supernatant was transferred to another flat-bottom 96-well plate, and already Add 50 μl / well of the prepared chromogenic substrate solution, shake gently and mix well, and leave at room temperature for 30 minutes. The light-blocking reaction occurred. Add 50 μl / well of stop solution and shake gently to mix well. Micropre The results were read using OD490 via a web reader.

[0074] As a result, compared to unmodified 4306 produced by parental CHO cells, in serum-free medium... BAT4306F, which contains N-polysaccharides produced from the CHO-2G8 cell clone, is R ADCC activity against aji cells and Wil2-s cells is significantly improved (Figure 11).

[0075] Example 9: Analysis of the affinity of antibodies produced from host cells for CD20.

[0076] The N-polysaccharide-modified antibody produced from the cells described in this invention is CD20-positive. To confirm whether it affects the ability to bind to cells, Klervi Even -Refer to the method of Desrumeaux et al (2012), and use the FASC method to detect the B affinity of AT4306F, 4306, and the control rituximab for CD20 on different cell surfaces was detected. Briefly, cells Wil2-s in the logarithmic growth phase were collected, centrifuged at 800 r pm for 5 minutes, and the supernatant was discarded. Washed once with PBS, the density was calculated, and then resuspended with PBS and placed into 1.5 mL centrifuge tubes to make 500,000 cells per tube . Centrifuged, at 1200 rpm for 5 minutes, and the supernatant was discarded. The antibody concentrations were respectively 30, 3.33, 1.11, 0.37, 0.1, 0.04, 0.014, 0.0046 μg / mL, and 200 μl of the antibody was added to the above cells in sequence, and the cells were resuspended and mixed well . At the same time, the same volume of PBS was added as a negative control. Incubated at 4°C for 2 hours in the dark . Centrifuged, at 1200 rpm for 5 minutes, the supernatant was discarded, and washed once with PBS . 100 μl of PBS was added to resuspend the cells, and 2 μl of the secondary antibody FITC-goat anti-human Ig G1 Fab was added, and incubated at 4°C for 30 minutes in the dark . Centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and washed once with PBS. Detected with a flow cytometer C6 . The result calculation formula: Kd = [Ab] * {Fmax / (F - Fback) - 1}. The results are shown in the following table

[0077] Table 2 Statistical results of IC50 values and Kd values of cell binding experiments by antibodies

Table 2

[0078] Example 10: Ability of BAT4306F to eliminate B cells in whole blood from different NHL patients. In Vitro Evaluation of Power

[0079] Regarding the mechanism of action of anti-CD20 antibodies in patients with B lymphoma, the ADCC and CDC have directly investigated the matter. There are various mechanisms, including B cell apoptosis induced by contact. However, a specific type of anti-CD20 Regarding the effects of antibodies, it is not that they enhance a single mechanism of action, but rather that the antibody eventually affects the disease This is reflected in the ability to eliminate B cells in the human body. The antibody having N-polysaccharide of the present invention To determine whether the modification enhances the ability to eliminate that B cell, In vitro elimination of B cells with BAT4306F in whole blood from different NHL patients. The bio-function was evaluated. To put it simply, in a heparin sodium anticoagulant tube, A blood sample of approximately 3 mL was taken from a person newly diagnosed with NHL disease. Until the worker came to pick it up... The samples were stored at room temperature. 90 μL was taken from each sample (likely from the patient's blood) and placed into a new FACS tube. After adding the sample, the final antibody concentrations in the test samples in each tube were 10 nM, 1 nM, and 0 Add the solution to each tube's sample to achieve concentrations of 0.1 nM, 0.01 nM, and 0.001 nM. 10 μL of BAT4306F antibody dilutions of different concentrations were added. 3°C incubator - Allow to stand for 4 hours, then take 50 μL of blood-like substance from each tube and count to BD TruCount In addition to tubes, a mixture of BD B cell count antibodies (anti-CD45 (lymphocyte population)) was added to the blood-like sample. ), anti-CD3 (T cells), or anti-CD19 (B cells) was added. Store at room temperature in the dark for 15 minutes. After standing, BD FACS dissolving solution was added, and the sample was placed on the instrument for measurement (BD C6). The results are shown in Figure 12.

[0080] As a result, at the three concentration levels tested, the BAT4306F antibody eliminated B cells. In all cases, the ability to do so is stronger with rituximab, an antibody in which the N-polysaccharide has not been modified.

[0081] Example 11: Enhancement of affinity between BAT4306F and FcγRIIIa molecule

[0082] The unique glycan spectrum produced by genome-edited CHO-BAT-KF cells To verify that the recombinant antibody possessed enhances affinity for FcγRIIIA, The affinity of BAT4306F, commercially available GAZYVA, and rituximab for FcγRIIIA Each was measured. The sensors were placed in PBS and prepared by wetting for 10 minutes. 2.5 μg of labeled FcγRIIIa 158V and FcγRIIIa 158F in AB solution. Diluted to / mL. Loading: Biotin-labeled FcγRIIIa 158V Load the diluted solution for 10 minutes (until the signal reaches approximately 1.3 nM), 3.6 .3 and FcγRIIIa 158V affinity test: The test drug is BAT4306F, Obinutuzumab was diluted to 500 nM with AB. Rituximab was diluted to 3000 nM with AB. It was then diluted to seven points with a 2x gradient using the same buffer. A B, FcγRIIa V158, regeneration buffer, pharmaceutical diluent, neutralization buffer, in that order. It was added to the corresponding column of the 96-well plate. The following steps were performed with the SA sensor: Bass Baseline: AB was detected. 150s, Association: Concentration gradient The drug dilution sample and blank (AB) of the gent were bonded for 90 seconds. Dissociation: Dissociated in AB for 120 s. Regeneration: Regenerated in NaOH with pH 10.5 for 5 s Neutralization: Neutralized in AB for 5 s. Regeneration and neutralization were repeated three times. After collecting data The data were analyzed by the equipment data analysis software Acquisition 8.2 At this time, with the signal collected at Baseline as the baseline, the reference signal (double subtraction of the sample blank and the sensor blank) was subtracted, and the obtained data were grouped and analyzed for fitting and analyzed for fitting Table 3 Statistical results of the affinity of BAT4306F for FcγRIIIa 158F

[0083]

Table 3

[0084] Example 12

[0085] In the above - mentioned CHO - BAT - KF host cells, in order to verify that the sugar chain spectra of antibodies expressed from other antibody sequences are stable and consistent, CHO - BAT - KF cells were used to express several other antibodies One of the antibodies is BAT4406F, which has two light chains shown in SEQ ID NO.22 and two heavy chains shown in SEQ ID NO.23 One of the antibodies is the anti - EGFR antibody BAT0206F, which has two light chains shown in SEQ ID NO.24 and two heavy chains shown in SEQ ID NO.25 One of the antibodies has two light chains shown in SEQ ID NO.26 and two heavy chains shown in SEQ ID N ​​​​BAT0808 is an anti-Trop2 antibody that has both heavy chains as shown in O.27. Specific experiments were conducted. Product details (LudgerTag TM PROC(procainamide)Glyca Refer to the n Labeling Kit, and after denaturing and reducing the sample, use glycosidase. The glycans in the sample were excised from the lycosylation site. Procainamide hydrochloride - fluorescein After labeling with an inconjugate, the molecules were isolated using a HILIC column, and the pH was used as mobile phase A. 4.5% 100 mM ammonium formate is used as mobile phase B with acetonitrile. Elution isolation was performed. The elution gradient was 28%A-38%A over 0-36 minutes, and fluorescence was observed. Detected by a detector. In the system-suitable solution, the separation degree of glycan types G1 and G1' was 1.0 or higher. It is necessary that it be above. The results are shown in Figure 13 and Table 4. The glycan types of the four antibodies are The sugar chains are highly uniform and have good homogeneity. The method or cells of the present invention have general applicability. It can be applied not only to the preparation of anti-CD20 antibodies, but also to the preparation of antibodies with other sites of action, A This method can produce homogeneous target antibodies that enhance DCC activity.

[0086] Table 4. Percentage of glycan types in four antibodies prepared using CHO-BAT-KF cells (%) [Table 4]

Claims

1. An antibody, wherein the antibody is a BAT4306F antibody, and the BAT4306F antibody is an anti-CD20 antibody comprising two light chains each containing the amino acid sequence of SEQ ID NO. 20 and two heavy chains each containing the amino acid sequence of SEQ ID NO. 21, wherein the G0 content of the BAT4306F antibody is 60% or more, it does not contain fucose, and the mannose content of the antibody is 5% or less.

2. A pharmaceutical composition comprising the antibody described in claim 1 and a pharmaceutically acceptable carrier.

3. A composition for treating non-Hodgkin lymphoma (NHL), comprising the antibody described in Claim 1.

4. The antibody according to claim 1, wherein the high mannose content of the antibody is 5% or less.

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