阅读说明：本技术 人源化单克隆抗体、其制备方法及其用途 (Humanized monoclonal antibody, preparation method and application thereof ) 是由 不公告发明人 于 2018-07-02 设计创作，主要内容包括：本发明公开一种抗人CD20人源化单克隆抗体、编码该抗体的核酸分子、包含该核酸分子的重组载体、包含所述重组载体的重组细胞以及所述抗人CD20人源化单克隆抗体的制备方法及其医药用途。其中,编码所述抗体的核酸分子包含如SEQ ID NO:1所示的编码轻链的核苷酸序列和如SEQ ID NO:2所示的编码重链的核苷酸序列；并分别在所述两个序列上设计信号肽和终止密码子。本发明由于对密码子进行优化,转基因后的CHO细胞表达抗人CD20人源化抗体的表达量高；且本发明提供的发酵方法,特别是在添加补料批培养基后,延长细胞生长时间,提高表达水平,降低生产成本,获得高纯度的目的蛋白。(The invention discloses an anti-human CD20 humanized monoclonal antibody, a nucleic acid molecule for coding the antibody, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, a preparation method of the anti-human CD20 humanized monoclonal antibody and medical application of the anti-human CD20 humanized monoclonal antibody. Wherein, the nucleic acid molecule for coding the antibody comprises a nucleotide sequence for coding a light chain shown as SEQ ID NO. 1 and a nucleotide sequence for coding a heavy chain shown as SEQ ID NO. 2; and designing a signal peptide and a stop codon on the two sequences respectively. According to the invention, because the codon is optimized, the expression quantity of the humanized antibody of anti-human CD20 expressed by the transgenic CHO cell is high; the fermentation method provided by the invention prolongs the cell growth time, improves the expression level, reduces the production cost and obtains the high-purity target protein particularly after the fed batch culture medium is added.)

1. A nucleic acid molecule encoding anti-human CD20 humanized monoclonal antibody SH006-2, characterized by comprising a nucleotide sequence encoding a light chain and a nucleotide sequence encoding a heavy chain; the nucleotide sequence of the coding light chain is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding heavy chain is shown as SEQ ID NO. 2.

2. The nucleic acid molecule of claim 1, further comprising a nucleotide sequence encoding a signal peptide at the 5' end of the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain, respectively; the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain further comprise a stop codon at the 3' end, respectively.

3. The nucleic acid molecule of claim 2, wherein the signal peptide consists of the amino acid sequence SEQ ID NO 6.

4. A recombinant vector comprising the nucleic acid molecule of any one of claims 1-3, wherein said nucleic acid molecule is operably linked to one or more regulatory elements.

5. A recombinant cell comprising the recombinant vector of claim 4; preferably, the recombinant cell is a CHO cell knock-out of the gene responsible for encoding fucosyltransferase 8, FUT 8; further preferably, the knockout refers to the knockout of the gene encoding FUT8 in CHO cells using CRISPR/Cas9 gene editing technology.

6. An anti-human CD20 humanized monoclonal antibody SH006-2, which is prepared from the nucleic acid molecule of any one of claims 1 to 3, the recombinant vector of claim 4 or the recombinant cell of claim 5, and has the amino acid sequence of the light chain as shown in SEQ ID NO. 3 and the amino acid sequence of the heavy chain as shown in SEQ ID NO. 4.

7. The method for producing the anti-human CD20 humanized monoclonal antibody SH006-2 of claim 6, which comprises culturing the recombinant cells of claim 5 under conditions such that the anti-human CD20 humanized monoclonal antibody SH006-2 is expressed, and collecting the expressed antibody SH 006-2.

8. The method of claim 7, comprising the steps of:

1) cloning the nucleotide sequence of any one of claims 1-3 into an expression vector to obtain a recombinant expression vector;

2) transferring the recombinant expression vector into a host cell to obtain a recombinant cell, wherein the host cell is a CHO cell in which a gene for encoding fucosyltransferase 8, namely FUT8, is knocked out;

3) carrying out pressurized screening on the recombinant cells on a screening culture medium to obtain a cell pool with stable growth;

4) screening high-expression cell strains from the cell pool in the step 3), then detecting the expression level of SH006-2 in monoclonal supernatant, further screening and culturing to obtain clones with higher expression level, and freezing and storing the cells for later use after amplification culture;

5) culturing the clone cells obtained in the step 4) in a target culture medium, and detecting the expression yield of the antibody; then selecting and culturing the clone with high expression to obtain a monoclonal cell strain with high expression, and harvesting and purifying the culture supernatant to obtain the anti-human CD20 humanized monoclonal antibody SH006-2 of claim 6.

9. The preparation method of claim 8, wherein the knockout in step 2) is that a gene responsible for encoding fucose, namely FUT8 gene, in CHO cells is knocked out by using CRISPR/Cas9 gene editing technology.

10. Use of the nucleic acid molecule of any one of claims 1 to 3 or the recombinant vector of claim 4 or the recombinant cell of claim 5 for the preparation of the anti-human CD20 humanized monoclonal antibody SH006-2 of claim 6.

11. The use of the anti-human CD20 humanized monoclonal antibody SH006-2 according to claim 6 for the preparation of a medicament for the prevention or treatment of a disease or disorder associated with the CD20 antigen, preferably a tumor, an autoimmune disease or an inflammatory disease; the tumor is more preferably selected from B cell type non-Hodgkin's lymphoma, chronic lymphocytic leukemia; the autoimmune disease is more preferably selected from autoimmune hemolytic anemia or idiopathic thrombocytopenic purpura; the inflammatory disease is more preferably selected from rheumatoid arthritis or multiple sclerosis.

12. Use of the anti-human CD20 humanized monoclonal antibody SH006-2 according to claim 6 for the preparation of a diagnostic agent related to CD20 antigen for use in a method for in vivo diagnosis of a disease or disorder related to CD20 antigen, said method comprising administering to a subject to be examined an effective amount of the anti-human CD20 humanized monoclonal antibody according to claim 6.

13. A pharmaceutical composition comprising the anti-human CD20 humanized monoclonal antibody SH006-2 according to claim 6, and optionally a pharmaceutically acceptable carrier or excipient.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-CD 20 humanized monoclonal antibody SH006-2, a nucleic acid molecule encoding the antibody, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, a preparation method of the anti-CD 20 humanized monoclonal antibody SH006-2 and medical application thereof.

Background

The CD20 antigen is a hydrophobic transmembrane protein on pre-B cells and mature B lymphocytes, which has a very important regulatory role in the proliferation and differentiation of B lymphocytes. CD20 is expressed during early pre-B cell development and continues to be expressed until plasma cell differentiation. It was found that over 90% of non-hodgkin lymphoma patients, chronic lymphoma patients and large B-cell lymphoma patients highly express CD20 antigen on the B-cell surface. The anti-CD 20 monoclonal antibody can bind to CD20 molecules on the surface of tumors and kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Since 1997 the first anti-CD 20 therapeutic mab rituximab (rituximab,

rituximab has been listed as the top of the global popular drug by FDA approval to come on the market to date, and also promoted the rapid development of this class of drugs. According to different structures, humanization degrees, Fc segment modification and the like of the antibody, 3 generations of research and development are approximately carried out: the first generation antibody is a murine antibody or a chimeric antibody (-ximab, most representative of rituximab); the second generation antibody is a humanized antibody (-zumab) or a fully human antibody (-mumab, obtained from ofatumumab (ofatumumab,) Representative), which is greatly reduced in immunogenicity as compared to first generation antibodies; the third generation antibody is to modify the Fc segment of the antibody by glycosylation and other engineering on the basis of the first generation and the second generation so as to increase the anti-antibodyThe body binds with affinity to Fc γ IIIa to enhance effector function, ultimately enhancing therapeutic activity (as described by obinutuzumab (obinutuzumab,GA101) is representative).

The existing rituximab belongs to a human-mouse chimeric antibody, and molecules contain more murine amino acid sequences, so that potential safety hazards caused by certain transfusion reaction and immunogenicity may exist. Current clinical practice has shown that one of the common adverse reactions of rituximab is infusion reactivity. Moreover, a great deal of clinical data has been available to show that, for low grade malignant lymphoma,

the single medicine has the curative effect of about 50 percent, about 50 percent of patients are invalid, and 60 percent of patients who are initially treated are invalid again; even if the chronic lymphocytic leukemia patient with high malignancy diffuse large B cell lymphoma and low CD20 expression is combined with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), half or more of the patients still have ineffective treatment or relapse.

In addition, the productivity of antibody drugs is currently far below the demand in the world. The yield of antibody is largely dependent on the preference of antibody genes and their expression level in mammalian cells, in addition to factors such as culture scale, production process, and purification process.

Disclosure of Invention

In order to overcome the defects of the prior art and obtain a new antibody with higher expression level and biological activity, the invention discloses a nucleic acid molecule of an anti-human CD20 humanized monoclonal antibody SH006-2 obtained by signal peptide and codon optimization and gene knockout technology, and then the recombinant anti-human CD20 humanized monoclonal antibody SH006-2 with fully knocked fucose is obtained by means of vector construction, cell transfection, cell strain screening, cell culture, active substance production, purification and the like. The invention specifically comprises the following aspects:

the first aspect of the present invention relates to a nucleic acid molecule encoding anti-human CD20 humanized monoclonal antibody SH006-2, characterized by comprising a nucleotide sequence encoding a light chain and a nucleotide sequence encoding a heavy chain.

The nucleotide sequence encoding the light chain is:

GATATTGTGATGACTCAGACTCCACTGTCACTGCCCGTGACACCTGGCGAGCCCGCCTCTATCTCCTGTAGGAGCTCTAAGTCCCTGCTGCATTCCAACGGCATCACCTACCTGTATTGGTACCTGCAGAAGCCTGGCCAGTCTCCTCAGCTGCTGATCTACCAGATGTCCAACCTGGTGTCTGGCGTGCCTGATAGGTTTTCCGGCTCTGGCTCCGGCACAGACTTTACCCTGAAGATCTCCAGAGTGGAGGCTGAGGATGTGGGCGTGTATTACTGCGCCCAGAATCTGGAGCTGCCATATACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGAGAACCGTGGCTGCCCCAAGCGTGTTTATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGCACAGCTAGCGTGGTGTGCCTGCTGAATAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGTCTGGCAACTCCCAGGAGTCTGTGACAGAGCAGGATTCCAAGGACAGCACATACTCCCTGTCTAGCACCCTGACACTGAGCAAGGCTGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTCACTCATCAGGGACTGTCATCTCCTGTCACTAAGAGTTTTAATCGCGGCGAGTGT(SEQ ID NO:1)；

the nucleotide sequence encoding the heavy chain is:

CAGGTCCAGCTGGTCCAGAGTGGTGCAGAAGTGAAGAAGCCAGGCTCCAGCGTGAAGGTGTCCTGTAAGGCCAGCGGCTACGCCTTTAGCTACTCCTGGATCAATTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCAGAATCTTCCCTGGCGATGGCGACACCGATTACAACGGCAAGTTCAAGGGCAGAGTGACCATCACAGCCGATAAGAGCACCTCCACAGCCTACATGGAGCTGTCTAGCCTGAGATCCGAGGACACCGCCGTGTATTACTGCGCTAGAAACGTGTTCGACGGCTATTGGCTGGTGTATTGGGGCCAGGGCACACTGGTGACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTGTTTCCACTGGCTCCCTCCTCTAAGAGCACAAGCGGCGGCACCGCTGCCCTGGGCTGTCTGGTGAAGGACTACTTTCCAGAGCCTGTGACAGTGAGCTGGAATTCCGGCGCTCTGACCTCTGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCTTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCTCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCTTCCAATACAAAGGTGGACAAGAAGGTGGAGCCAAAGTCCTGTGACAAGACCCATACATGCCCCCCATGTCCTGCTCCCGAGCTGCTGGGCGGCCCTTCCGTGTTCCTGTTTCCCCCAAAGCCCAAGGATACCCTGATGATCAGCAGAACCCCAGAGGTGACATGCGTGGTGGTGGACGTGTCCCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAACGCCAAGACAAAGCCTAGAGAGGAGCAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCTAATAAGGCCCTGCCTGCTCCAATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGCCTCGGGAGCCCCAGGTGTATACCCTGCCTCCATCCAGAGACGAGCTGACCAAGAATCAGGTGTCTCTGACATGCCTGGTGAAGGGCTTCTATCCATCCGATATCGCTGTGGAGTGGGAGAGCAATGGCCAGCCTGAGAACAATTATAAGACAACCCCACCTGTGCTGGATTCTGACGGCAGCTTTTTCCTGTATTCCAAGCTGACCGTGGATAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTGTAGCGTGATGCACGAGGCACTGCATAATCACTACACCCAGAAGTCACTGTCACTGAGTCCAGGCAAA(SEQ ID NO:2)。

further, such nucleotide sequences may be fused to polynucleotides encoding signal peptides or heterologous signal peptides native to the original antibody. Specifically, the nucleic acid molecule may further comprise a nucleotide sequence encoding a signal peptide, which may be a natural signal peptide or a heterologous signal peptide, at the 5' end of the nucleotide sequence encoding its light chain and the nucleotide sequence encoding its heavy chain, respectively; the nucleotide sequence encoding the light chain and the nucleotide sequence encoding the heavy chain further comprise a stop codon at the 3' end, respectively.

Still further, the signal peptide consists of the amino acid sequence: MGWSCIILFLVATATGVHS (SEQ ID NO: 6).

The nucleotide sequence encoding the signal peptide is:

ATGGGATGGTCCTGTATTATCCTGTTCCTGGTCGCAACCGCAACTGGTGTCCACTCA(SEQ ID NO:5)。

the stop codon that may be included on the light chain is TGA and the stop codon that may be included on the heavy chain is TAA.

The second aspect of the present invention relates to a vector, such as an expression vector, which comprises a nucleotide sequence encoding the heavy chain or the light chain or both of the anti-human CD20 humanized monoclonal antibody SH 006-2. In such vectors, the nucleotide sequence of the present invention may be operably linked to one or more regulatory elements. Wherein the regulatory element is selected from expression regulatory sequences, such as promoters, enhancers, and the like.

The vector of the present invention comprises a regulatory element (e.g., a promoter or enhancer) operably linked to a nucleic acid sequence encoding the heavy or light chain of SH006-2 of the humanized monoclonal antibody against human CD 20. "operably linked" refers to an arrangement of nucleic acid sequences that are configured such that their normal function is performed. Thus, regulatory elements operably linked to a nucleotide sequence encoding a polypeptide can direct transcription, replication, and/or translation to produce the antibody. In one embodiment, the vector encodes the light or heavy chain amino acid sequence of the humanized monoclonal antibody SH 006-2.

In the present invention, the expression vector is, for example, a prokaryotic expression vector, a eukaryotic expression vector, a phage vector, or a viral vector. Further, the vector is selected from eukaryotic vectors. Further, the vector was selected from commercially available vectors pcDNA3.4-DHFR and pcDNA3.4-G418. The heavy and light chains of the antibody can be expressed in pcDNA3.4-DHFR vector and pcDNA3.4-G418, respectively. pcDNA3.4-DHFR also contains eukaryotic selection marker DHFR label and prokaryotic selection label Ampicilline, and can be used for screening high-expression heavy chain cell strain by methotrexate pressurization, and pcDNA3.4-G418 contains eukaryotic selection marker G418 label and prokaryotic selection label Ampicilline, and can be used for screening high-expression light chain cell strain by neomycin pressurization.

In a specific embodiment of the present invention, a kozak sequence, HindIII and a signal peptide sequence are added to the 5 'end of the nucleotide sequence (SEQ ID NO:1) encoding the light chain, respectively, and a stop codon and an XhoI cleavage site are added to the 3' end, and the product is inserted into pcDNA3.4-G418 by cleavage ligation; the 5 'end of the nucleotide sequence (SEQ ID NO:2) for coding the heavy chain is respectively and sequentially added with a kozak sequence, a HindIII and a signal peptide sequence, the 3' end is added with a stop codon and an XhoI enzyme cutting site, the recombinant plasmids containing the SH006-2 full-length heavy chain and light chain genes finally obtained are named as pcDNA3.4-DHFR-006-2 and pcDNA3.4-G418-006-2 after being inserted into a pcDNA3.4-DHFR vector through enzyme cutting connection. The plasmid maps of pcDNA3.4-G418-SH006-2 and pcDNA3.4-DHFR-SH006-2 are shown in FIG. 2.

The third aspect of the present invention relates to a recombinant cell containing the recombinant vector of any one of the second aspects of the present invention. The recombinant cells of the present invention are cells in which the gene responsible for encoding fucosyltransferase 8, FUT8, has been knocked out.

Further, the cell is a CHO cell selected from the group consisting of CHO-S, CHO-K1, CHO/DG44 cells. The knocking-out refers to knocking out a gene responsible for encoding fucose in CHO cells, namely FUT8 gene, by using CRISPR/Cas9 gene editing technology.

The fourth aspect of the present invention relates to an anti-human CD20 humanized monoclonal antibody SH006-2, which is prepared from the nucleic acid molecule of any one of the first aspect of the present invention, the recombinant vector of any one of the second aspect of the present invention or the recombinant cell of any one of the third aspect of the present invention.

Furthermore, the amino acid sequence of the antibody light chain is shown as SEQ ID NO. 3, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 4.

Further, the antibody comprises a CH2 domain; the CH2 domain has little or no fucose glycosylation modification.

In a specific embodiment of the invention, the anti-human CD20 humanized monoclonal antibody SH006-2 has enhanced ADCC activity compared with rituximab.

In a specific embodiment of the invention, the ADCC activity of the anti-human CD20 humanized monoclonal antibody SH006-2 is stronger than that of GA101 monoclonal antibody.

The fifth aspect of the invention relates to a preparation method of the anti-human CD20 humanized monoclonal antibody SH006-2, which specifically comprises the following steps:

1) cloning the sequence of the nucleic acid molecule of the first aspect of the invention into an expression vector to obtain a recombinant expression vector;

2) transferring the recombinant expression vector into a host cell, namely a CHO cell in which a gene for encoding fucosyltransferase 8, namely FUT8, is knocked out to obtain a recombinant cell;

3) carrying out pressurized screening on the recombinant cells on a screening culture medium to obtain a cell pool with stable growth;

4) screening high-expression cell strains from the cell pool in the step 3), then detecting the expression level of SH006-2 in monoclonal supernatant, screening and culturing to obtain clones with higher expression level, and freezing and storing the cells for later use after amplification culture;

5) culturing the clone cells obtained in the step 4) in a target culture medium, and detecting the expression yield of the antibody; then selecting the clone with high expression for culturing to obtain the monoclonal cell strain with high expression, and harvesting and purifying the culture supernatant to obtain the anti-human CD20 humanized monoclonal antibody SH006-2 according to the fourth aspect of the invention.

In the step 1) above, the recombinant expression vectors may be pcDNA3.4-G418 and pcDNA3.4-DHFR, respectively. A kozak sequence, a HindIII and a signal peptide sequence are respectively and sequentially added to the 5 'end of a nucleotide sequence (SEQ ID NO:1) for encoding a light chain, a stop codon and an XhoI enzyme cutting site are added to the 3' end, and the product is inserted into pcDNA3.4-G418 through enzyme cutting connection; the nucleotide sequence encoding the heavy chain (SEQ ID NO:2) was sequentially added with a kozak sequence, HindIII and a signal peptide sequence at the 5 'end, and a stop codon and an XhoI cleavage site at the 3' end, respectively. After the recombinant plasmid containing the SH006-2 full-length heavy chain and light chain genes is inserted into a pcDNA3.4-DHFR vector through enzyme digestion connection, the finally obtained recombinant plasmids containing the SH006-2 full-length heavy chain and light chain genes are named as pcDNA3.4-DHFR-SH006-2 and pcDNA3.4-G418-SH006-2 respectively.

In the step 2), the knockout refers to the knockout of the gene responsible for encoding fucosyltransferase 8, i.e., FUT8, in CHO cells by using CRISPR/Cas9 gene editing technology.

In the step 3), the pressurized screening adopts Methotrexate (MTX) gradient pressurization; the stably growing cell pool was one that was stably growing at 1000nM MTX pressure.

In the step 4), the detection refers to detection by an ELISA method.

In the above step 5), the target medium is the same as the screening medium in the step 3), and is a medium not containing hypoxanthine and thymidine, i.e., HT^-The medium is preferably a mixed medium containing CD OptiCHO medium, GlutaMAX (100 ×) medium, and 10% F-68(100 ×) medium. And, the selected high expression clone is fed batch cultured until collection.

The sixth aspect of the present invention relates to the use of the nucleic acid molecule of the first aspect of the present invention, the recombinant vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention in the preparation of the anti-human CD20 humanized monoclonal antibody SH006-2 of the fourth aspect of the present invention.

The seventh aspect of the present invention relates to a medicament containing the anti-human CD20 humanized monoclonal antibody SH006-2 as an active ingredient, which optionally contains a pharmaceutically acceptable carrier or excipient.

The invention also relates to the use of the anti-human CD20 humanized monoclonal antibody SH006-2 in the preparation of medicines for preventing or treating diseases or symptoms related to CD20 antigen, such as tumors, autoimmune diseases or inflammatory diseases; preferably, the tumor is selected from B-cell non-hodgkin's lymphoma, chronic lymphocytic leukemia; the autoimmune disease is selected from autoimmune hemolytic anemia or idiopathic thrombocytopenic purpura; the inflammatory disease is selected from rheumatoid arthritis or multiple sclerosis.

The present invention also relates to the use of the anti-human CD20 humanized monoclonal antibody SH006-2 for the preparation of a diagnostic agent related to the CD20 antigen for use in a method for the in vivo diagnosis of a disease or disorder related to the CD20 antigen, said method comprising administering to a subject to be examined an effective amount of the anti-human CD20 humanized monoclonal antibody according to the fourth aspect of the present invention.

The invention also relates to a reagent, a composition or a kit containing the anti-human CD20 humanized monoclonal antibody SH006-2 as an active ingredient.

Advantageous effects of the invention

The research designs and obtains a new coding gene of the anti-human CD20 humanized monoclonal antibody SH006-2 through the techniques of codon optimization, molecular biology and the like on the basis of GA101 antibody, the coded antibody amino acid sequence is the same as GA101, but the expression level is higher in an expression system compared with other sequences. And meanwhile, the fucose glycosylation modification of the antibody is completely removed through a gene knockout technology, the ADCC activity of the antibody is enhanced, and the purposes of improving the tumor treatment effect and/or expanding the tumor application range are achieved.

Because the monoclonal antibody SH006-2 is a fully humanized antibody, a lethal immune response (HAMA) reaction easily generated by other antibodies can not occur, the affinity with a receptor is greatly enhanced, the competitive inhibition of normal IgG in serum is avoided, B lymphocytes can be effectively eliminated in vivo, and the possible drug resistance and ineffective conditions of the target molecule can be compensated. The antibody has a fucose modification that is nearly or completely knocked out and has higher ADCC activity, which alteration will be clinically beneficial to reduce the chance of therapeutic resistance to the antibody by the patient. Meanwhile, compared with the method for modifying cells through genetic engineering (such as those disclosed in Chinese patents CN1902231A and CN 101291954A), the preparation method is more convenient, and the antibody yield is also obviously increased.

Drawings

FIG. 1 is a technical scheme for constructing CHO-DG44Fut 8-/-cell line.

FIG. 2 is a map of plasmid pcDNA3.4-G418-SH006-2 containing SH006-2 light chain and plasmid pcDNA3.4-DHFR-SH006-2 containing SH006-2 heavy chain, in which LC represents SH006-2 light chain and HC represents SH006-2 heavy chain.

FIG. 3 shows the electrophoresis chart of plasmid linearized digestion verification: lane 1 shows the PvuI-HF cleavage of SH 006-2; lanes 2 and 3 are Maker125 and Maker15000, respectively.

FIGS. 4 and 5 are graphs showing the binding of SH006-2 antibody to Fc γ RIIIa (CD16a, 158F).

FIG. 6 is an ADCC effect curve of SH006-2 induced NK-92MI-CD16a cells on Raji cells and Daudi cells.

FIG. 7 is a drawing showing

SH006-2 andinhibition rate of B-lymphoma cells Daudi cells.

Fig. 8 is a photograph of tumors of mice tested in each group (Daudi solid tumor model): from top to bottom are respectively: SH006-230mg/kg, SH006-23 mg/kg, SH 006-20.3 mg/kg,

3mg/kg、

3mg/kg、0.3mg/kg and PBS group.

Detailed Description

The present invention will be further described with reference to the following embodiments and drawings, and the present invention is not limited to the following embodiments. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge of those skilled in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.