Nucleic acid molecules encoding anti-her2 antibodies and uses thereof

Abstract

The application belongs to the field of biology and relates to a nucleic acid molecule encoding an anti-HER2 antibody, a vector containing the nucleic acid molecule and an expression method, wherein the nucleic acid molecule contains codons optimized for expression cells. By using the nucleic acid molecule of the application, the expression amount of the anti-HER2 antibody in cells can be improved, the yield of the anti-HER2 antibody is increased, and the production cost is reduced; and the anti-HER2 antibody produced has similar performance to that of the original research drug and has a good drug-making prospect.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biology, and particularly relates to nucleic acid molecules encoding anti-HER2 antibodies and applications thereof. BACKGROUND

[0002] HER2 (Human Epidermal Growth Factor Receptor 2), also known as ERBB2, is a member of the growth factor receptor family. The receptor protein is usually only expressed in the fetal period, and is only expressed at a low level in a very small number of normal tissues after adulthood, but is overexpressed in a variety of human tumor tissues (such as breast cancer, gastric cancer, ovarian cancer, lung cancer, primary renal cell carcinoma, endometrial cancer, etc.), and indicates poor prognosis.

[0003] Pertuzumab (also known as 2C4, Perjeta, and Patjet) is a monoclonal antibody that blocks the heterodimerization of HER2 with other HER receptors by binding to HER2, thereby slowing the growth of tumors. Pertuzumab inhibits ligand-initiated intracellular signal transduction through two main signaling pathways, mitogen-activated protein (MAP) kinase and phosphatidylinositol 3 kinase (PI3K), and inhibition of these signaling pathways can lead to cell growth arrest and apoptosis. Pertuzumab can also mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Pertuzumab has been approved for the treatment of early breast cancer and metastatic breast cancer, and the combination of pertuzumab and trastuzumab can increase the anti-tumor effect.

[0004] However, the existing pertuzumab has problems such as high price, limited cell expression, which greatly affects the yield, leading to insufficient supply, etc. Therefore, it is urgent to provide a technical solution for optimizing the method of producing pertuzumab, nucleic acid, etc. SUMMARY

[0005] The purpose of the present application is to optimize and analyze the codons for producing anti-HER2 antibodies in multiple systems and multiple rounds, and to screen out codons that can be highly expressed in host cells, so as to improve the expression of anti-HER2 antibodies in cells, and the relative binding activity, quality parameters, etc. of the obtained anti-HER2 antibodies to HER2 are similar to those of the original drug Patjet.

[0006] In this document, the amino acid sequences of the heavy chain and light chain of the anti-HER2 antibody are set forth in SEQ ID NOs: 21 and 23, respectively, the amino acid sequences of the heavy chain variable region and the light chain variable region are set forth in SEQ ID NOs: 22 and 24, respectively; the sequences of the three CDR regions of the heavy chain variable region are set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the sequences of the three CDR regions of the light chain variable region are set forth in SEQ ID NOs: 28, 29, and 30, respectively.

[0007] In a first aspect, a nucleic acid molecule is provided, the nucleic acid molecule comprising a nucleic acid sequence encoding HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of an anti-HER2 antibody, and a nucleic acid sequence encoding LCDR1, LCDR2, and LCDR3 of a light chain variable region of an anti-HER2 antibody, wherein:

[0008] the nucleic acid sequence encoding HCDR1 is set forth in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is set forth in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is set forth in SEQ ID NO: 27; and

[0009] the nucleic acid sequence encoding LCDR1 is set forth in SEQ ID NO: 28, the nucleic acid sequence encoding LCDR2 is set forth in SEQ ID NO: 29, and the nucleic acid sequence encoding LCDR3 is set forth in SEQ ID NO: 30.

[0010] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding a heavy chain variable region and a light chain variable region of an anti-HER2 antibody, wherein:

[0011] the nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 22, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 22; and

[0012] the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 24, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24.

[0013] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding a heavy chain variable region and a light chain variable region of an anti-HER2 antibody, wherein:

[0014] The nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 22, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 24.

[0015] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding a human antibody heavy chain constant region and a light chain constant region. In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding a human antibody heavy chain and a light chain, wherein:

[0016] the nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 21, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 21;

[0017] and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 23, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 23.

[0018] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding an anti-HER2 antibody heavy chain and a light chain, wherein:

[0019] the nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 21, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 23.

[0020] In some embodiments, the aforementioned nucleic acid molecule comprises a signal peptide sequence encoding an anti-HER2 antibody heavy chain and a light chain. In some embodiments, the signal peptide sequence of the anti-HER2 antibody heavy chain and light chain is set forth in SEQ ID NO: 41. In some embodiments, the nucleic acid sequence encoding the signal peptide sequence of the anti-HER2 antibody heavy chain and light chain is set forth in SEQ ID NO: 42.

[0021] In a second aspect, the present application provides a vector comprising the nucleic acid molecule of the present application.

[0022] In some embodiments, the vector is an expression vector.

[0023] In a third aspect, the present application provides a vector system for expressing an anti-HER2 antibody, comprising a first vector and a second vector, wherein:

[0024] The first vector comprises a nucleic acid sequence encoding HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of an anti-HER2 antibody, wherein the nucleic acid sequence encoding HCDR1 is set forth in SEQ ID NO: 25, the nucleic acid sequence encoding HCDR2 is set forth in SEQ ID NO: 26, and the nucleic acid sequence encoding HCDR3 is set forth in SEQ ID NO: 27; and

[0025] The second vector comprises a nucleic acid sequence encoding LCDR1, LCDR2, and LCDR3 of a light chain variable region of an anti-HER2 antibody, wherein the nucleic acid sequence encoding LCDR1 is set forth in SEQ ID NO: 28, the nucleic acid sequence encoding LCDR2 is set forth in SEQ ID NO: 29, and the nucleic acid sequence encoding LCDR3 is set forth in SEQ ID NO: 30.

[0026] In some embodiments, the foregoing anti-HER2 expressing vector system, wherein the first vector comprises a nucleic acid sequence encoding a heavy chain variable region of an anti-HER2 antibody, and the second vector comprises a nucleic acid sequence encoding a light chain variable region of an anti-HER2 antibody, wherein:

[0027] the nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 22, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 22; and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 24, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24.

[0028] In some embodiments, the foregoing anti-HER2 expressing vector system, wherein the first vector comprises a nucleic acid sequence encoding a heavy chain variable region of an anti-HER2 antibody, and the second vector comprises a nucleic acid sequence encoding a light chain variable region of an anti-HER2 antibody, wherein:

[0029] the nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID NO: 22, and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID NO: 24.

[0030] In some embodiments, the foregoing anti-HER2 expressing vector system, wherein the first vector comprises a nucleic acid sequence encoding a heavy chain constant region of an anti-HER2 antibody, and the second vector comprises a nucleic acid sequence encoding a light chain constant region of an anti-HER2 antibody.

[0031] In some embodiments, the aforementioned vector system for expressing an anti-HER2 antibody, wherein the first vector comprises a nucleic acid sequence encoding a heavy chain of the anti-HER2 antibody, and the second vector comprises a nucleic acid sequence encoding a light chain of the anti-HER2 antibody, wherein:

[0032] the nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 21, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 21; and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 23, or has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 23.

[0033] In some embodiments, the aforementioned vector system for expressing an anti-HER2 antibody, wherein the first vector comprises a nucleic acid sequence encoding a heavy chain of the anti-HER2 antibody, and the second vector comprises a nucleic acid sequence encoding a light chain of the anti-HER2 antibody, wherein:

[0034] the nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 21, and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO: 23.

[0035] In some embodiments, the aforementioned vector system for expressing an anti-HER2 antibody, wherein the first vector comprises a signal peptide sequence encoding a heavy chain of the anti-HER2 antibody, and the second vector comprises a signal peptide sequence encoding a light chain of the anti-HER2 antibody; the signal peptide can be any signal peptide known in the art that is suitable for antibody expression. In some embodiments, the amino acid sequence of the signal peptide of the heavy chain of the anti-HER2 antibody and the signal peptide of the light chain of the anti-HER2 antibody are both set forth in SEQ ID NO: 41. In some embodiments, the nucleic acid sequence encoding the signal peptide of the heavy chain of the anti-HER2 antibody is set forth in SEQ ID NO: 42.

[0036] In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule or the vector or the vector system for expressing an anti-HER2 antibody of the present application.

[0037] In some embodiments, the host cell is obtained by transfecting the vector for expressing an anti-HER2 antibody or the vector system for expressing an anti-HER2 antibody of the present application.

[0038] In some embodiments, the host cell is a CHO, ExpiCHO-S, CHO-S, CHO-K1, or CHO DG44 cell. In some embodiments, the host cell is an ExpiCHO-S cell.

[0039] In a fifth aspect, there is provided use of the nucleic acid molecule, the vector, the vector system or the host cell of the present application in the manufacture of an anti-HER2 antibody.

[0040] In a sixth aspect, there is provided a method of producing an anti-HER2 antibody, comprising culturing the host cell of the present application to express the anti-HER2 antibody. In some embodiments, the method further comprises purification and recovery of the antibody.

[0041] In some embodiments, the method of producing an anti-HER2 antibody comprises the steps of:

[0042] a. introducing the nucleic acid molecule, the vector or the vector system of the present application into a host cell;

[0043] b. culturing the host cell to express the anti-HER2 antibody; and optionally,

[0044] c. purifying and recovering the anti-HER2 antibody.

[0045] The nucleic acid molecule, the vector or the vector system can be introduced into the host cell using any method known in the art. Such procedures are well known to those skilled in the art and enable the expression of the anti-HER2 antibody. For example, the vector system of the present application can be introduced using the following methods:

[0046] In one embodiment, the first vector and the second vector of the vector system of the present application for expressing an anti-HER2 antibody are introduced into the host cell simultaneously.

[0047] In one embodiment, the first vector and the second vector of the vector system of the present application for expressing an anti-HER2 antibody are introduced into the host cell sequentially:

[0048] the first vector of the vector system of the present application for expressing an anti-HER2 antibody is introduced into the host cell first, and then the second vector of the vector system of the present application for expressing an anti-HER2 antibody is transfected into the same host cell; or

[0049] the second vector of the vector system of the present application for expressing an anti-HER2 antibody is introduced into the host cell first, and then the first vector of the vector system of the present application for expressing an anti-HER2 antibody is transfected into the same host cell.

[0050] The present application is directed to the expression characteristics of the anti-HER2 antibody, and a nucleic acid molecule capable of efficiently expressing the anti-HER2 antibody is designed, the expression amount of the anti-HER2 antibody is as high as 0.172 g / L, and in the expressed product, the content of the complete antibody is relatively high, and the fragments of the antibody are relatively few. And the relative binding activity of the antibody prepared by the present application to HER2, the affinity to FcRn and the affinity to FcγRlla are very close to the original research drug, that is, the original research drug has biological equivalence. Therefore, the anti-HER2 antibody produced by using the codon of the present application not only has high yield, but also has consistency and biological equivalence of quality characteristics, and has good drug-making prospects, which provides strong support for the industrialized production of the antibody. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The plasmid map of pcDNA3.4-HC in the embodiment of the present application;

[0052] Figure 2 The plasmid map of pcDNA3.4-LC in the embodiment of the present application.

[0053] Figure 3 The columnar diagram of the expression amount of the antibody of different codon groups in the embodiment of the present application. DETAILED DESCRIPTION

[0054] DEFINITIONS

[0055] The term "comprising" encompasses the meaning of "including" as well as "consisting of", for example, a composition "comprising" X can consist only of X, or can include some other elements, such as X+Y.

[0056] In the present application, the term "antibody" is an immunoglobulin capable of specifically recognizing and binding to an antigen, which encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies or antibody fragments. In particular, the amino acid sequence of the heavy chain of the anti-HER2 antibody of the present application is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain is shown in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24; the three CDR sequences of the heavy chain are shown in SEQ ID NO: 25, 26 and 27, respectively, and the three CDR sequences of the light chain are shown in SEQ ID NO: 28, 29 and 30, respectively.

[0057] The term "variable region" refers to the domain of the heavy chain or light chain of the antibody that recognizes and specifically binds to the epitope of the antigen.

[0058] CDR regions, or "complementarity determining regions," refer to the regions of an antibody variable region that are hypervariable in sequence and form structurally defined loops and / or contain the amino acid residues that contact the antigen. The CDRs are primarily responsible for binding to an epitope of an antigen and determine the specificity of an antibody. Within a given heavy or light chain variable region amino acid sequence, the particular amino acid sequence of each CDR is determined using any of a number of well-known numbering schemes, or combinations thereof, including, for example, Kabat, Contact, AbM, and Chothia.

[0059] In the present application, "sequence identity" refers to the percentage of amino acids (or bases) that are the same between two sequences when the sequences are aligned for comparison. The alignment and percent homology or sequence identity can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP. By way of example, "a nucleic acid sequence encoding a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 22" means that the nucleic acid sequence encoding a heavy chain variable region comprises HCDR1 set forth in SEQ ID NO: 25, HCDR2 set forth in SEQ ID NO: 26, HCDR3 set forth in SEQ ID NO: 27, and that codon mutations are allowed in regions outside of the CDRs such that the nucleic acid sequence has at least 90% sequence identity to SEQ ID NO: 22, and the like.

[0060] In the present application, "nucleic acid molecule" refers to an oligomer or polymer of at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds. The nucleic acid molecules of the present application can be isolated nucleic acid molecules, meaning nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule, such as by recombinant techniques essentially free of other cellular material, or culture medium when produced by chemical synthesis, essentially free of chemical precursors or other chemicals. The nucleic acid molecules of the present application are not limited to the sequences shown, but also include the complements thereof and modifications to the nucleic acid molecules that are well known in the art.

[0061] In the present application, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector on its own as well as the vector incorporating a heterologous nucleic acid that it has been engineered to carry. One or more heterologous proteins can be expressed from the vector when the vector is transformed into an appropriate expression cell. Vectors with respect include those into which a nucleic acid encoding a polypeptide or fragment thereof can be inserted by restriction enzyme digestion and ligation. Vectors with respect also include those comprising a nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are generally episomal, but can be designed to integrate the gene or part thereof into the chromosome of the genome. Vectors of artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The choice and use of such vehicles is well within the skill of those in the art.

[0062] An "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences capable of affecting expression of such DNA fragments, such as a promoter region. Such additional fragments can include a promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into an appropriate expression cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain episomal or integrate into the expression cell genome. Commercially available expression vectors, particularly for expression in mammalian cells, can be selected, for example, from the pIRES vector (from Clontech, Palo Alto, USA), the pCI-neo vector (from Promega, Madison, USA), the pCMV-Script vector (from Stratagene, La Jolla, USA), and the pCDNA vector (from Invitrogen, Paisley, UK).

[0063] In the present application, "codon" has the meaning well known in the art, and refers to a set of three nucleotide residues on mRNA (or DNA), each triplet encoding a specific amino acid. Codons are degenerate, in that there are at least two codons for each amino acid, except methionine and tryptophan. Different species have preferences for multiple codons for the same amino acid.

[0064] In the present application, "codon optimization" is a technique to increase the expression level of a protein in an organism by increasing the translation efficiency of a target gene. The expression sequence of a protein is redesigned by avoiding rare codons, using biased codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating restriction enzyme sites, adjusting GC content, etc. to improve the translation efficiency and thus increase the expression level of the protein.

[0065] "Vector system" refers to a combination of two or more vectors. The vector system is usually used to express a specific antibody or fusion protein, such as the anti-HER2 antibody used in the present application. It is well known to those skilled in the art that a double vector expression system can be used to express an antibody. Generally, in a double vector expression system, a first vector comprising an antibody light chain and a second vector comprising an antibody heavy chain are introduced into a host cell simultaneously or sequentially for antibody expression. Sequentially introduced into the host cell means that the first vector is introduced first and then the second vector is introduced, or the second vector is introduced first and then the first vector is introduced. Methods for introducing nucleic acids or vectors into cells are well known to those skilled in the art, such as electroporation, injection, transfection and / or transformation.

[0066] "Host cell" refers to a cell into which a foreign nucleic acid has been introduced and its progeny, regardless of the number of passages of the progeny. The progeny is allowed to be not completely identical to the parent cell in terms of nucleic acids, but can contain mutations, as long as the progeny with mutations has the same desired function or activity as the original cell.

[0067] The host cell cannot develop into a complete animal or plant individual.

[0068] The host cell includes prokaryotic and eukaryotic host cells. The eukaryotic host cell includes but is not limited to: mammalian cells, insect cells, plant cells and fungal cells. The mammalian cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, hamster cells. Exemplary host cells include but are not limited to CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells and HEK-293 cells. The fungal cells include yeast, such as but not limited to Pichia, Saccharomyces, Hensenula, Kluyveromyces.

[0069] "Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur. The singular forms "a", "an" and "the" include their corresponding plural referents unless the content clearly dictates otherwise.

[0070] The present application will be described in detail below with reference to specific examples, but the content of the present application is not limited thereto.

[0071] Unless otherwise indicated, the reagents and instruments used in the following methods are commercially available and are used according to the manufacturer's instructions; the methods used are conventional methods in the art, which can be performed without any doubt by the person skilled in the art and the corresponding results can be obtained according to the description in the examples.

[0072] Unless otherwise indicated, the molecular biology experimental methods and immunoassay methods used in the present application are basically performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995; and the use of restriction enzymes is in accordance with the recommended conditions of the product manufacturer. The person skilled in the art knows that the examples describe the present application by way of example and do not mean to limit the scope of the present application as claimed.

[0073] The room temperature described in the examples is the room temperature conventionally used in the art, which is generally 18-26°C.

[0074] Unless otherwise indicated, the reagents and cells used in the following examples are provided by Shanghai Genomics Co., Ltd.

[0075] In the following examples, the original drug used is Pertuzumab, also known as 2C4, with the trade name Perjeta, and the name Paget. The amino acid sequences of the heavy chain and light chain of Pertuzumab are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

[0076] Example 1. Optimization of anti-HER2 antibody nucleic acid sequences

[0077] The heavy chain, heavy chain variable region, light chain and light chain variable region of the anti-HER2 antibody in this example are as follows:

[0078] Heavy chain of Pertuzumab:

[0079] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRF TLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

[0080] Heavy chain variable region of Pertuzumab:

[0081] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 2)

[0082] Light chain of Pertuzumab:

[0083] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3)

[0084] Light chain variable region of Pertuzumab:

[0085] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: 4)

[0086] The CDR regions of Pertuzumab are shown in Table 1 below.

[0087] Table 1: CDR sequences of Pertuzumab

[0088]

[0089] The Pertuzumab heavy chain and light chain amino acid sequences were reverse-transcribed to generate nucleotide sequences encoding the same original amino acid sequences. Different codons were used to generate multiple nucleotide sequences with the highest theoretical expression level in the selected host cells (e.g. CHO cells) for subsequent screening. And the codon set 1 in the prior art (see SEQ ID NO: 1 and 2 in CN104726462A) was used as a comparative set.

[0090] After multiple rounds of optimization in multiple systems, two groups of high expression nucleic acid sequences with different sequences were finally obtained, which were referred to as codon set 2 and codon set 3, respectively.

[0091] The heavy chain, heavy chain variable region, light chain and light chain variable region of codon set 1, codon set 2 and codon set 3 are shown below, and their CDR encoding sequences are shown in Tables 2, 3 and 4.

[0092] Codon set 1 heavy chain:

[0093] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGTCCTGCGC CGCCAGCGGCTTCACCTTTACCGACTACACCATGGACTGGGTGCGCCAGGCTCCCGGCAAGGGCCTGGAGTGGGTG GCCGACGTGAACCCCAACAGCGGCGGCAGCATCTACAACCAGCGCTTCAAGGGCCGCTTCACCCTGAGCGTGGACC GCAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCAA CCTGGGCCCCAGCTTCTACTTCGACTATTGGGGGCAGGGCACCCTGGTCACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 1)NO:11)

[0094] Codon Set 1 Heavy Chain Variable Region:

[0095] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGTCCTGCGCCGCCAGCGGCTTCACCTTTACCGACTACACCATGGACTGGGTGCGCCAGGCTCCCGGCAAGGGCCTGGAGTGGGTGGCCGACGTGAACCCCAACAGCGGCGGCAGCATCTACAACCAGCGCTTCAAGGGCCGCTTCACCCTGAGCGTGGACCGCAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCAACCTGGGCCCCAGCTTCTACTTCGACTATTGGGGGCAGGGCACCCTGGTCACCGTGAGCAGC (SEQ ID NO: 12)

[0096] Codon Set 1 Light Chain:

[0097] GATATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCAGCGTGGGCGACCGCGTGACCATCACCTG CAAGGCCAGCCAGGACGTGAGCATCGGCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC TACAGCGCCTCCTACCGCTACACCGGCGTGCCCTCCCGCTTCAGCGGCTCCGGCAGCGGCACCGACTTTACCCTGA CCATCTCCAGCCTGCAGCCCGAGGACTTTGCCACCTACTACTGCCAGCAGTACTACATCTATCCCTATACCTTCGG CCAGGGCACCAAGGTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 13)

[0098] Codon Set 1 Light Chain Variable Region:

[0099] GATATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCAAGGCCAGCCAGGACGTGAGCATCGGCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCTCCTACCGCTACACCGGCGTGCCCTCCCGCTTCAGCGGCTCCGGCAGCGGCACCGACTTTACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTTGCCACCTACTACTGCCAGCAGTACTACATCTATCCCTATACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG (SEQ ID NO: 14)

[0100] Table 2: CDR-encoding sequences for Codon Set 1

[0101]

[0102] Codon Set 2 Heavy Chain:

[0103] GAGGTGCAACTGGTGGAATCTGGCGGAGGCCTCGTGCAGCCAGGCGGCTCTCTGCGGCTGTCCTGTGC CGCCTCTGGATTTACCTTCACTGACTATACCATGGACTGGGTCAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTG GCCGACGTGAATCCTAACAGCGGCGGCTCCATCTACAACCAGCGGTTCAAGGGCAGATTCACCCTGTCCGTGGACC GCTCCAAGAACACCCTGTACCTGCAGATGAACTCTCTGAGAGCCGAGGACACCGCCGTGTATTACTGCGCCAGAAA CCTGGGACCTTCCTTCTACTTCGACTACTGGGGCCAAGGTACACTGGTTACAGTGTCCTCC GCCTCCACCAAGGGCCCTTCCGTGTTTCCTCTGGCTCCTAGTTCTAAGAGCACCTCTGGCGGAACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCCGGCGCTCTGACCTCCGGCGTGCACACCTTTCCCGCTGTGCTGCAGTCCAGCGGCCTGTACTCTCTGAGCTCTGTGGTGACAGTGCCATCTTCCTCCCTGGGCACACAGACCTACATCTGCAACGTGAACCACAAGCCATCTAACACCAAAGTCGACAAGAAAGTGGAACCTAAGTCTTGCGACAAAACACATACCTGCCCCCCTTGTCCTGCTCCCGAGCTGCTCGGCGGACCTAGCGTGTTCCTGTTCCCTCCAAAGCCCAAGGATACACTGATGATCTCCCGGACACCTGAAGTGACATGCGTGGTCGTGGATGTGTCTCACGAGGATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAAACCAAGCCTCGGGAGGAACAGTACAACTCTACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCTCAAGTGTACACCCTGCCCCCATCTAGAGAAGAGATGACCAAGAACCAGGTATCCTTGACCTGTCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTCGAGTGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAGTCTCTGAGCCTGTCTCCTGGCAAG(SEQ IDNO:21)

[0104] Codon Set 2 heavy chain variable region:

[0105] GAGGTGCAACTGGTGGAATCTGGCGGAGGCCTCGTGCAGCCAGGCGGCTCTCTGCGGCTGTCCTGTGCCGCCTCTGGATTTACCTTCACTGACTATACCATGGACTGGGTCAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGACGTGAATCCTAACAGCGGCGGCTCCATCTACAACCAGCGGTTCAAGGGCAGATTCACCCTGTCCGTGGACCGCTCCAAGAACACCCTGTACCTGCAGATGAACTCTCTGAGAGCCGAGGACACCGCCGTGTATTACTGCGCCAGAAACCTGGGACCTTCCTTCTACTTCGACTACTGGGGCCAAGGTACACTGGTTACAGTGTCCTCC (SEQ ID NO: 22)

[0106] Codon Set 2 light chain:

[0107] GACATCCAGATGACCCAGTCTCCCTCCTCTCTGTCCGCTTCCGTGGGCGACAGAGTGACCATCACCTG CAAGGCTTCTCAGGACGTGTCCATCGGCGTGGCCTGGTACCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATC TATTCTGCTTCCTACCGGTACACCGGCGTGCCTTCTAGATTCAGCGGATCTGGCTCCGGCACCGACTTTACACTGA CCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAACAGTACTACATCTACCCCTACACCTTCGG CCAGGGCACAAAAGTGGAAATCAAG CGGACCGTGGCTGCTCCTAGCGTGTTCATCTTTCCTCCATCTGATGAACAGCTGAAATCTGGCACAGCTTCTGTGGTGTGCCTGCTGAACAACTTCTACCCTAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAATTCCCAAGAGTCCGTCACCGAGCAGGATAGCAAGGACTCCACCTATTCCCTGTCTAGTACCCTGACCCTCTCCAAGGCCGATTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTCACCAAGTCCTTCAACAGAGGCGAGTGT (SEQ ID NO: 23)

[0108] Codon Set 2 light chain variable region:

[0109] GACATCCAGATGACCCAGTCTCCCTCCTCTCTGTCCGCTTCCGTGGGCGACAGAGTGACCATCACCTGCAAGGCTTCTCAGGACGTGTCCATCGGCGTGGCCTGGTACCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTATTCTGCTTCCTACCGGTACACCGGCGTGCCTTCTAGATTCAGCGGATCTGGCTCCGGCACCGACTTTACACTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAACAGTACTACATCTACCCCTACACCTTCGGCCAGGGCACAAAAGTGGAAATCAAG (SEQ ID NO: 24)

[0110] Table 3: CDR-encoding sequences for Codon Set 2

[0111]

[0112] Codon Set 3 Heavy Chain:

[0113] GAGGTTCAGTTGGTGGAGTCCGGTGGAGGATTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGC TGCTTCTGGATTTACCTTTACAGATTACACAATGGATTGGGTGAGGCAGGCCCCTGGAAAGGGACTGGAGTGGGTG GCTGATGTGAACCCTAATAGCGGCGGGTCCATTTACAACCAGAGGTTCAAGGGCAGATTCACACTGAGCGTGGACA GGAGCAAGAACACACTGTATCTGCAGATGAACTCCTTGAGGGCCGAGGATACCGCCGTTTACTACTGTGCCAGGAA CCTGGGGCCCTCTTTCTATTTCGACTACTGGGGCCAGGGAACACTGGTGACAGTCTCTTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 1)NO:31)

[0114] Codon Set 3 heavy chain variable region:

[0115] GAGGTTCAGTTGGTGGAGTCCGGTGGAGGATTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCTGCTTCTGGATTTACCTTTACAGATTACACAATGGATTGGGTGAGGCAGGCCCCTGGAAAGGGACTGGAGTGGGTGGCTGATGTGAACCCTAATAGCGGCGGGTCCATTTACAACCAGAGGTTCAAGGGCAGATTCACACTGAGCGTGGACAGGAGCAAGAACACACTGTATCTGCAGATGAACTCCTTGAGGGCCGAGGATACCGCCGTTTACTACTGTGCCAGGAACCTGGGGCCCTCTTTCTATTTCGACTACTGGGGCCAGGGAACACTGGTGACAGTCTCTTCT (SEQ ID NO: 32)

[0116] Codon Set 3 light chain:

[0117] GACATCCAGATGACCCAGTCCCCATCCTCCCTGTCTGCTTCTGTGGGAGACAGGGTGACCATCACCTG CAAGGCATCCCAGGACGTGTCCATCGGAGTGGCTTGGTACCAGCAGAAGCCTGGAAAAGCCCCCAAGCTGCTCATC TACTCTGCCTCTTACAGGTATACCGGAGTGCCCAGCCGGTTTAGCGGAAGCGGAAGCGGCACCGACTTCACCCTGA CCATTTCCTCCCTGCAGCCCGAAGATTTTGCTACCTACTACTGCCAGCAGTACTACATCTACCCCTACACCTTCGG ACAGGGTACCAAGGTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 33)

[0118] Codon Set 3 light chain variable region:

[0119] GACATCCAGATGACCCAGTCCCCATCCTCCCTGTCTGCTTCTGTGGGAGACAGGGTGACCATCACCTGCAAGGCATCCCAGGACGTGTCCATCGGAGTGGCTTGGTACCAGCAGAAGCCTGGAAAAGCCCCCAAGCTGCTCATCTACTCTGCCTCTTACAGGTATACCGGAGTGCCCAGCCGGTTTAGCGGAAGCGGAAGCGGCACCGACTTCACCCTGACCATTTCCTCCCTGCAGCCCGAAGATTTTGCTACCTACTACTGCCAGCAGTACTACATCTACCCCTACACCTTCGGACAGGGTACCAAGGTGGAGATCAAG (SEQ ID NO: 34)

[0120] Table 4: CDR-encoding sequences of codon group 3

[0121]

[0122] By sequence alignment, the sequence identity of different codon groups is as follows: for the antibody full-length nucleic acid sequence, the identity of codon group 2 heavy chain to codon group 1 heavy chain is 82.2%, and the identity of codon group 3 heavy chain to codon group 1 heavy chain is 93.7%. The identity of codon group 2 light chain to codon group 1 light chain is 83.2%, and the identity of codon group 3 light chain to codon group 1 light chain is 91.7%.

[0123] For the variable region, the identity of codon group 2 heavy chain variable region to codon group 1 heavy chain variable region is 83.2%, and the identity of codon group 3 heavy chain variable region to codon group 1 heavy chain variable region is 77.3%. The identity of codon group 2 light chain variable region to codon group 1 light chain variable region is 86.6%, and the identity of codon group 3 light chain variable region to codon group 1 light chain variable region is 83.8%.

[0124] Example 2. Plasmid construction

[0125] The nucleic acid sequences encoding the light chain and heavy chain amino acids of the anti-HER2 antibody were synthesized respectively, and the nucleic acid sequences encoding the light and heavy chains containing the signal peptide were recombined into the pcDNA3.4 vector (signal peptide+heavy chain, signal peptide+light chain) using restriction enzyme sites AflII and EcoRV, to construct the recombinant plasmids pcDNA3.4-HC and pcDNA3.4-LC. The plasmid maps of pcDNA3.4-HC and pcDNA3.4-LC are shown in Figure 1 and Figure 2The signal peptide sequence used for the light chain and the heavy chain of the antibody is the same, and the sequence of the signal peptide is as follows:

[0126] Signal peptide of the anti-HER2 antibody:

[0127] MGWSCIILFLVATATGVHS (SEQ ID NO: 41)

[0128] Nucleic acid sequence encoding the signal peptide:

[0129] ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCA (SEQ ID NO: 42).

[0130] The six different recombinant plasmids are named pcDNA3.4-codon set 1-HC, pcDNA3.4-codon set 1-LC, pcDNA3.4-codon set 2-HC, pcDNA3.4-codon set 2-LC, pcDNA3.4-codon set 3-HC, and pcDNA3.4-codon set 3-LC, and the light and heavy chain plasmid frameworks are consistent. The recombinant plasmids are transformed into E. coli cells DH5a (Takara, 9057) respectively, and are amplified and cultured. After the recombinant plasmids are extracted and sequencing is confirmed to be correct, the obtained plasmids are transformed into E. coli, and are stored at -20°C for subsequent transient transfection experiments.

[0131] Example 3. Plasmid amplification and extraction

[0132] The recombinant plasmids obtained in Example 2 are transformed into E. coli, and are cultured using LB medium. According to the resistance of the pcDNA3.4 plasmid, ampicillin (Shanghai Generay, A100339) is added to the LB medium for large-scale culture.

[0133] The E. coli cells after large-scale culture are collected by centrifugation, and a plasmid extraction kit (Axygen, AP-MX-P-25) is used to lyse the E. coli cells according to the alkaline lysis method. Endotoxin is removed (Shanghai Generay, B641718) during the process, and the plasmid is extracted. The plasmid concentration is detected by a microspectrophotometer (Hangzhou Aosheng, Nano-300), and is stored at -20°C for subsequent transient transfection experiments.

[0134] Example 4. Transfection of plasmids into ExpiCHO-S cells

[0135] One vial of ExpiCHO-S (Thermo, item number A29133) cryopreserved cells is taken out and thawed at 37°C. The cells are counted, and 0.5×10 6Cell / mL viable cell density passage, culture medium is Dynamis (Gibco, A26175) complete medium (Dynamis medium + 6 mmol / L L-Gln) containing 6 mmol / L L-Gln (Sigma, V900419-500G). ExpiCHO-S cells were cultured in a humidified carbon dioxide incubator (Kuhner, ISF4-XC) at a culture condition of 37°C and 5% carbon dioxide. Subculture was performed every 2-4 days, and the subculture viable cell density was 0.2 x 10 6 ~0.5 x 10 6 cells / mL. After the cells recovered activity, they were transferred to Dynamis complete medium for subculture. 24 hours before transfection, subculture was performed in Dynamis complete medium, and the subculture viable cell density was 3 x 10 6 ~4 x 10 6 cells / mL.

[0136] On the day of transfection, the viable cell density was adjusted to 6 x 10 6 cells / mL using Dynamis complete medium. The transfection system was 10.5 pg PEI (Polyscoences, 23966), 1.75 pg recombinant heavy chain plasmid, 1.75 pg recombinant light chain plasmid, and 2.625 pg inert DNA (Sigma, 31149-10G-F) added per milliliter of cell culture solution. 4 hours after transfection, a total volume of 4% Cell Boost 7a solution (Cytiva, SH31026.02), 0.4% Cell Boost 7b solution (Cytiva, SH31027.07CN), and 0.125% DMA was supplemented. On the first day of transfection, D-glucose was supplemented to 7 g / L, and the temperature was lowered to 32°C for culture; on the second day of transfection, 2 mM L-Gln was supplemented; on the fifth day of transfection, 6% Cell Boost 7a solution, 0.6% Cell Boost 7b solution, and 4 mM L-Gln were supplemented; and on the seventh day of transfection, the transient transfection cell culture supernatant was harvested. The harvested supernatant was subjected to antibody expression detection and one-step affinity purification.

[0137] Example 5. Purification of antibodies

[0138] The antibody was purified using one-step affinity chromatography. The steps were as follows: centrifugation (12000 g for 10 min) to collect the supernatant of the cell culture of the transiently transfected recombinant plasmid, purification using a Protein A affinity chromatography column (Cytiva, 17543803) on a protein purification chromatography system (Cytiva, AKTA avant), and elution of the target protein using a pH 2.8 acetic acid solution (Macklin, A801295). The antibody obtained after purification was subjected to SEC-HPLC and IEC-HPLC quality testing (see Example 6 for specific methods).

[0139] Example 6. Antibody quality testing

[0140] 6.1 SEC-HPLC

[0141] The SEC-HPLC sample was diluted to 2.0 mg / mL with the mobile phase.

[0142] Instrument: high-performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic column: TOSOH G3000 SWXL chromatographic column, specifications 7.8*300 mm, 5 μm.

[0143] Detection mobile phase: 0.06 M potassium dihydrogen phosphate (Guojia 10017518), 0.14 M potassium dihydrogen phosphate (Guojia 10017618), 0.25 M potassium chloride (Guojia 10016318), pH 6.2.

[0144] Injection volume: 25 μL; column temperature: 25.0 ± 5.0 °C. Sample room temperature: 2-8 °C. Detection wavelength: 280 nm, detection time 40 min.

[0145] During detection, first perform column equilibration, i.e., equilibrate the system to the baseline with 100% mobile phase at a flow rate of 0.5 mL / min, then inject the sample, perform sample determination, and record the chromatogram.

[0146] Integrate the chromatogram according to the chromatogram, and obtain the content of the target peak and the content of the monomer peak by analyzing and calculating the peak area of the target peak.

[0147] 6.2 IEC-HPLC

[0148] The IEC-HPLC sample was diluted to 1.0 mg / mL with mobile phase A, 1.5% CPB enzyme was added to a total volume, and mixed uniformly, and then placed in a 37 °C water bath for 30 min.

[0149] Instrument: High performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Column: ThermoPropac WCX-10, 054993 column, 4*250 mm.

[0150] Mobile phase A: 0.02M MES (Macklin, M813152), 0.001M Na2EDTA (National Pharmaceutical, 10009717), pH 6.0; flow rate: 0.8 mL / min.

[0151] Mobile phase B: 0.02M MES (Macklin, M813152), 0.001M Na2EDTA (National Pharmaceutical, 10009717), 0.25M sodium chloride (National Pharmaceutical, 10019318), pH 6.0; flow rate: 0.8 mL / min.

[0152] Injection volume: 50 μL; column temperature: 25.0±5.0℃. Sample room temperature: 2-8℃. Detection wavelength: 280 nm, detection time 95 min.

[0153] At the time of detection, first perform column equilibration, i.e., equilibrate the system to baseline stability with 100% mobile phase A at a flow rate of 1.0 mL / min, then inject the sample, perform sample determination, and record the chromatogram. Sample determination uses gradient elution, and the elution gradient is shown in Table 5 below:

[0154] Table 5: Chromatographic elution gradient for protein charge variant determination by IEC-HPLC method

[0155]

[0156] Integrate the chromatogram according to the chromatogram, and obtain the contents of the acidic peak, main peak, and basic peak of the charge variant by analyzing and calculating the peak area of the target peak of the test sample solution. The original drug, pertuzumab injection (Pertuzumab, Roche, batch number H0641B08), is used as the reference group.

[0157] The quality of the anti-HER2 antibodies expressed by different codon groups is shown in Table 6 below.

[0158] Table 6: Antibody quality results of different codon groups

[0159]

[0160] From the SEC-HPLC data, it can be seen that the main peak content of codon group 2 is significantly higher than that of codon groups 1 and 2. From the IEC-HPLC data, it can be seen that the main peak content of codon group 2 is significantly higher than that of the other groups.

[0161] Example 7. Antibody expression amount detection

[0162] The expression amount of the antibody was detected by HPLC:

[0163] According to the Protein A one-step affinity chromatography method, the antibody was eluted by using a low pH solution, the areas of the elution peaks of the standard and sample were counted, the linear relationship between the standard concentration and the chromatographic peak area was established, and the antibody concentration in the sample was calculated according to the linear relationship of the standard and the peak area of the sample.

[0164] Instrument: high performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic column: ThermoFish POROS A / 20, 1502412 chromatographic column, 2.1*30 mm in size.

[0165] Detection mobile phase A: 50 mM disodium hydrogen phosphate dodecahydrate (National Pharmaceutical, 10020318) and 150 mM sodium chloride (National Pharmaceutical, 10019318), pH 7.0; flow rate: 3.0 mL / min.

[0166] Detection mobile phase B: 150 mM sodium chloride (National Pharmaceutical, 10019318), pH 1.9; flow rate: 3.0 mL / min.

[0167] Injection volume: 80 μL; column temperature: 25.0±5.0℃. Sample temperature: 2-8℃. Detection wavelength: 280 nm, detection time 3 min.

[0168] During detection, first, the chromatographic column was equilibrated, i.e., 100% mobile phase A was used to equilibrate the system to the baseline until it was stable, then the sample was injected, the sample concentration was determined, and the chromatogram was recorded. Gradient elution was used for sample determination, and the elution gradient is shown in Table 7, wherein A (%) and B (%) refer to the volume percentages of A phase and B phase, respectively:

[0169] Table 7: Chromatographic elution gradient for protein content determination by HPLC method

[0170]

[0171] According to the chromatogram, the peak areas of the target peaks of the standard curve solution and the test sample solution were calculated to obtain the protein content. The results are shown in Table 8 and Figure 3 .

[0172] Table 8: Antibody expression amounts of different codon groups

[0173]

[0174] From the results of Table 8 and Figure 3 , it can be seen that the expression amount of codon group 2 is the highest, reaching 0.172 g / L, which is higher than that of codon 1 and significantly higher than that of codon group 3.

[0175] Based on the comparison of the expression levels, codon group 2 is preferred.

[0176] Example 8. Detection of the binding activity of the antibody to HER2

[0177] 8.1 Binding of the antibody to the HER2 protein

[0178] In this experiment, enzyme-linked immunosorbent assay (ELISA) was used. HER2 antigen was connected to a solid carrier. The antibody to be detected in the sample was combined with the HER2 antigen to form a solid-phase antigen-antibody complex. Enzyme-labeled secondary antibody was combined with the antibody in the solid-phase immune complex to form a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex. The degree of color development after adding the substrate was determined. The absorbance value was positively correlated with the binding activity of the antibody.

[0179] Specifically, HER2 antigen (Yiqiao God, 10004-H08H) was diluted with coating buffer to 0.5 μg / mL and added to the enzyme-labeled plate, which was coated at 4°C for 12-18 hours. Then 350 μL of blocking solution was added to each well, and the blocking was performed at 37°C for 1.5 hours. The antibody to be detected was diluted with diluent and incubated at 37°C for 1.5 hours. Peroxidase-labeled goat anti-human IgG Fc antibody (Sigma, A0170) was diluted with diluent and incubated at room temperature for 1 hour. After incubation, TMB color developing solution was incubated at room temperature for 30 minutes. After color development, 100 μL of stop solution (1M sulfuric acid) was added to each well to stop the substrate reaction. The absorbance was measured at an enzyme marker detection wavelength of 450 nm to read the absorbance results of each well. According to Prism software analysis, the EC 50 values of each group were calculated, and the calculation formula was as follows:

[0180] ;

[0181] The reference was the original drug pertuzumab injection (Pertuzumab, Roche, batch number H0641B08), and the experimental group was the antibody prepared using codon group 1, codon group 2, and codon group 3.

[0182] The relative binding activity of the antibody of the present disclosure to human HER2 antigen is shown in Table 9.

[0183] Table 9 Relative binding activity of the antibody to human HER2 antigen

[0184]

[0185] From the results of Table 9, it can be seen that the relative binding activity of the antibody prepared by codon group 2 to human HER2 antigen is very close to that of the reference (i.e. the original drug Pertuzumab), while the relative binding activity of the antibody prepared by codon 1 and codon group 3 to human HER2 antigen is quite different from that of the reference, therefore, the biological performance of the antibody prepared by codon 2 is very close to that of the original drug, and codon 2 is more optimal.

[0186] 8.2 Detection of antibody affinity to FcRn

[0187] The affinity of the antibody to FcRn was detected by using HIS1K sensor (Sartorius, 18-5120) on a fortébio BID molecular interaction instrument (Sartorius, OCTET(RED)96), and the running parameters are shown in Table 10 below.

[0188] Table 10

[0189]

[0190] Note: R / N is Regeneration / Neutralization, which is the abbreviation of the reduction and regeneration cycle at the beginning and end of the test.

[0191] The affinity of the antibody to FcRn was detected by using HIS1K sensor (Sartorius, 18-5120) on a fortébio BID molecular interaction instrument (Sartorius, OCTET(RED)96), and the running parameters are shown in Table 10 below.

[0192] Table 11

[0193]

[0194] From Table 11, it can be seen that the binding activity of the antibody prepared by codon group 2 to FcRn is very close to that of the original drug, indicating that the biological performance of the antibody is very close to that of the original drug.

[0195] 8.3 Detection of antibody affinity to FcγRlla

[0196] The affinity of the antibody to FcγRlla was detected by using ProA sensor (Sartorius, 18-5010) on a fortébio BID molecular interaction instrument (Sartorius, OCTET(RED)96), and the running parameters are shown in Table 12 below.

[0197] Table 12

[0198]

[0199] Note: R / N is Regeneration / Neutralization, which is the abbreviation of the reduction and regeneration cycle at the beginning and end of the test.

[0200] The Steady-State method was used to analyze the affinity of the antibody to FcyRIla, and the results are shown in Table 13 below.

[0201] Table 13

[0202]

[0203] From the FcyRIla affinity data, it can be seen that the antibody prepared by using the codon group 2 has a binding activity to FcyRIla very close to that of the original drug, indicating that the biological performance of the antibody is very close to that of the original drug.

[0204] In summary, the codon group 2 of the present application is a codon optimized for expression cells. By using the nucleic acid molecule of the present application, the expression amount of the anti-HER2 antibody in cells can be improved, the yield of the anti-HER2 antibody is increased, and the production cost is reduced; and the anti-HER2 antibody produced has similar performance to the original drug, and has a good drug development prospect.

Claims

1. A nucleic acid molecule comprising nucleic acid sequences encoding anti-HER2 antibody heavy and light chains, wherein: the nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 21 and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO:

23.

2. A vector comprising the nucleic acid molecule of claim 1.

3. An anti-HER2 antibody-expressing vector system comprising a first vector and a second vector, wherein: the first vector comprises a nucleic acid sequence encoding an anti-HER2 antibody heavy chain and the second vector comprises a nucleic acid sequence encoding an anti-HER2 antibody light chain, the nucleic acid sequence encoding the heavy chain is set forth in SEQ ID NO: 21 and the nucleic acid sequence encoding the light chain is set forth in SEQ ID NO:

23.

4. A host cell comprising the nucleic acid molecule of claim 1, or the vector of claim 2, or the vector system of claim 3.

5. The host cell of claim 4, wherein, the host cell is an ExpiCHO-S, CHO-S, CHO-K1 or CHO DG44 cell.

6. The host cell of claim 5, wherein, the host cell is an ExpiCHO-S cell.

7. Use of the nucleic acid molecule of claim 1, or the vector of claim 2, or the vector system of claim 3, or the host cell of any one of claims 4-6 in the manufacture of an anti-HER2 antibody.

8. A method of making an anti-HER2 antibody, comprising culturing the host cell of any one of claims 4-6 to express the anti-HER2 antibody.

9. The method of claim 8, wherein, the method further comprises purification and recovery of the antibody.

10. The method of claim 8, wherein: the method comprises the following steps: a. introducing the nucleic acid molecule of claim 1, the vector of claim 2 or the vector system of claim 3 into a host cell; b. culturing the host cell to express the anti-HER2 antibody; and optionally, c. purifying and recovering the anti-HER2 antibody.

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