Nucleic acid molecules encoding zebetuzumab and their applications

Abstract

The present invention belongs to the field of biology, and relates to a nucleic acid molecule encoding zebetuzumab, a recombinant vector comprising the nucleic acid molecule, and an expression method, wherein the nucleic acid molecule comprises codons optimized for expression cells. By using the nucleic acid molecule of the present invention, the expression amount of zebetuzumab in cells can be increased, the yield of zebetuzumab can be increased, and the production cost can be reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biology and relates to a nucleic acid molecule encoding zebetuzumab. Background Art

[0002] Zebetuzumab (also known as HS006) is a monoclonal antibody targeting CD20. It can specifically bind to the CD20 antigen on the surface of B cells and quickly, thoroughly, durably and reversibly eliminate B cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0003] Preclinical research data show that compared with other similar CD20 antibodies, zebetozumab has a stronger cytotoxic effect (ADCC) and a larger steady-state distribution volume, which can produce a more sustained clearance effect on B cells, thereby exerting a better drug effect. Zebetozumab has been approved for the treatment of CD20-positive diffuse large B-cell lymphoma, providing a new option for lymphoma patients.

[0004] However, the existing cell expression of Zebetuzumab is limited, which greatly affects the production of Zebetuzumab. Therefore, how to increase the production of Zebetuzumab is a major problem that needs to be solved in this field. Summary of the invention

[0005] The purpose of the present invention is to optimize the codons of zebetuzumab to increase the expression level of the monoclonal antibody in cells.

[0006] In a first aspect, a nucleic acid molecule is provided, comprising nucleic acid sequences encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of zebetuin, and nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of zebetuin, wherein:

[0007] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO:21, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO:22, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO:23, the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:24, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:25, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:26; or

[0008] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO:27, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO:28, the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO:29, the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:30, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:31, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:32.

[0009] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of zebetuin, wherein:

[0010] The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO:15, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO:16, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0011] In some embodiments, the aforementioned nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of zebetuin, wherein:

[0012] The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO:19, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO:20, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0013] In some embodiments, the aforementioned nucleic acid molecules comprise nucleic acid sequences encoding the heavy and light chains of zebetuin, wherein:

[0014] The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO:13, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO:14, or has at least 85% sequence identity thereto.

[0015] In some embodiments, the aforementioned nucleic acid molecules comprise nucleic acid sequences encoding the heavy and light chains of zebetuin, wherein:

[0016] The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO:17, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO:18, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0017] In a second aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule of the present invention.

[0018] In some embodiments, the starting vector of the recombinant vector is an expression vector.

[0019] In some embodiments, the starting vector of the recombinant vector is a pcDNA3.4-TOPO vector.

[0020] In a third aspect, the present invention provides a vector system for expressing zebetuin, comprising a first vector and a second vector, wherein:

[0021] The first vector comprises a nucleic acid sequence encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of zebetuin, wherein:

[0022] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO:21, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO:22, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO:23; or

[0023] The nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO:27, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO:28, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO:29; and

[0024] The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of zebetozumab, wherein:

[0025] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:24, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:25, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:26; or

[0026] The nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:30, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:31, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:32.

[0027] In some embodiments, in the aforementioned vector system for expressing zebetuzumab, the first vector comprises nucleic acid sequences encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of zebetuzumab, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 21, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 22, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 23; and

[0028] The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of Zebetuzumab, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:24, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:25, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:26.

[0029] In some embodiments, in the aforementioned vector system for expressing zebetuzumab, the first vector comprises nucleic acid sequences encoding HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of zebetuzumab, wherein the nucleic acid sequence encoding HCDR1 is shown in SEQ ID NO: 27, the nucleic acid sequence encoding HCDR2 is shown in SEQ ID NO: 28, and the nucleic acid sequence encoding HCDR3 is shown in SEQ ID NO: 29; and

[0030] The second vector comprises nucleic acid sequences encoding LCDR1, LCDR2 and LCDR3 of the light chain variable region of Zebetuzumab, wherein the nucleic acid sequence encoding LCDR1 is shown in SEQ ID NO:30, the nucleic acid sequence encoding LCDR2 is shown in SEQ ID NO:31, and the nucleic acid sequence encoding LCDR3 is shown in SEQ ID NO:32.

[0031] In some embodiments, the aforementioned vector system for expressing zebetuzumab, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of zebetuzumab, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of zebetuzumab, wherein:

[0032] The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO:15 or SEQ ID NO:19, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:15 or SEQ ID NO:19; and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO:16 or SEQ ID NO:20, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:16 or SEQ ID NO:20.

[0033] In some embodiments, the aforementioned vector system for expressing zebetuzumab, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of zebetuzumab, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of zebetuzumab, wherein:

[0034] The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO:15, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:15; and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO:16, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:16.

[0035] In some embodiments, the aforementioned vector system for expressing zebetuzumab, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain variable region of zebetuzumab, and the second vector comprises a nucleic acid sequence encoding the light chain variable region of zebetuzumab, wherein:

[0036] The nucleic acid sequence encoding the heavy chain variable region is as shown in SEQ ID NO:19, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:19; and the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO:20, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:20.

[0037] In some embodiments, the aforementioned vector system for expressing zabetuzumab, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of zabetuzumab, and the second vector comprises a nucleic acid sequence encoding the light chain of zabetuzumab, wherein:

[0038] The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO:13 or SEQ ID NO:17, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:13 or SEQ ID NO:17; and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO:14 or SEQ ID NO:18, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:14 or SEQ ID NO:18.

[0039] In some embodiments, the aforementioned vector system for expressing zabetuzumab, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of zabetuzumab, and the second vector comprises a nucleic acid sequence encoding the light chain of zabetuzumab, wherein:

[0040] The nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO:13, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:13; and the nucleic acid sequence encoding the light chain is as shown in SEQ ID NO:14, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:14.

[0041] In some embodiments, the aforementioned vector system for expressing zabetuzumab, wherein the first vector comprises a nucleic acid sequence encoding the heavy chain of zabetuzumab, and the second vector comprises a nucleic acid sequence encoding the light chain of zabetuzumab, wherein:

[0042] The nucleic acid sequence encoding the heavy chain variable region is as set forth in SEQ ID NO:17, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:17; and the nucleic acid sequence encoding the light chain is as set forth in SEQ ID NO:18, or has at least 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:18.

[0043] In a fourth aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule or recombinant vector of the present invention or a vector system expressing zebetuzumab.

[0044] In some embodiments, the recombinant cell is obtained by transfecting the vector system expressing zebetuzumab of the present invention.

[0045] In some embodiments, the host cell of the recombinant cell is a CHO cell. In some embodiments, the host cell of the recombinant cell is a CHO-K1 cell.

[0046] In a fifth aspect, provided is the use of the nucleic acid molecule, recombinant vector, vector system of zebetutumab or recombinant cell of the present invention in the preparation of zebetutumab.

[0047] In a sixth aspect, a method for preparing zebetutumab is provided, comprising culturing the recombinant cell of the present invention to express zebetutumab. Preferably, the method further comprises purification and recovery of the antibody.

[0048] In a seventh aspect, a method for preparing zebetuzumab is provided, comprising the following steps:

[0049] a. introducing the aforementioned nucleic acid molecule, recombinant vector or vector system for expressing zebetuzumab into a host cell;

[0050] b. culturing the host cells to express zebetozumab; and optionally,

[0051] c. Purification and recovery of zebetuin. Nucleic acid molecules, recombinant vectors or vector systems can be introduced into host cells using any method known in the art. Such operations are well known to those skilled in the art and can enable expression of zebetuin.

[0052] For example, a vector system expressing zebetuin can be introduced in the following manner:

[0053] In one embodiment, the first vector and the second vector in the vector system for expressing zabetuzumab described in the present invention are introduced into the host cell at the same time.

[0054] In one embodiment, the first vector and the second vector in the vector system for expressing zabetuzumab according to the present invention are sequentially introduced into a host cell:

[0055] First, the first vector in the vector system for expressing zabetutumab of the present invention is introduced into a host cell, and then the second vector in the vector system for expressing zabetutumab is transfected into the same host cell; or

[0056] The second vector in the vector system for expressing zabetutumab of the present invention is first introduced into a host cell, and then the first vector in the vector system for expressing zabetutumab is transfected into the same host cell.

[0057] The present invention optimizes the codons for expressing these amino acids without changing the amino acid sequence of Zebetuzumab and without affecting the performance of Zebetuzumab, so that the expression amount of the optimized nucleic acid molecule in the host cell is significantly improved, which can greatly increase the yield of Zebetuzumab, reduce the production cost, and improve the quality of the produced antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a schematic diagram of the structure of the HS006 heavy chain expression vector;

[0059] Figure 2 This is a schematic diagram of the structure of the HS006 light chain expression vector;

[0060] Figure 3 This is the result diagram of the expression amount of different codons in Example 6. DETAILED DESCRIPTION

[0061] definition

[0062] In the present invention, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and covers a variety of 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 Zebetuzumab (HS006 monoclonal antibody) of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; the three CDR sequences of the heavy chain are shown in SEQ ID NO: 5, 6, and 7, respectively, and the three CDR sequences of the light chain are shown in SEQ ID NO: 8, 9, and 10, respectively.

[0063] The term "variable region" refers to the domain of an antibody heavy or light chain that recognizes and specifically binds an antigenic epitope.

[0064] CDR region or "complementarity determining region" refers to the region in the variable region of an antibody that is highly variable in sequence and forms a loop that is structurally determined and / or contains antigen contact amino acid residues. CDR is primarily responsible for the binding of the antibody to the antigen epitope and determines the specificity of the antibody. In a given heavy chain or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of many well-known numbering conventions, including, for example, Kabat, Contact, AbM and Chothia.

[0065] In the present invention, "identity" refers to the percentage of amino acids (or bases) in the two sequences being compared that are identical when the sequences are aligned between two peptides or between two nucleic acid molecules. The alignment and homology percentage or sequence identity can be determined using software programs known in the art, such as the software programs described by Ausubel et al. (2007) in Current Protocols in Molecular Biology. 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.

[0066] In the present invention, "nucleic acid molecule" refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) usually linked together by phosphodiester bonds. The nucleic acid molecule of the present invention can be an isolated nucleic acid molecule, which means a nucleic acid molecule separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule, such as being substantially free of other cellular materials or culture medium when prepared by recombinant technology, or being substantially free of chemical precursors or other chemical components when chemically synthesized. The nucleic acid molecule of the present invention is not limited to the sequence shown, but also includes its complementary sequence and modifications known in the art made to the nucleic acid molecule.

[0067] In the present invention, "expression cells" refer to cells that can be used to receive, maintain, replicate and amplify vectors, and may also be referred to as "host cells". Expression cells can also be used to express polypeptides encoded by vectors. When expression cells divide, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Expression cells can be eukaryotic cells or prokaryotic cells. Suitable expression cells include, but are not limited to, CHO cells such as CHO-K1 cells, HeLa cells, HEK cells such as HEK 293 cells, and the like.

[0068] In the present invention, "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate expression cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into expression cells, for amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

[0069] "Expression vector" includes a vector capable of expressing DNA, and the DNA is operably connected to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate expression cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the genome of expression cells. Commercially available expression vectors, particularly those expressing in mammalian cells, include, for example, pIRES (from Clontech, Palo Alto, USA), pCI-neo vectors (from Promega, Madison, USA), pCMV-Script (from Stratagene, La Jolla, USA) and pCDNA vectors (from Invitrogen, Paisley, UK).

[0070] In the present invention, "codon optimization" is a technology that improves the protein expression level in an organism by increasing the translation efficiency of the target gene. The protein expression sequence is redesigned by avoiding rare codons, using preferred 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 improve the protein expression level.

[0071] In the present invention, "codon" has a well-known meaning in the art, and refers to a triplet of nucleotide residues on mRNA (or DNA), each triplet encoding a specific amino acid. Codons are degenerate, and except for methionine and tryptophan, each amino acid has at least two codons, and different species have preferences for multiple codons for the same amino acid.

[0072] A "vector system" refers to a combination of two or more vectors. A vector system is generally used to express a specific antibody or fusion protein, such as for expressing zebetuzumab in the present invention. It is well known to those skilled in the art that a dual vector expression system can be used to express antibodies. Typically, in a dual vector expression system, a first vector expressing an antibody light chain and a second vector expressing an antibody heavy chain are included, and the first vector and the second vector are introduced into a host cell simultaneously or sequentially to express the antibody.

[0073] 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.

[0074] "Host cell" and "cell line" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced and their progeny, regardless of the number of generations of the progeny. The progeny are not allowed to be completely identical to the parent cell in terms of nucleic acid, but may contain mutations, as long as the progeny with the mutation have the same desired function or activity as the original cell.

[0075] The host cell cannot develop into a complete animal or plant individual.

[0076] Host cells include prokaryotic and eukaryotic host cells. Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and 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. Fungal cells include yeast, such as, but not limited to, Pichia, Saccharomyces, Hansenula polymorpha, and Kluyveromyces.

[0077] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur. As used in the specification and claims, the singular forms "a", "an" include the corresponding plural referents unless the content clearly dictates otherwise.

[0078] The present invention will be described in detail below through specific implementations, but the content of the present invention is not limited thereto. Any technical solution based on the idea of ​​the present invention falls within the scope of the present invention.

[0079] Unless otherwise specified, the instruments and reagents used in the following examples are commonly used in the art and can be obtained through commercial channels; the methods used are conventional technical methods in the art, and those skilled in the art can repeat the experiments without any problems based on the contents described in the examples.

[0080] Example 1. Optimization of the nucleic acid sequence of Zebetuzumab

[0081] The full-length amino acid sequence of Zebetuzumab (shown in SEQ ID NO: 1, 3) was reverse transcribed to generate various nucleotide sequences encoding the same original amino acid sequence. Different codons were used to generate multiple nucleotide sequences with the highest theoretical expression levels in the selected host (e.g., CHO cells) for subsequent screening. After multiple rounds of multi-system optimization, two highly expressed nucleic acid sequences with different sequences were finally obtained, respectively called JSR and GWZ, and the relevant sequences are as follows:

[0082] Zebetuin heavy chain:

[0083] EVQLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKA TLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

[0084] Zebetuin heavy chain variable region:

[0085] EVQLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKA TLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSS (SEQ ID NO:2)

[0086] Zebetuin light chain:

[0087] DIELSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:3)

[0088] Zebetuin light chain variable region:

[0089] DIELSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLEIK (SEQ ID NO:4)

[0090] Table 1: CDR sequences of zebetozumab

[0091]

[0092]

[0093] Original nucleic acid sequence encoding the heavy chain of Zebetuzumab:

[0094] HS006-HC( VH 、HCDR1、HCDR2、HCDR3)

[0095] GAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCCTCAGTGAAGATGTCCTGCAA GGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTGAAGCAGACACCTAGACAGGGCCTGGAATGGATT GGAGCTATTTATCCAGGAAATGGCGATACTTCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGTGGACA AATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAAGACTCTGCCGTCTATTTCTGTGCAAGAGT GGTGTACTATAGTAACTCTTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTCCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCTTAAGTCCGGGAAAATAA(SEQ IDNO:11)

[0096] HS006-LC (VL, LCDR1, LCDR2, LCDR3)

[0097] GATATCGAGCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTG CAGGGCCAGCTCAAGTGTGAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTAT GCCCCATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAA TCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTTTTAACCCACCCACGTTCGGCGC TGGGACCAAGCTCGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCACCGGTGACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQ ID NO:12);

[0098] The nucleic acid coding sequence of Zebetuzumab obtained after optimization is as follows:

[0099] JSR-HC:

[0100] GAAGTCCAGCTGCAGCAATCTGGCGCTGAACTGGTGCGGCCTGGAGCTAGTGTGAAGATGTCCTGCAA GGCCTCCGGCTACACCTTCACCTCTTATAATATGCACTGGGTGAAGCAGACCCCTAGACAGGGACTGGAATGGATC GGCGCCATCTACCCTGGCAATGGCGACACCTCCTACAATCAGAAGTTCAAGGGCAAGGCTACCCTGACCGTGGATA AGTCCTCTTCCACCGCCTACATGCAGCTGAGCAGTCTGACTTCCGAGGACTCTGCTGTGTACTTCTGCGCCAGAGT TGTCTACTACTCCAACTCCTACTGGTATTTCGACGTCTGGGGCACAGGCACAACTGTGACCGTGAGCTCT GCCTCTACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCCTCCAGCAAGAGCACCTCTGGCGGCACCGCTGCTCTGGGTTGCCTGGTCAAGGACTACTTTCCTGAGCCCGTGACCGTGTCTTGGAACTCCGGCGCTCTGACCTCTGGAGTGCATACCTTTCCAGCCGTGCTGCAGTCCTCTGGCCTGTACTCTCTGTCTTCCGTCGTGACCGTGCCTAGCAGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCTAACACCAAGGTGGACAAGAAGGTGGAACCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCTTGCCCTGCTCCTGAGCTGCTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCTAAACCTAAGGATACCCTGATGATCTCCAGAACCCCAGAAGTCACCTGCGTGGTGGTCGATGTGTCTCACGAGGATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCCCGCGAGGAACAGTACAACTCTACCTACAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAAGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTTAGCAACAAGGCCCTGCCTGCCCCTATCGAGAAGACCATCTCCAAAGCTAAGGGCCAGCCTCGGGAGCCTCAAGTGTACACACTGCCTCCATCTCGGGACGAGCTGACCAAGAACCAGGTGTCCCTCACCTGTCTGGTGAAAGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAAACCACACCACCTGTGCTGGACTCCGATGGCTCTTTCTTCCTGTACTCCAAGCTGACAGTGGACAAATCTAGATGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCCTGCATAACCACTACACCCAGAAAAGCCTGTCCCTGTCCCCTGGCAAGTGATAA(SEQID NO:13)

[0101] JSR-LC:

[0102] GACATCGAGCTGTCTCAGTCTCCCGCCATCCTGTCTGCTTCCCCTGGAGAAAAAGTGACCATGACCTG CAGAGCCTCCTCTAGCGTGTCCTACATGCACTGGTACCAGCAAAAGCCCGGATCTTCTCCTAAGCCTTGGATCTAC GCCCCTTCCAACCTGGCCTCTGGCGTTCCTGCCAGATTCAGCGGCTCTGGCTCCGGCACCTCGTATTCCCTGACCA TCTCCAGAGTGGAAGCCGAGGATGCTGCTACCTACTACTGCCAGCAGTGGTCCTTCAACCCTCCTACCTTCGGCGC TGGCACCAAGCTGGAAATCAAG CGGACCGTGGCCGCTCCATCCGTGTTCATCTTCCCCCCATCTGATGAACAGCTGAAGTCCGGCACCGCTTCTGTGGTGTGTCTCCTGAACAACTTCTACCCTCGGGAGGCCAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAAGAGTCCGTCACAGAGCAGGACTCCAAGGACTCTACCTACTCTCTGTCCTCCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGAGCAGCCCTGTGACAAAATCCTTTAATAGAGGCGAGTGCTGATAA(SEQ ID NO:14)

[0103] JSR-VH:

[0104] GAAGTCCAGCTGCAGCAATCTGGCGCTGAACTGGTGCGGCCTGGAGCTAGTGTGAAGATGTCCTGCAA GGCCTCCGGCTACACCTTCACCTCTTATAATATGCACTGGGTGAAGCAGACCCCTAGACAGGGACTGGAATGGATC GGCGCCATCTACCCTGGCAATGGCGACACCTCCTACAATCAGAAGTTCAAGGGCAAGGCTACCCTGACCGTGGATA AGTCCTCTTCCACCGCCTACATGCAGCTGAGCAGTCTGACTTCCGAGGACTCTGCTGTGTACTTCTGCGCCAGAGT TGTCTACTACTCCAACTCCTACTGGTATTTCGACGTCTGGGGCACAGGCACAACTGTGACCGTGAGCTCT (SEQ IDNO:15)

[0105] JSR-VL:

[0106] GACATCGAGCTGTCTCAGTCTCCCGCCATCCTGTCTGCTTCCCCTGGAGAAAAAGTGACCATGACCTG CAGAGCCTCCTCTAGCGTGTCCTACATGCACTGGTACCAGCAAAAGCCCGGATCTTCTCCTAAGCCTTGGATCTAC GCCCCTTCCAACCTGGCCTCTGGCGTTCCTGCCAGATTCAGCGGCTCTGGCTCCGGCACCTCGTATTCCCTGACCA TCTCCAGAGTGGAAGCCGAGGATGCTGCTACCTACTACTGCCAGCAGTGGTCCTTCAACCCTCCTACCTTCGGCGC TGGCACCAAGCTGGAAATCAAG (SEQ ID NO:16)

[0107] JWZ-HC:

[0108] GAGGTGCAGCTGCAACAGAGCGGAGCCGAGCTGGTGAGACCTGGCGCTAGCGTGAAGATGAGCTGCAA GGCTAGCGGCTACACCTTCACAAGCTACAACATGCACTGGGTGAAGCAGACCCCTAGACAAGGCCTGGAGTGGATC GGCGCCATCTACCCTGGCAACGGCGACACAAGCTACAATCAGAAGTTCAAGGGCAAGGCCACCCTGACCGTCGACA AAAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGTGTACTTCTGCGCTAGAGT GGTGTACTACAGCAACAGCTACTGGTACTTCGACGTGTGGGGCACCGGCACCACCGTGACCGTCAGCAGCGCTAGCACCAAGGGACCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGCGGCGGCACCGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGAGCTGGAACAGCGGCGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTCGTGACCGTGCCTAGCAGCAGCCTGGGCACACAGACCTACATCTGCAACGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGCCCTCCTTGCCCTGCCCCTGAGCTGCTGGGCGGCCCTAGCGTGTTTCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTCGTGGACGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCTGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGACAGCCTAGAGAGCCTCAAGTGTACACCCTGCCTCCTAGCAGAGACGAGCTGACCAAGAACCAAGTGAGCCTGACCTGTCTGGTCAAGGGATTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGACAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTCACCGTGGACAAAAGCAGATGGCAGCAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCCTGGCAAGTGATAA(SEQID NO:17)

[0109] JWZ-LC:

[0110] GACATCGAGCTGAGCCAAAGCCCTGCCATCCTGAGCGCTAGCCCTGGCGAGAAGGTGACCATGACCTG CAGAGCTAGCAGCAGCGTGAGCTACATGCACTGGTATCAGCAGAAGCCTGGCAGCAGCCCTAAGCCTTGGATCTAC GCCCCTAGCAACCTGGCTAGCGGCGTGCCTGCTAGATTCAGCGGCAGCGGCAGCGGCACAAGCTACAGCCTGACCA TCAGCAGAGTGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCTTCAACCCTCCTACCTTCGGCGC CGGCACCAAGCTGGAGATCAAG AGAACCGTGGCCGCCCCTAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAAGAGAGC GTGACCGAGCAAGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAAGGCCTGAGCAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAGTGCTGATAA(SEQ ID NO:18)

[0111] JWZ-VH:

[0112] GAGGTGCAGCTGCAACAGAGCGGAGCCGAGCTGGTGAGACCTGGCGCTAGCGTGAAGATGAGCTGCAA GGCTAGCGGCTACACCTTCACAAGCTACAACATGCACTGGGTGAAGCAGACCCCTAGACAAGGCCTGGAGTGGATC GGCGCCATCTACCCTGGCAACGGCGACACAAGCTACAATCAGAAGTTCAAGGGCAAGGCCACCCTGACCGTCGACA AAAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGTGTACTTCTGCGCTAGAGT GGTGTACTACAGCAACAGCTACTGGTACTTCGACGTGTGGGGCACCGGCACCACCGTGACCGTCAGCAGC (SEQ ID NO: 19)

[0113] JWZ-VL:

[0114] GACATCGAGCTGAGCCAAAGCCCTGCCATCCTGAGCGCTAGCCCTGGCGAGAAGGTGACCATGACCTG CAGAGCTAGCAGCAGCGTGAGCTACATGCACTGGTATCAGCAGAAGCCTGGCAGCAGCCCTAAGCCTTGGATCTAC GCCCCTAGCAACCTGGCTAGCGGCGTGCCTGCTAGATTCAGCGGCAGCGGCAGCGGCACAAGCTACAGCCTGACCA TCAGCAGAGTGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCTTCAACCCTCCTACCTTCGGCGC CGGCACCAAGCTGGAGATCAAG (SEQ ID NO:20)

[0115] Table 2: Zebetuzumab CDR coding sequence - JSR

[0116]

[0117] Table 3: Zebetuzumab CDR coding sequence - JWZ

[0118]

[0119] After sequence comparison, for the full-length nucleic acid sequence of the antibody, the homology between JSR-LC and HS006-LC is only 78.8%, the homology between JWZ-LC and HS006-LC is only 76.5%, the homology between JSR-HC and HS006-HC is only 81.6%, and the homology between JWZ-HC and HS006-HC is only 81.4%.

[0120] For the variable region, the homology between JSR-VL and HS006-VL is only 78.6%, the homology between JWZ-VL and HS006-VL is only 73.9%, the homology between JSR-VH and HS006-VH is only 82.2%, and the homology between JWZ-VH and HS006-VH is only 80.1%.

[0121] Example 2. Plasmid construction

[0122] The nucleic acid sequences encoding the light chain and heavy chain amino acids of the HS006 antibody (original sequence, JSR sequence and JWZ sequence) were synthesized respectively, and the light chain and heavy chain nucleic acid sequences containing the signal peptide were recombined into the pcDNA3.4-TOPO vector (Thermo fisher, A14697) using the restriction enzyme cutting sites AflII and EcoRV to construct the expression vectors pcDNA3.4-TOPO-HS006HC and pcDNA3.4-TOPO-HS006LC expressing the light chain and heavy chain of HS006, respectively. The maps are shown in Figure 1 and Figure 2 The expression vectors were transformed into E. coli cells DH5α (Takara, 9057) for amplification and culture. The expression vectors were extracted, and after sequencing confirmed to be correct, the transformed E. coli were stored at -20°C for subsequent transient transfection experiments.

[0123] Example 3. Plasmid amplification and extraction

[0124] The transformed E. coli obtained in Example 2 was cultured in LB medium. According to the resistance of the pcDNA3.4-TOPO plasmid, ampicillin (Shanghai Biotechnology, A100339) was added to the LB medium for expansion.

[0125] The E. coli cultured after expansion was collected by centrifugation, and the E. coli was lysed by alkaline lysis using a plasmid extraction kit (Axygen, AP-MX-P-25). Endotoxins were removed during the process (Shanghai Biotech, B641718), and plasmids were extracted. The plasmid concentration was detected by a micro-spectrophotometer (Hangzhou Aosheng, Nano-300) and stored at -20°C for subsequent transient transfection experiments.

[0126] Example 4. Plasmid transfection into CHO cells

[0127] Take out 1 CHO-K1 (ATCC, catalog number CCL-61 TM , batch number: 70014310) and revived in a 37°C water bath. Cell count (Count Star, IC1000) was performed at 0.5×10 6 The cells were subcultured at a viable cell density of 0.2-0.5 × 10 cells / mL in CD CHO complete medium (Gibco, 10743029) containing 6 mmol / L L-Gln (Sigma, V900419-500G). CHO-K1 cells were cultured in a humidified carbon dioxide shaker incubator (Kuhner, ISF4-XC) at 37°C and 5% carbon dioxide. Subculture was performed every 2-4 days, and the viable cell density was 0.2-0.5 × 10 6 cells / mL. After the cells recovered, they were transferred to Dynamis complete medium (Dynamis medium + 6mmol / L L-Gln, Gibco, A26175) for subculture. 24 hours before transfection, subcultured in Dynamis complete medium, the subculture viable cell density was 3-4×10 6 cells / mL.

[0128] On the day of transient transfection, the viable cell density was adjusted to 6 × 10 cells / mL using Dynamis complete medium. 6 cells / mL. The transient transfection system was to add 10.5 μg PEI (Polyscoences, 23966), 1.75 μg heavy chain recombinant plasmid, 1.75 μg light chain recombinant plasmid and 2.625 μg inert DNA (Sigma, 31149-10G-F) per ml of cell culture medium. 4 h after transient transfection, the total volume was supplemented with 4% Cell Boost 7a solution (Cytiva, SH31026.02), 0.4% Cell Boost 7b solution (Cytiva, SH31027.07CN) and 0.125% DMA. On the first day of transient transfection, D-glucose was supplemented to 7 g / L and the temperature was lowered to 32°C for culture; on the second day of transient transfection, 2 mM L-Gln was supplemented; on the fifth day of transient transfection, 6% Cell Boost 7a solution, 0.6% Cell Boost 7b solution and 4 mM L-Gln were supplemented; on the seventh day of transient transfection, the culture supernatant of transient transfected cells was harvested. The supernatant was harvested for antibody expression detection and one-step affinity purification (see Example 5 for details), and the purified samples were subjected to SEC-HPLC, nrCE-SDS and IEC-HPLC quality detection (see Example 7 for details).

[0129] Example 5. Purification of Antibodies

[0130] In Example 4, a one-step affinity chromatography method was used to purify the antibody, and the steps were as follows: centrifugation (12000g for 10 min) was used to collect the cell culture supernatant of the transiently transfected recombinant vector, purification was performed using a Protein A affinity chromatography column (Cytiva, 17543803) on a protein purification chromatography system (Cytiva, AKTAavant), and the target protein was eluted using a pH 3.0 acetic acid solution (McLean, A801295).

[0131] Example 6. Detection of Antibody Expression

[0132] Detect the expression level of the antibody using HPLC:

[0133] According to the Protein A one-step affinity chromatography method, the antibody was eluted using a low pH solution, the areas of the elution peaks of the standard (commercially available zebetozumab) and the sample were counted, and a linear relationship between the standard concentration and the chromatographic peak area was established. The antibody concentration in the sample was calculated based on the linear relationship of the standard and the sample peak area.

[0134] Instrument: High performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic column: POROS A / 20, 1502412 chromatographic column of Thermo Fish, specification is 2.1*30 mm.

[0135] Detection mobile phase A: 50 mM Na2HPO4 (Sinopharm, 10020318) and 150 mM NaCl (Sinopharm, 10019318), pH 7.0; flow rate: 3.0 mL / min.

[0136] Detection mobile phase B: 150 mM NaCL (Sinopharm, 10019318), pH 1.9; flow rate: 3.0 mL / min.

[0137] Injection volume: 30 μL; Column temperature: 25.0±5.0℃. Sample room temperature: 2-8℃. Detection wavelength: 280nm, detection time: 3 minutes.

[0138] During the test, the chromatographic column is first balanced, that is, 100% mobile phase A is used to balance the system at a flow rate of 3.0 mL / min until the baseline is stable, and then the sample is injected, the sample concentration is measured, and the chromatogram is recorded. The sample is measured using gradient elution, and the elution gradient is shown in the following table:

[0139] Table 4. Chromatographic elution gradient for protein content determination by HPLC

[0140]

[0141] According to the chromatogram, the protein content was obtained by analyzing and calculating the peak area of ​​the target peak of the standard curve solution and the test solution. The results are shown in Table 5 and Figure 3 :

[0142] Table 5. Antibody expression levels of nucleic acids with different sequences

[0143]

[0144]

[0145] From the expression results, we can see that the original codon expression level in HS006 antibody hybridoma is the lowest, and the expression level of the optimized codon is significantly improved.

[0146] Example 7. Antibody quality detection

[0147] 7.1SEC-HPLC

[0148] Instrument: High performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic column: TOSOH G3000SWxL chromatographic column, specification: 7.8*300mm, 5um.

[0149] Detection mobile phase: 0.2M sodium chloride (Sinopharm, 10019318), 0.04M sodium dihydrogen phosphate dihydrate (Sinopharm, 20040718) and 0.04M disodium hydrogen phosphate dodecahydrate (Sinopharm, 10020318), pH 7.0.

[0150] Injection volume: 25 μL; Column temperature: 25.0 ± 5.0 °C. Sample room temperature: 2-8 °C. Detection wavelength: 280 nm, detection time: 30 minutes.

[0151] During the detection, the chromatographic column was first balanced, that is, 100% mobile phase was used to balance the system at a flow rate of 0.5 mL / min until the baseline was stable, and then the sample was injected, the sample was measured, and the chromatogram was recorded.

[0152] The chromatogram was integrated and the peak area of ​​the target peak was analyzed and calculated to obtain the aggregate content and monomer peak content.

[0153] 7.2 Non-reduced CE-SDS

[0154] The sample was diluted to 1.0 mg / mL with non-reducing CE-SDS sample diluent (pH 6.5, containing 0.04 M sodium dihydrogen phosphate dihydrate, 0.04 M sodium dihydrogen phosphate dodecahydrate and 1% SDS), and 5% of the total volume of 800 mM iodoacetamide (TCI, I0044) was added and mixed evenly, and then incubated at 70°C for 5 min. After cooling, centrifuged at 6000 rpm for 1 min, and 75 μL of the supernatant was taken as the loading sample.

[0155] Instrument: Capillary electrophoresis instrument (Agilent, 7100). Chromatographic column: CE-SDS capillary (Agilent, G1600-6411), column length 224 mm / 310 mm.

[0156] Sample buffer: 0.04 M sodium dihydrogen phosphate dihydrate, 0.04 M disodium hydrogen phosphate dodecahydrate and 1% SDS, pH 6.5.

[0157] Running buffer: SCIEX Gel.

[0158] Injection: injection'-10KV, 40S, operation'-15KV, 40min; column temperature: 20℃.

[0159] Sample plate temperature: 18-22°C. DAD detection wavelength: 220nm / 360nm, detection time: 40 minutes.

[0160] The chromatogram was integrated and the low molecular weight fragment content and monomer peak content were obtained by analyzing and calculating the peak area ratio of the target peak.

[0161] 7.3IEC-HPLC

[0162] Instrument: High performance liquid chromatograph (Agilent, 1260 Infinity II Prime). Chromatographic column: ThermoMabPac SCX-10, 074625 chromatographic column, specification: 4*250 mm.

[0163] Detection mobile phase A: 0.0175 M sodium dihydrogen phosphate dihydrate and 0.005 M disodium hydrogen phosphate dodecahydrate, pH 6.1; flow rate: 1.0 mL / min.

[0164] Mobile phase B: 0.075 M sodium dihydrogen phosphate dihydrate and 0.003 M disodium hydrogen phosphate dodecahydrate, pH 6.0; flow rate: 1.0 mL / min.

[0165] Injection volume: 10 μL; column temperature: 25.0±5.0℃. Sample room temperature: 2-8℃. Detection wavelength: 214nm, detection time: 70 minutes.

[0166] During the test, the chromatographic column is first balanced, that is, 100% mobile phase A is used to balance the system at a flow rate of 1.0 mL / min until the baseline is stable, and then the sample is injected, the sample is measured, and the chromatogram is recorded. The sample is measured using gradient elution, and the elution gradient is shown in the following table:

[0167] Table 6. Chromatographic elution gradient for protein charge variant determination by IEC-HPLC

[0168]

[0169] The chromatogram was integrated and the contents of the charge variant acidic peak, main peak and basic peak were obtained by analyzing and calculating the peak area of ​​the target peak of the test solution.

[0170] The quality of antibodies expressed by different codons is shown in the table below:

[0171] Table 7. Antibody quality results expressed by different codons

[0172]

[0173] From the SEC-HPLC and CE-SDS data, we know that the polymer ratio and low molecular weight fragment ratio of the antibody expressed by the original codon of HS006 are the highest. After codon optimization, the content of polymers and fragments in the antibody can be significantly reduced. Among them, the polymer and fragment ratio of the antibody expressed by the HS006-JSR codon is the lowest, followed by the HS006-JWZ codon. From the IEC-HPLC data, we know that the ratios of the three codon charge variants are not much different.

[0174] In summary, the expression level and purity of the original codons of HS006 were lower than those of the optimized codons.

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain and a light chain of zebetuin, wherein: The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO:13, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO:

14.

2. A recombinant vector comprising the nucleic acid molecule according to claim 1, wherein the starting vector of the recombinant vector is an expression vector.

3. The recombinant vector according to claim 2, wherein The starting vector of the recombinant vector is pcDNA3.4-TOPO vector.

4. A vector system for expressing zebetuin, comprising a first vector and a second vector, wherein: The first vector comprises a nucleic acid sequence encoding the heavy chain of zabetutumab, and the second vector comprises a nucleic acid sequence encoding the light chain of zabetutumab, wherein: The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO:13, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO:

14.

5. A host cell comprising the nucleic acid molecule of claim 1, or the recombinant vector of claim 2 or 3, or the vector system for expressing zebetuin as claimed in claim 4.

6. The host cell according to claim 5, wherein The host cell is a CHO cell.

7. The host cell according to claim 6, wherein The host cell is CHO-K1 cell.

8. Use of the nucleic acid molecule according to claim 1, the recombinant vector according to claim 2 or 3, the vector system for expressing zebetuzumab according to claim 4, or the host cell according to any one of claims 5 to 7 in the preparation of zebetuzumab.

9. A method for preparing zebetutinumab, comprising the following steps: a. introducing the nucleic acid molecule of claim 1, the recombinant vector of claim 2 or 3, or the vector system for expressing zebetuzumab of claim 4 into a host cell; b. Cultivating host cells to express zebetuin.

10. A method for preparing zebetuin, comprising the following steps: a. introducing the nucleic acid molecule of claim 1, the recombinant vector of claim 2 or 3, or the vector system for expressing zebetuzumab of claim 4 into a host cell; b. culturing the host cells to express zebetozumab; and c. Purification and recovery of zebetuin.

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