A recombinant CHO cell, a method for constructing the same, a detection system and method using the same

Abstract

Provided are a recombinant CHO cell, a construction method thereof, a detection system comprising the recombinant CHO cell, and a detection method using the recombinant CHO cell. The recombinant CHO cell stably expresses NGF on the cell surface thereof and can be used as a target cell for ADCC effect and CDC effect detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immune effect detection, and particularly relates to a recombinant CHO cell, a method for constructing the recombinant CHO cell, a detection system and a detection method for detecting ADCC effect and CDC effect of an antibody by using the cell. BACKGROUND

[0002] ADCC (antibody-dependent cell-mediated cytotoxicity) is antibody-dependent cell-mediated cytotoxicity, which refers to that after specific binding of an antibody to an antigen determinant on the surface of a target cell through a Fab segment, the Fc segment of the antibody is combined with an effector cell having FcγR, triggering the killing activity of the effector cell, and directly killing the target cell. The ADCC effect is an important immune mechanism of the body against infection and tumor, and the effector cells participating in ADCC mainly include natural killer (NK) cells, phagocytes, eosinophils, basophils and mast cells, etc.

[0003] CDC (complement dependent cytotoxicity) is complement-dependent cytotoxicity, which refers to complement-involved cytotoxicity, that is, specific antibodies are combined with corresponding antigens on the surface of a cell membrane to form a complex to activate the classical pathway of complement, the complement protein C1q initiates the classical pathway of complement first, and then the complement proteins C2-C9 are activated to form a membrane attack complex (MAC), which exerts a lytic effect on the target cell.

[0004] Considering the important role of ADCC effect and CDC effect in tumor immunity, in the current development of anti-tumor antibodies, many researchers hope to obtain improved ADCC effect and CDC effect by specific design of antibody structure. However, in the development of other types of antibodies, antibody-mediated ADCC effect and CDC effect are not always desirable.

[0005] Nerve growth factor (NGF) is the first discovered neurotrophic factor, which is produced in the neocortex and hippocampus, and is composed of two α subunits, one β subunit and two γ subunits. NGF can promote the growth, development, differentiation and maturation of central and peripheral neurons, maintain the normal function of the nervous system, and accelerate the repair after nervous system injury. At present, recombinant NGF is mainly used for treating various neuropathies and nerve injuries.

[0006] Chinese hamster ovary (CHO) cells are one of the most widely used mammalian cell expression systems, which have good post-translational processing ability to maintain the natural structure and activity of the expression product. Currently, CHO has been used to express NGF and secrete recombinant NGF outside the cell, so that NGF can be produced on a large scale (e.g., Xu L et al., Expression, purification, and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells. Protein Expr Purif, 2014, 104: 41-49; Wang XY et al., Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells. Genet Mol Res, 2015, 14(3): 9191-9199). However, there is no report on the stable expression of NGF on the surface of recombinant CHO cells. SUMMARY

[0007] In order to accurately detect the ADCC effect and the CDC effect of the anti-NGF antibody, the inventors developed a recombinant CHO cell that can stably express NGF on its surface, which can be effectively used as a target cell to evaluate the ADCC effect and the CDC effect of the anti-NGF antibody.

[0008] In one aspect, the present application relates to a recombinant CHO cell, wherein NGF is stably expressed on the surface of the cell.

[0009] In another aspect, the present application relates to a method of constructing the above-mentioned recombinant CHO cell, comprising:

[0010] (1) cloning a nucleic acid sequence of NGF, a hinge region and a transmembrane region in series into an expression vector to obtain a recombinant expression vector comprising the nucleic acid sequence;

[0011] (2) introducing the recombinant expression vector into a CHO cell to obtain a recombinant CHO cell.

[0012] In another aspect, the present application relates to a system for detecting the ADCC effect of an antibody, wherein the system comprises:

[0013] the recombinant CHO cell described above, and

[0014] effector cells.

[0015] In another aspect, the present application relates to a system for detecting the CDC effect of an antibody, wherein the system comprises:

[0016] the recombinant CHO cell described above, and

[0017] complement.

[0018] In yet another aspect, the present application relates to the use of the recombinant CHO cell described above in detecting the ADCC effect or the CDC effect of an antibody.

[0019] In yet another aspect, the present application relates to a method for detecting the ADCC effect of an antibody, wherein the method comprises incubating the recombinant CHO cell described above as target cells with the antibody to be tested and effector cells.

[0020] In yet another aspect, the present application relates to a method for detecting the CDC effect of an antibody, wherein the method comprises incubating the recombinant CHO cell described above as target cells with the antibody to be tested and complement.

[0021] In conventional recombinant NGF expression studies, mammalian cells such as CHO cells are usually used as expression systems to express and secrete NGF outside the cells, so that it exists in the supernatant in a free form similar to the natural form. In addition, some researchers have also tried to use prokaryotic systems (e.g. E. coli) to express NGF, and the expression product exists in insoluble inclusion bodies (Jiang Jing et al., Comparison of Properties of Recombinant Human β-NGF from E. coli and CHO Expression Systems, Chinese Journal of Bioengineering, 2006, 26(2): 8-12). There is great uncertainty as to whether NGF itself as a free protein rather than a transmembrane protein can be expressed on the cell surface. Therefore, so far there has been no relevant report on stable expression of NGF on the surface of CHO cells. In the present application, by linking the nucleic acid sequence of NGF with the nucleic acid sequence of an appropriate hinge region and transmembrane region, and then introducing it into CHO cells, NGF is stably expressed on the surface of recombinant CHO cells, so that NGF exists on the cell surface rather than being secreted into the culture medium, and the expressed NGF can be properly post-translationally processed (including correct folding, glycosylation, etc.) to maintain the natural conformation and activity. The recombinant CHO cells thus obtained have NGF on the cell surface and can be effectively used as target cells for detecting the ADCC effect and the CDC effect of anti-NGF antibodies, thereby being used for detecting the ADCC effect and the CDC effect of anti-NGF antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 SDS-PAGE detection results of the positive control antibody HAB20-6-2 are shown. According to the results in Table 1, the purity of the positive control antibody HAB20-6-2 prepared in Example 3 is greater than 90%. Figure 1 SDS-PAGE detection results of the positive control antibody HAB20-6-2 are shown. According to the results in Table 1, the purity of the positive control antibody HAB20-6-2 prepared in Example 3 is greater than 90%.

[0023] Figure 2 Flow cytometry identification results of CHO-NGF recombinant cells are shown. In the results, Figure 2 A in the results shows flow cytometry detection results obtained by incubating CHO-S cells as a blank control with the anti-NGF positive control antibody HAB20-6-2, and the positive rate is 2%; Figure 2 B in the results shows flow cytometry detection results obtained by incubating the CHO-NGF cells constructed in Example 2 with the anti-NGF positive control antibody HAB20-6-2, and the positive rate is 82.67%.

[0024] Figure 3 ADCC effect detection results are shown. In the results, A: superimposed plot of ADCC effect detection results of Tanezumab (4 detections) and the positive control antibody HAB20-6-2; B: detection results of the positive control antibody HAB20-6-2; C: first detection results of Tanezumab; D: second detection results of Tanezumab; E: third detection results of Tanezumab; F: fourth detection results of Tanezumab.

[0025] Figure 4 CDC effect detection results are shown. In the results, A: superimposed plot of CDC effect detection results of Tanezumab (4 detections) and the positive control antibody HAB20-6-2; B: detection results of the positive control antibody HAB20-6-2; C: first detection results of Tanezumab; D: second detection results of Tanezumab; E: third detection results of Tanezumab; F: fourth detection results of Tanezumab. DETAILED DESCRIPTION

[0026] The advantages and features of the present application will become more apparent with the description of specific embodiments. However, these embodiments are only exemplary and do not limit the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0027] Unless defined otherwise, each technical and scientific term used herein has the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (2012); Abbas et al., Cellular and Molecular Immunology, Elsevier Science Health Science div (2009); He Wei et al., Medical Immunology (2nd Edition), People's Medical Publishing House, 2010.

[0028] In the present text, the terms "comprise", "comprises" and "comprising" or equivalents thereof, contain, contain, contain, include, include, include, are open expressions that mean the inclusion of the elements, components or steps listed, but not excluding the inclusion of other unspecified elements, components or steps.

[0029] In the present text, unless otherwise stated, all numbers expressing amounts, measurements or conditions used herein are to be understood as being modified in all instances by the term "about". The term "about", when used in connection with a percentage, can mean, for example, ± 1 %, preferably ± 0.5 %, more preferably ± 0.1 %.

[0030] Unless the context clearly indicates otherwise, a singular term includes a plural connotation, and vice versa. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to encompass "and".

[0031] In the present text, the percentage of identity (degree of homology) between sequences can be determined by comparing two sequences, for example using freely available computer programs commonly used for this purpose on the World Wide Web (e.g. BLASTp or BLASTn with default settings) (www.ncbi.nlm.nih.gov).

[0032] In the present context, the term "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the natural sequence with a codon that is more frequently or most frequently used in the host cell while maintaining the natural amino acid sequence to improve expression in the host cell. Various species have specific preferences for specific codons for specific amino acids, and this codon preference (differences in codon usage between organisms) is often correlated with the efficiency of translation of mRNA.

[0033] In the present application, "expression vector" refers to a vector comprising a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. The expression vector can preferably include one or more marker genes. For the purpose of expressing the sequences of the present application, any of a variety of expression control sequences such as, for example, the following can be used in the vector: replication origin, polyadenylation signal, promoter, enhancer, terminator, etc.

[0034] Since the U.S. FDA approved the first monoclonal antibody drug OKT3 (muromonab-CD3) in 1986, antibody drugs have been increasingly studied as an option for disease treatment, and to date, the FDA has approved hundreds of antibody drugs (https: / / www.nature.com / articles / d41573-021-00079-7). The mechanism by which antibodies activate the human immune system is complex, and the most important ones include antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0035] In general, since NGF itself is a free protein, the recombinant cells constructed to express NGF secrete the expressed NGF to the outside of the cells, allowing it to exist in a free form substantially equivalent to the natural form in the culture medium, thereby making the conventional recombinant cells constructed to express NGF unsuitable as target cells used in ADCC and CDC assays. There is great uncertainty regarding expression of NGF on the surface of cells, and even if NGF is expressed on the surface of recombinant cells, it is unknown whether the expressed NGF can maintain the same natural conformation and activity as free NGF. Therefore, prior to this, there has been no report of recombinant CHO cells or other recombinant cells that stably express NGF on their surfaces.

[0036] In some embodiments, the present application relates to a recombinant CHO cell, wherein NGF is stably expressed on the surface of the cell.

[0037] In the present disclosure, any known sequence of NGF can be used, such as those available in NCBI, ENSMBL or UniProt databases, or those codon-optimized according to the codon bias of the host cell (using available software such as codon W) from known sequences. In some preferred embodiments, the NGF can be a mammalian NGF, such as human NGF, mouse NGF, rat NGF, bovine NGF, horse NGF, pig NGF, etc., but not limited thereto. Exemplary amino acid sequences and nucleic acid sequences of NGF can be found in the descriptions of the following: Katherine A. Fitzgerald et al., The Cytokine Facts Book and Webfacts (Second Edition), Academic Press, 2001; Wei MA et al., Cloning and Sequencing of Matured Fragment of Human Never Growth Factor Gene, ACAD J XJTU, 15(1), 2003: 62-65; YA Barde et al., The nerve growth factor family, Progress in Growth Factor Research, 2, 1990: 237-248; Wiesmann, C. et al., Nerve growth factor: structure and function. CMLS, Cell. Mol. Life Sci., 58, 2001: 748-759; Eric Adriaenssens et al., Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer, Cancer Res., 68(2), 2008: January 15, 2008: 346-351; WO2021120900A1, WO2021093134A1, WO2021091363A1, WO2019201133A1, US2021079053A1, WO2010128398A1, US2014170137A1, etc.

[0038] In some preferred embodiments, the amino acid sequence of NGF comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-3 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0039] SEQ ID NO: 1: Amino acid sequence of NGF

[0040] MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA

[0041] SEQ ID NO: 2: Amino acid sequence of mature NGF 1 (mature NGF 1)

[0042] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA

[0043] SEQ ID NO: 3: Amino acid sequence of mature NGF 2 (mature NGF 2)

[0044] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR

[0045] In some preferred embodiments, the nucleic acid sequence of NGF is integrated in the genome of the cell.

[0046] In some preferred embodiments, the nucleic acid sequence of the NGF comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 4-SEQ ID NO: 6 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0047] SEQ ID NO: 4: nucleic acid sequence of NGF

[0048] ATGAGCATGCTGTTCTATACCCTGATCACCGCCTTTCTGATCGGCATCCAGGCCGAGCCTCACAGCGAGTCTAATGTGCCTGCCGGCCACACAATCCCTCAGGCTCACTGGACAAAGCTGCAGCACAGCCTGGATACAGCTCTGCGGAGAGCCAGATCTGCTCCTGCCGCTGCTATTGCCGCTAGAGTGGCTGGCCAGACCAGAAACATCACCGTGGATCCCCGGCTGTTCAAGAAGCGGAGACTGAGAAGCCCCAGAGTGCTGTTCAGCACCCAGCCTCCAAGAGAGGCCGCCGATACACAGGACCTGGATTTTGAAGTTGGCGGCGCTGCCCCTTTCAACAGAACCCACAGAAGCAAGCGGAGCAGCTCTCACCCCATCTTCCACAGAGGCGAGTTCAGCGTGTGCGACTCCGTGTCTGTGTGGGTCGGAGATAAGACCACCGCCACCGACATCAAGGGCAAAGAAGTGATGGTCCTGGGCGAAGTGAACATCAACAACAGCGTGTTCAAGCAGTACTTCTTCGAGACAAAGTGCAGGGACCCCAATCCTGTGGACAGCGGCTGTAGAGGCATCGACAGCAAGCACTGGAACTCCTACTGCACCACCACACACACCTTCGTGAAGGCCCTGACCATGGATGGAAAACAGGCCGCCTGGCGGTTCATCAGAATCGATACCGCTTGCGTGTGCGTGCTGAGCAGAAAAGCCGTTAGAAGGGCC

[0049] SEQ ID NO: 5: nucleic acid sequence of mature NGF 1

[0050] AGCAGCTCTCACCCCATCTTCCACAGAGGCGAGTTCAGCGTGTGCGACTCCGTGTCTGTGTGGGTCGGAGATAAGACCACCGCCACCGACATCAAGGGCAAAGAAGTGATGGTCCTGGGCGAAGTGAACATCAACAACAGCGTGTTCAAGCAGTACTTCTTCGAGACAAAGTGCAGGGACCCCAATCCTGTGGACAGCGGCTGTAGAGGCATCGACAGCAAGCACTGGAACTCCTACTGCACCACCACACACACCTTCGTGAAGGCCCTGACCATGGATGGAAAACAGGCCGCCTGGCGGTTCATCAGAATCGATACCGCTTGCGTGTGCGTGCTGAGCAGAAAAGCCGTTAGAAGGGCC

[0051] SEQ ID NO: 6: nucleic acid sequence of mature NGF 2

[0052] AGCAGCTCTCACCCCATCTTCCACAGAGGCGAGTTCAGCGTGTGCGACTCCGTGTCTGTGTGGGTCGGAGATAAGACCACCGCCACCGACATCAAGGGCAAAGAAGTGATGGTCCTGGGCGAAGTGAACATCAACAACAGCGTGTTCAAGCAGTACTTCTTCGAGACAAAGTGCAGGGACCCCAATCCTGTGGACAGCGGCTGTAGAGGCATCGACAGCAAGCACTGGAACTCCTACTGCACCACCACACACACCTTCGTGAAGGCCCTGACCATGGATGGAAAACAGGCCGCCTGGCGGTTCATCAGAATCGATACCGCTTGCGTGTGCGTGCTGAGCAGAAAAGCCGTTAGA

[0053] In some preferred embodiments, the nucleic acid sequence of the NGF, the hinge, and the transmembrane region (also referred to as “transmembrane domain”) in tandem is integrated in the genome of the cell.

[0054] In further preferred embodiments, the amino acid sequence of the hinge region has 3 or more amino acids, 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, or 100 or fewer amino acids, 90 or fewer amino acids, 80 or fewer amino acids, 70 or fewer amino acids, 60 or fewer amino acids, or an amino acid sequence within any two of the above ranges, e.g., 3-100 amino acids, 10-90 amino acids, 10-80 amino acids, 10-70 amino acids, 10-60 amino acids, 10-50 amino acids, 12-45 amino acids.

[0055] In further preferred embodiments, the amino acid sequence of the transmembrane region has 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, or 60 or fewer amino acids, 50 or fewer amino acids, 40 or fewer amino acids, or an amino acid sequence within any two of the above ranges, e.g., 10-60 amino acids, 10-50 amino acids, 10-40 amino acids, 10-30 amino acids, 15-30 amino acids, 20-30 amino acids, 20-25 amino acids.

[0056] In further preferred embodiments, the hinge region can be a CD8 hinge region (e.g., a CD8a hinge region) or a human IgGl hinge region.

[0057] In further preferred embodiments, the transmembrane region can be a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region.

[0058] In some preferred embodiments, the amino acid sequence of the CD8 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0059] SEQ ID NO: 7: Amino acid sequence of a CD8 hinge region

[0060] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0061] In some preferred embodiments, the amino acid sequence of the human IgGl hinge region comprises the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0062] SEQ ID NO: 8: Amino acid sequence of human IgGl hinge region

[0063] EPKSCDKTHTCP

[0064] In some preferred embodiments, the nucleic acid sequence encoding the CD8 hinge region comprises the nucleic acid sequence set forth in SEQ ID NO: 9 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0065] SEQ ID NO: 9: Nucleic acid sequence of CD8 hinge region

[0066] ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGtGGTCTTGACTTCGCCTGCGAT

[0067] In some preferred embodiments, the amino acid sequence of the CD8 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0068] SEQ ID NO: 10: Amino acid sequence of CD8 transmembrane region

[0069] IYIWAPLAGTCGVLLLSLVITLYC

[0070] In some preferred embodiments, the amino acid sequence of the PGFRA transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0071] SEQ ID NO: 11: Amino acid sequence of PGFRA transmembrane region

[0072] AAVLVLLVIVIISLIVLVVIW

[0073] In some preferred embodiments, the amino acid sequence of the CD80 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0074] SEQ ID NO: 12: Amino acid sequence of CD80 transmembrane region

[0075] LLPSWAITLISVNGIFVICCL

[0076] In some preferred embodiments, the nucleic acid sequence encoding the CD8 transmembrane region comprises the nucleic acid sequence set forth in SEQ ID NO: 13 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0077] SEQ ID NO: 13: Nucleic acid sequence of CD8 transmembrane region

[0078] ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGT

[0079] In some preferred embodiments, the hinge region is a CD8 hinge region and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some cases, the hinge region is a CD8 hinge region and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a CD8 hinge region and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a CD8 hinge region and the transmembrane region is a CD80 transmembrane region.

[0080] In some preferred embodiments, the hinge region is a human IgGl hinge region and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some cases, the hinge region is a human IgGl hinge region and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a human IgGl hinge region and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a human IgGl hinge region and the transmembrane region is a CD80 transmembrane region.

[0081] In some preferred embodiments, the nucleic acid sequence of the tandem NGF, hinge region, and transmembrane region is a nucleic acid sequence of a tandem NGF, CD8 hinge region, and CD8 transmembrane region.

[0082] In some embodiments, the present application relates to a method for constructing the recombinant CHO cell described above, comprising:

[0083] (1) cloning the nucleic acid sequence of the NGF, the hinge region and the transmembrane region in series into an expression vector to obtain a recombinant expression vector comprising the nucleic acid sequence;

[0084] (2) introducing the recombinant expression vector into a CHO cell to obtain a recombinant CHO cell.

[0085] In some preferred embodiments, the NGF can be a mammalian NGF, such as human NGF, mouse NGF, rat NGF, bovine NGF, horse NGF, pig NGF, etc., but is not limited thereto.

[0086] In some preferred embodiments, the amino acid sequence of the NGF comprises the amino acid sequence shown in any one of SEQ ID NO: 1-SEQ ID NO: 3 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0087] In some preferred embodiments, the nucleic acid sequence of the NGF comprises the nucleic acid sequence shown in any one of SEQ ID NO: 4-SEQ ID NO: 6 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0088] In further preferred embodiments, the amino acid sequence of the hinge region has more than 3 amino acids, more than 10 amino acids, more than 20 amino acids, more than 30 amino acids, more than 40 amino acids, more than 50 amino acids, or less than 100 amino acids, less than 90 amino acids, less than 80 amino acids, less than 70 amino acids, less than 60 amino acids, or an amino acid within any two of the above-mentioned numerical ranges, such as 3-100 amino acids, 10-100 amino acids, 10-90 amino acids, 10-80 amino acids, 10-70 amino acids, 10-60 amino acids, 10-50 amino acids, 12-45 amino acids.

[0089] In further preferred embodiments, the transmembrane region has an amino acid sequence length of more than 10 amino acids, more than 20 amino acids, more than 30 amino acids, or less than 60 amino acids, less than 50 amino acids, less than 40 amino acids, or an amino acid sequence length within any of the above-mentioned ranges, e.g., 10-60 amino acids, 10-50 amino acids, 10-40 amino acids, 10-30 amino acids, 15-30 amino acids, 20-30 amino acids, 20-25 amino acids.

[0090] In some preferred embodiments, the hinge region can be a CD8 hinge region (e.g., a CD8a hinge region) or a human IgGl hinge region.

[0091] In some preferred embodiments, the transmembrane region can be a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region.

[0092] In some preferred embodiments, the amino acid sequence of the CD8 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto. In some preferred embodiments, the amino acid sequence of the human IgGl hinge region comprises the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0093] In some preferred embodiments, the nucleic acid sequence encoding the CD8 hinge region comprises the nucleic acid sequence set forth in SEQ ID NO: 9 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0094] In some preferred embodiments, the amino acid sequence of the CD8 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto. In some preferred embodiments, the amino acid sequence of the PGFRA transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto. In some preferred embodiments, the amino acid sequence of the CD80 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0095] In some preferred embodiments, the nucleic acid sequence encoding the CD8 transmembrane region comprises the nucleic acid sequence set forth in SEQ ID NO: 13 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) sequence identity thereto.

[0096] In some preferred embodiments, the hinge region is a CD8 hinge region and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some cases, the hinge region is a CD8 hinge region and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a CD8 hinge region and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a CD8 hinge region and the transmembrane region is a CD80 transmembrane region.

[0097] In some preferred embodiments, the hinge region is a human IgGl hinge region and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some cases, the hinge region is a human IgGl hinge region and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a human IgGl hinge region and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a human IgGl hinge region and the transmembrane region is a CD80 transmembrane region.

[0098] In some preferred embodiments, the nucleic acid sequence of the tandem NGF, hinge region, and transmembrane region is a nucleic acid sequence of a tandem NGF, CD8 hinge region, and CD8 transmembrane region.

[0099] In some preferred embodiments, the expression vector can be a lentivirus expression vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a poxvirus vector, or a herpes simplex virus vector, but is not limited thereto. As an example of a lentivirus expression vector, the lentivirus expression vector can be, for example, a Lenti-CMV-puro lentivirus expression vector, a pCDH series lentivirus expression vector, or a pLenti series lentivirus expression vector, but is not limited thereto.

[0100] In some preferred embodiments, in step (2), the recombinant expression vector and the viral packaging plasmid are co-transfected into a packaging cell, and after cultivation, a virus containing the nucleic acid sequence is prepared, and then the virus is used to transfect CHO cells to obtain the recombinant CHO cells.

[0101] In further preferred embodiments, the viral packaging plasmid can be pMD2.G, psPAX2, Lenti-packaging Mix, pCMV-dR8.91, pCMV-dR8.74, pLP1, pLP2, pVSV-G, etc., but is not limited thereto.

[0102] In further preferred embodiments, the packaging cell can be a 293T cell, a 293FT cell, etc., but is not limited thereto.

[0103] In this document, unless otherwise specified, the cultivation of cells is carried out using conventional cultivation conditions and common cell culture media (e.g., 1640 medium, DMEM medium, MEM medium, F12 medium, etc.) in the art, which can be found, for example, in the following: Ran Rong et al., Cell Culture Technology, Chemical Industry Press, 2007; R.I. Fershney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (Sixth Edition), John Wiley & Sons, Inc; 2010; Liu Bin et al., Cell Culture (3rd Edition), World Publishing Company, 2018; the contents of each of which are incorporated herein by reference in their entirety.

[0104] Transfection is one of the common means known in the art for introducing a target exogenous DNA (extracted from a donor of interest or directly synthesized) into a host cell. Virus-mediated transfection is one of the commonly used transfection methods in the art, which uses a method of infecting cells with a virus packaged with a desired exogenous gene to allow the exogenous gene of interest to enter the cells, which has the advantages of the highest transfection efficiency and very low cytotoxicity.

[0105] In some embodiments, the present application relates to a system for detecting ADCC effect of an antibody, wherein the system comprises:

[0106] the recombinant CHO cell described above, and

[0107] an effector cell.

[0108] In the present context, the effector cell can be any conventional effector cell known in the art. By way of example, in some preferred embodiments, the effector cell can include, but is not limited to, effector T cells, NK cells, phagocytes, eosinophils, basophils, and mast cells, etc., such as Jurkat-NFAT-Luciferase-CD16 cells, primary NK cells, etc.

[0109] In some preferred embodiments, the effector cell and the recombinant CHO cell can have an effector-to-target ratio (E:T) of 1 : 1-1 :20, such as 1 :5.

[0110] In some preferred embodiments, the system further comprises a luciferase substrate. As a typical luciferase, firefly luciferase is a protein with a molecular weight of about 61 kDa, which is capable of catalyzing the oxidation of luciferin to oxyluciferin in the presence of ATP, magnesium ions and oxygen, and emitting bioluminescence in the process of oxidation.

[0111] In some embodiments, the present application relates to a system for detecting CDC effect of an antibody, wherein the system comprises:

[0112] the recombinant CHO cell described above, and

[0113] complement.

[0114] Complement, also known as complement system, is a protein existing in human and animal serum or tissue fluid, which can mediate immune response and inflammatory reaction, including more than 30 soluble proteins and membrane-bound proteins. In some preferred embodiments, the complement is complement serum comprising complement proteins C1-C9.

[0115] In some preferred embodiments, the system further comprises a luciferase substrate.

[0116] In some embodiments, the present application relates to the use of the recombinant CHO cell described above in detecting ADCC effect or CDC effect of an antibody.

[0117] In some embodiments, the present application relates to a method for detecting the ADCC effect of an antibody, wherein the method comprises incubating the recombinant CHO cell described above as target cells with the antibody to be tested and effector cells. In further preferred embodiments, the effector cells and the target cells can be in an effector-to-target ratio of 1 : 1 to 1 :20, such as 1 :5. In some preferred embodiments, the method further comprises adding luciferase substrate to the system after incubation.

[0118] In some embodiments, the present application relates to a method for detecting the CDC effect of an antibody, wherein the method comprises incubating the recombinant CHO cell described above as target cells with the antibody to be tested and complement. In further preferred embodiments, the complement is complement serum comprising complement proteins C1-C9. In some preferred embodiments, the method further comprises adding luciferase substrate to the system after incubation.

[0119] Examples

[0120] The present application will be described in detail below with reference to Examples, but is not intended to be limited to the scope of the present application. Various changes, modifications and improvements, which are based on the description of the following Examples, can be made by those skilled in the art without departing from the spirit and scope of the present application, and the resulting schemes still fall within the scope of the present application.

[0121] It should be noted that the design, synthesis and cloning of genes, the ligation of gene fragments, the construction of expression vectors, sequence analysis and identification, the construction of recombinant cells, and the isolation and purification of expression products and other operation steps involved in the present application can be carried out according to the known techniques in the art (for example, see the description of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY). If not specifically indicated, the technical means used in the Examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, materials and equipment used in the following Examples are commercially available reagents, materials and equipment.

[0122] The main reagents, consumables and equipment used in the following Examples and their sources are shown in the table below, and those skilled in the art can use any other reagents, consumables and equipment with equivalent effects.

[0123]

[0124]

[0125] Example 1 Construction of NGF overexpression lentiviral vector and preparation of lentivirus

[0126] The nucleic acid sequence of NGF can be concatenated with the nucleic acid sequence encoding the hinge region and the transmembrane region and inserted into a suitable expression vector, thereby achieving the heterologous expression of the above-mentioned nucleic acid sequence. For NGF, NGF from mammals can be selected, such as human NGF, mouse NGF, rat NGF, bovine NGF, horse NGF, pig NGF, etc., and NGF and its nucleic acid sequence can be synthesized according to known sequences disclosed in commonly used nucleic acid sequence and protein sequence databases, such as NCBI, ENSMBL or UniProt.

[0127] Here, human NGF is taken as an example, and CD8 hinge region and CD8 transmembrane region are used in concatenation with the NGF. Specifically, the full-length sequence of human NGF is codon-optimized according to the codon bias of the host cell CHO-S cell, obtaining NGF with the amino acid sequence shown in SEQ ID NO: 1 (the corresponding nucleic acid sequence is shown in SEQ ID NO: 4), which is concatenated with the CD8 hinge region (the amino acid sequence is shown in SEQ ID NO: 7; the nucleic acid sequence is shown in SEQ ID NO: 9) and the CD8 transmembrane region (the amino acid sequence is shown in SEQ ID NO: 10; the nucleic acid sequence is shown in SEQ ID NO: 13). After concatenation, the synthesized gene fragment is subcloned into the Lenti-CMV-puro lentivirus expression vector (Ekon Biotech (Suzhou) Co., Ltd.) to prepare the target gene overexpression vector Lenti-CMV-NGF-Full(CD8-TM) vector. After sequencing verification that the sequence is correct, a endotoxin-free plasmid is prepared for standby.

[0128] Next, the lentivirus is prepared according to the following steps:

[0129] 1. Prepare 15 cm cell culture dishes, inoculate 293T cells (Genewiz) at 5 x 10 6 cells / dish, add complete culture medium (DMEM high glucose culture medium supplemented with 10% FBS), and incubate in a 37°C, 5% CO2 incubator overnight.

[0130] 2. Take out the transfection reagent LVTransm, Lenti-CMV-NGF-Full(CD8-TM) expression vector and lentivirus packaging plasmid Lenti-packaging Mix from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Prepare the transfection complex according to the following procedure: take 1x PBS or HBSS buffer and warm to room temperature, then take 2 mL of PBS or HBSS buffer and add to one well of a 6-well plate, add 10 μg of Lenti-CMV-NGF-Full(CD8-TM) expression vector and 30 μL of Lenti-packaging Mix, mix thoroughly by pipetting up and down, then add 50 μL of LVTransm, immediately mix by pipetting up and down, and stand at room temperature for 10-15 minutes.

[0131] 3. Add the transfection complex dropwise to the 15 cm cell culture dish from step 1, gently shake the dish to mix thoroughly. After incubating the dish at 37°C in a 5% CO2 incubator for 6-8 hours, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium, then return the dish to the incubator for continued culture.

[0132] 4. After continuous culture for 24 hours, collect the virus-containing culture medium supernatant from the dish into a 50 mL centrifuge tube; add about 25 mL of fresh complete culture medium to the dish and continue to culture for 24 hours.

[0133] 5. Collect the virus-containing culture medium supernatant from the dish into a 50 mL centrifuge tube; add about 25 mL of fresh complete culture medium to the dish and continue to culture for 24 hours.

[0134] 6. Collect the virus-containing culture medium supernatant from the dish and mix it with the supernatant from the previous two culture steps.

[0135] 7. Filter the culture medium supernatant described above using a 0.45 μm PES filter membrane, then transfer the filtrate to a sterile centrifuge tube and centrifuge at 50000 x g at 4°C for 2 hours. After centrifugation, carefully remove the liquid from the centrifuge tube in a biological safety cabinet, resuspend the precipitate with 1 mL of PBS buffer, and store the obtained Lenti-CMV-NGF-Full(CD8-TM) overexpression lentivirus at -80°C.

[0136] Example 2 Construction of recombinant CHO cells

[0137] Using the Lenti-CMV-NGF-Full(CD8-TM) overexpression lentivirus prepared in Example 1, the recombinant CHO cells capable of stably expressing NGF on the cell surface (also referred to as "CHO-NGF recombinant cells" or "CHO-NGF cells") were prepared according to the following procedure:

[0138] 1. The CHO-S cells were resuscitated from liquid nitrogen and cultured at 37°C in a 5% CO2 incubator using CHO Grow CD1 serum-free medium. The cells were subcultured for 5 times in succession to make them in logarithmic growth phase.

[0139] 2. A new 6-well plate was taken and the above-mentioned CHO-S cells in logarithmic growth phase were inoculated into the 6-well plate at a density of 5x10 6 cells / well, and 3 mL of CHO Grow CD1 serum-free medium was added. The required virus amount was calculated according to the MOI = 5, the number of inoculated cells and the virus titer, and the calculation formula was as follows:

[0140] Virus amount (mL) = (cell number x MOI) / virus titer

[0141] 3. The Lenti-CMV-NGF-Full(CD8-TM) overexpression lentivirus prepared in Example 1 was added into the 6-well plate in an amount calculated according to the above formula, and the 6-well plate was placed in a centrifuge for centrifugation at 800xg for 1 hour at room temperature.

[0142] 4. After centrifugation, the 6-well plate was taken out and placed in a 37°C, 5% CO2 incubator for continuous culture for 24 hours.

[0143] 5. After 24 hours of culture, the culture medium in the 6-well plate was replaced with fresh CHO Grow CD1 serum-free medium, and the culture was continued for another 24 hours.

[0144] 6. The culture medium in the 6-well plate was replaced with CHO Grow CD1 serum-free medium containing 8 μg / ml Puromycin, and the culture was continued for 5 days until all the uninfected CHO-S cells were killed.

[0145] 7. The remaining living cells were further expanded, and the cells were collected to obtain the constructed recombinant CHO cells.

[0146] Example 3 Flow cytometric identification of recombinant CHO cells

[0147] The recombinant CHO cells constructed in Example 2 were identified by flow cytometry using a positive control antibody. The positive control antibody HAB20-6-2 was prepared according to the following procedure:

[0148] 1. Starting from the anti-NGF antibody Tanezumab of IgG2 subtype developed by Pfizer, according to the sequence information of Tanezumab antibody (the sequence of its heavy chain is shown in SEQ NO ID: 14; the sequence of its light chain is shown in SEQ NO ID: 15), the Fc segment constant region sequence of Tanezumab antibody is replaced by the constant region sequence of human IgG1 subtype (the amino acid sequence is shown in SEQ NO ID: 16; the nucleotide sequence is shown in SEQ NO ID: 17), and the rest remains unchanged, and a pcDNA3.1-hIgG1 vector (prepared by Aikangde Biotech (Suzhou) Co., Ltd.) is used to construct a human IgG1 subtype recombinant antibody expression vector, which is stored in the refrigerator for standby.

[0149] 2. Take the transfection reagent LVTransm and the human IgG1 subtype recombinant antibody expression vector from the refrigerator, and after thawing at room temperature, blow them up and down with a pipette gun. Take 1xPBS or HBSS buffer and warm it to room temperature. Then, take 2 mL of PBS or HBSS buffer and add it to one well of a 6-well plate, and add 90 μg of the human IgG1 subtype recombinant antibody expression vector (the ratio of light chain to heavy chain is 2:1) to it, and mix it well by blowing it up and down with a pipette gun. Then add 270 μL of LVTransm to it, and immediately mix it well by blowing it up and down with a pipette, and let it stand at room temperature for 10 minutes to obtain a complex.

[0150] 3. Add the complex obtained above to 50 mL of 293F cells, and mix them well by gently shaking. Place the cells in a 37°C, 5% CO2 incubator and incubate at 130 RPM for 6-8 hours, then add 50 mL of fresh 293F medium to the cells and place them back in the incubator for further culture.

[0151] 4. After continuous culture for 7 days, centrifuge to collect the supernatant of the culture medium, filter it with a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube. Use a Protein A affinity column to purify the antibody to obtain the positive control antibody HAB20-6-2.

[0152] 5. Detect the purity of the protein by SDS-PAGE.

[0153] The detection results are shown in Figure 1 According to the SDS-PAGE detection results, the purity of the positive control antibody HAB20-6-2 prepared above is greater than 90%, and it can be used for ADCC effect detection.

[0154] SEQ NO ID: 14: Heavy chain sequence of Tanezumab

[0155] QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0156] SEQ NO ID: 15: Light chain sequence of Tanezuab

[0157] DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0158] SEQ NO ID: 16: Amino acid sequence of human IgGl constant region

[0159] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0160] SEQ NO ID: 17: nucleic acid sequence of human IgGl constant region

[0161] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCTAAGAACCAGGACACCGCACGTGTACACC ATCGAGACCGAGGAGAAAGAGGCTTCGGCGCGGTGGAGCAGTACACCGTGATAGAGCGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGG

[0162] Based on the positive control antibody HAB20-6-2 prepared above, the recombinant CHO cells can be identified by flow cytometry, for example, the recombinant CHO cells (CHO-NGF cells) constructed in Example 2 can be identified according to the following procedure:

[0163] (1) The CHO-NGF cells constructed in Example 2 and CHO-S cells as a blank control were cultured in CHO Grow CD1 serum-free medium at 37°C in a 5% CO2 incubator, and the cell state was adjusted to the logarithmic growth phase.

[0164] (2) 5 x 10 5 cells of each of the above two cells were added to one well of a 6-well plate, 100 μl of 1 x PBS buffer was added to each well with the above cells to resuspend the cells, and then 1 μg of the positive control antibody HAB20-6-2 was added to each well, which was mixed well and incubated at room temperature for 45 min; at the same time, a well without the addition of the positive control antibody was set as a negative control.

[0165] (3) Centrifugation at 800 x g for 5 min at room temperature to remove the supernatant containing the antibody, and the cells were washed 3 times with 1 x PBS buffer;

[0166] (4) PE anti-human IgG Fc secondary antibody (1:200 dilution) was added, mixed well, and incubated at room temperature for 30 min in the dark;

[0167] (5) Centrifugation at 800 x g for 5 min at room temperature to remove the supernatant containing the secondary antibody, and the cells were washed 3 times with 1 x PBS buffer;

[0168] (6) The cells were resuspended with 500 uL of 1 x PBS buffer and subjected to flow cytometry analysis.

[0169] The flow cytometry identification results are shown in Figure 2 . According to the flow cytometry detection results, Figure 2 the positive rate of the positive control antibody of the CHO-NGF cells constructed in Example 2 was 82.67%, and therefore, the cells can be used as target cells for ADCC effect detection.

[0170] Example 4 ADCC effect detection

[0171] The CHO-NGF recombinant cells stably expressing human NGF on cell surface constructed in Example 2 above were used as target cells, and the Jurkat-NFAT-Luciferase-CD16 cell line stably transfected with luciferase was used as effector cells. After the Fab fragment of the sample to be tested or the positive control antibody binds to the antigen on the target cells, the Fc fragment of the sample to be tested or the positive control antibody binds to CD16 on the surface of the effector cells Jurkat-NFAT-Luciferase-CD16, the NFAT-related signal pathway in the cells is activated, which in turn leads to an increase in the expression level of luciferase. By detecting the change in luciferase activity, the ADCC effect of the sample to be tested and the positive control antibody can be evaluated.

[0172] The sample to be tested Tanezumab is a recombinant anti-NGF humanized monoclonal antibody developed by Pfizer, which targets human nerve growth factor and is synthesized using a CHO cell expression system. The relevant information of Tanezumab and the positive control antibody HAB20-6-2 used in this example is shown in the table below.

[0173] Sample Name Concentration Storage Tanezumab 10 mg / mL -70±10℃ Positive Control Antibody HAB20-6-2 3.5 mg / mL -70±10℃

[0174] The ADCC effect detection experiment was performed using the ADCC Reporter Bioassay, and the experimental steps were as follows.

[0175] 1. Jurkat-NFAT-Luciferase-CD16 overexpressing cells were used as effector cells, and CHO-NGF cells constructed in Example 2 were used as target cells. The ADCC experimental system was established according to the effector-to-target ratio of 1:5 (a total of 100 μL), wherein the number of effector cells was 2 x 10 4 cells, and the number of target cells was 1 x 10 5 cells / well, which were added to a 96-well plate as the subsequent experimental system.

[0176] 2. Tanezumab and the positive control antibody HAB20-6-2 were first diluted to a concentration of 200 μg / mL with RPMI 1640 medium to obtain their respective stock solutions, and then 10-fold gradient dilution was performed (20 μL of each stock solution obtained by dilution was added to 180 μL of RPMI 1640 medium, and gradient dilution was performed in sequence). Nine gradients were continuously diluted (200, 20, 2, 0.2, 0.02, 0.002, 0.0002, 0.00002, 0.000002, 0 μg / ml).

[0177] 3. In the above experimental system, the different concentrations of the test sample Tanezumab and the positive control antibody HAB20-6-2 obtained by dilution were added, 100 μL of the test sample and the positive control antibody (with a final concentration of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0 μg / ml in each well) were inoculated in each well, 2 replicates for each concentration, and incubated at 37°C in a 5% CO2 incubator for 18 hours. Then, 20 μL of One-Glo reagent was added, and the reaction was carried out at room temperature for 5 minutes. The ADCC effect of the antibody was evaluated by detecting the change in luciferase activity. The detection of Tanezumab was repeated 4 times.

[0178] In this example, the CHO-NGF recombinant cells stably expressing NGF on the cell surface constructed in Example 2 were used as target cells, and the existing effector cells were used to evaluate the ability of the positive control antibody HAB20-6-2 and Tanezumab to mediate ADCC effect by the reporter gene method. The detection results of the ADCC effect are shown in Figure 3 Figure 3 According to the detection results in

[0179] It can be seen that under the above experimental conditions, the positive control antibody produced a significant ADCC effect, while Tanezumab, which has the same variable region as the positive control antibody (thus it can be expected that the antibody Tanezumab can recognize and specifically bind to the same antigen, i.e. NGF expressed by CHO-NGF recombinant cells), did not produce a significant ADCC effect. Therefore, the CHO-NGF recombinant cells of the present application can be effectively used as target cells for detecting the ADCC effect mediated by anti-NGF antibodies in vitro, so as to accurately detect whether the test antibody can cause the effector cells to produce an ADCC effect.

[0180] Example 5 CDC effect detection

[0181] ​The CHO-NGF recombinant cells stably expressing human NGF on cell surface constructed in Example 2 were used as target cells. The target cells, human complement serum, the sample to be tested or the positive control antibody were incubated together. After the Fab fragment of the sample to be tested or the positive control antibody bound to the antigen on the target cells, the Fc fragment of the sample to be tested or the positive control antibody combined with the complement in the human serum and initiated the CDC effect, causing the target cells to be killed. ATP is an important indicator of cell metabolism, and its content directly reflects the number of living cells. After the incubation was completed, the Cell Counting-Lite 3D Luminescent reagent was added to the reaction system. The reagent contains heat-stable luciferase and its substrate luciferin, and can promote the lysis of living cells in the reaction system to release ATP. ATP promotes luciferase to act on the substrate to make it emit light. By detecting the light intensity, the number of living cells in the detection system can be reflected, and the lysis effect of the sample to be tested or the positive control antibody on the target cells through CDC can be reflected.

[0182] The relevant information of the sample to be tested Tanezumab and the positive control antibody HAB20-6-2 was the same as in Example 4. The CDC effect detection experiment was carried out according to the following steps:

[0183] 1. The CHO-NGF cells constructed in Example 2 were used as target cells, and the cell amount for inoculation was 4x10 4 cells / well (90 μL), which were inoculated into a 96-well plate as the subsequent experimental system.

[0184] 2. The sample to be tested Tanezumab and the positive control antibody HAB20-6-2 were first diluted with RPMI 1640 medium to a concentration of 200 μg / mL to obtain the respective stock solutions, and then 10-fold gradient dilution was carried out (20 μL of each stock solution obtained by dilution was added to 180 μL of RPMI 1640 medium, and gradient dilution was carried out in turn). Nine gradients were continuously diluted (200, 20, 2, 0.2, 0.02, 0.002, 0.0002, 0.00002, 0.000002, 0 μg / ml).

[0185] 3. In the above experimental system, the different concentrations of the test sample Tanezumab and the positive control antibody HAB20-6-2 (the final concentrations in each well were 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0 μg / ml, respectively) obtained by dilution were added, 10 μL of Human Complement Serum was added, 2 replicates for each concentration, and incubated at 37°C in a 5% CO2 incubator for 18 hours. Then, 50 μL of Cell Counting-Lite 3D reagent was added, and the cell clusters were fully lysed by shaking for 2 min. After incubation at room temperature for 25 min, the luminescence value was detected using a multifunctional microplate reader. The lysis of target cells was reflected by the change in luminescence value, and the CDC effect of the antibody was evaluated. The Tanezumab was detected 4 times.

[0186] 4. The formula for calculating the percentage of target cell lysis (Lysis %) is (RLU max : the fluorescence intensity of the cell blank control well; RLU sample : the fluorescence intensity of the well with the test sample or the positive control antibody added)

[0187]

[0188] In this example, the CHO-NGF recombinant cells stably expressing NGF on the cell surface constructed in Example 2 were used as target cells to evaluate the ability of the positive control antibody HAB20-6-2 and Tanezumab to mediate the CDC effect. According to the detection results in Example 1, Figure 4 According to the detection results in Example 1, the positive control antibody HAB20-6-2 produced a significant CDC effect on CHO-NGF target cells, and there was a clear dose-effect relationship within a certain dose range. However, the 4 detections of Tanezumab, which only differed from the positive control antibody in the Fc constant region sequence, did not detect a significant CDC effect.

[0189] It can be seen that under the above experimental conditions, the positive control antibody HAB20-6-2 produced a significant CDC effect, while Tanezumab (which has the same variable region as the positive control antibody, so it can be expected that the antibody Tanezumab can also recognize and specifically bind to the NGF expressed on the surface of the CHO-NGF recombinant cells) did not produce a significant CDC effect. Therefore, the CHO-NGF recombinant cells of the present application can be effectively used as target cells for detecting the CDC effect mediated by anti-NGF antibodies in vitro, thereby accurately detecting whether the test antibody can produce a CDC effect on target cells.

[0190] For purposes of United States patent practice, the contents of all U.S. patent and

[0191] Those skilled in the art will recognize that the scope of the present application is not intended to be limited to the various specific embodiments and examples described above, but rather only by the claims below. Various modifications, substitutions, or reconfigurations of the various specific embodiments and examples, which are within the spirit of the present application, are intended to fall within the scope of the present application. SEQUENCE LISTING <110> Chengdu Youlu Biotechnology Co., Ltd. <120> A recombinant CHO cell, a construction method thereof, a detection system and method using the same <130> SPIC218044-26 / 18 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 241 <212> PRT <213> Homo sapiens <400> 1 Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile 1 5 10 15 Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile 20 25 30 Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu 35 40 45 Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala 50 55 60 Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg 65 70 75 80 Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu 85 90 95 Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro 100 105 110 Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe 115 120 125 His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly 130 135 140 Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu 145 150 155 160 Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu 165 170 175 Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile 180 185 190 Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val 195 200 205 Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg 210 215 220 Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg 225 230 235 240 Ala <210> 2 <211> 120 <212> PRT <213> Homo sapiens <400> 2 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys 20 25 30 Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val 35 40 45 Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val 50 55 60 Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80 Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln 85 90 95 Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu 100 105 110 Ser Arg Lys Ala Val Arg Arg Ala 115 120 <210> 3 <211> 118 <212> PRT <213> Homo sapiens <400> 3 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys 20 25 30 Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val 35 40 45 Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val 50 55 60 Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80 Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln 85 90 95 Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu 100 105 110 Ser Arg Lys Ala Val Arg 115 <210> 4 <211> 723 <212> DNA <213> Homo sapiens <400> 4 atgagcatgc tgttctatac cctgatcacc gcctttctga tcggcatcca ggccgagcct 60 cacagcgagt ctaatgtgcc tgccggccac acaatccctc aggctcactg gacaaagctg 120 cagcacagcc tggatacagc tctgcggaga gccagatctg ctcctgccgc tgctattgcc 180 gctagagtgg ctggccagac cagaaacatc accgtggatc cccggctgtt caagaagcgg 240 agactgagaa gccccagagt gctgttcagc acccagcctc caagagaggc cgccgataca 300 caggacctgg attttgaagt tggcggcgct gcccctttca acagaaccca cagaagcaag 360 cggagcagct ctcaccccat cttccacaga ggcgagttca gcgtgtgcga ctccgtgtct 420 gtgtgggtcg gagataagac caccgccacc gacatcaagg gcaaagaagt gatggtcctg 480 ggcgaagtga acatcaacaa cagcgtgttc aagcagtact tcttcgagac aaagtgcagg 540 gaccccaatc ctgtggacag cggctgtaga ggcatcgaca gcaagcactg gaactcctac 600 tgcaccacca cacacacctt cgtgaaggcc ctgaccatgg atggaaaaca ggccgcctgg 660 cggttcatca gaatcgatac cgcttgcgtg tgcgtgctga gcagaaaagc cgttagaagg 720 gcc 723 <210> 5 <211> 360 <212> DNA <213> Homo sapiens <400> 5 agcagctctc accccatctt ccacagaggc gagttcagcg tgtgcgactc cgtgtctgtg 60 tgggtcggag ataagaccac cgccaccgac atcaagggca aagaagtgat ggtcctgggc 120 gaagtgaaca tcaacaacag cgtgttcaag cagtacttct tcgagacaaa gtgcagggac 180 cccaatcctg tggacagcgg ctgtagaggc atcgacagca agcactggaa ctcctactgc 240 accaccacac acaccttcgt gaaggccctg accatggatg gaaaacaggc cgcctggcgg 300 ttcatcagaa tcgataccgc ttgcgtgtgc gtgctgagca gaaaagccgt tagaagggcc 360 <210> 6 <211> 354 <212> DNA <213> Homo sapiens <400> 6 agcagctctc accccatctt ccacagaggc gagttcagcg tgtgcgactc cgtgtctgtg 60 tgggtcggag ataagaccac cgccaccgac atcaagggca aagaagtgat ggtcctgggc 120 gaagtgaaca tcaacaacag cgtgttcaag cagtacttct tcgagacaaa gtgcagggac 180 cccaatcctg tggacagcgg ctgtagaggc atcgacagca agcactggaa ctcctactgc 240 accaccacac acaccttcgt gaaggccctg accatggatg gaaaacaggc cgcctggcgg 300 ttcatcagaa tcgataccgc ttgcgtgtgc gtgctgagca gaaaagccgt taga 354 <210> 7 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the CD8 hinge region <400> 7 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 8 <211> 12 <212> PRT <213> Homo sapiens <400> 8 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 1 5 10 <210> 9 <211> 135 <212> DNA <213> Artificial Sequence <220> Nucleic acid sequence of CD8 hinge region <400> 9 accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60 tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccgtggtctt 120 gacttcgcct gcgat 135 <210> 10 <211> 24 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of CD8 transmembrane region <400> 10 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 11 <211> 21 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of PGFRA transmembrane region <400> 11 Ala Ala Val Leu Val Leu Leu Val Ile Val Ile Ile Ser Leu Ile Val 1 5 10 15 Leu Val Val Ile Trp 20 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CD80 transmembrane region <400> 12 Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe 1 5 10 15 Val Ile Cys Cys Leu 20 <210> 13 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence of CD8 transmembrane region <400> 13 atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60 actctttact gt 72 <210> 14 <211> 447 <212> PRT <213> Unknown <220> <223> Heavy chain sequence of tanezumab <400> 14 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr 20 25 30 Asp Leu Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly lie lie Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser Ala Val Lys 50 55 60 Ser Arg Val Thr lie Ser Lys Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Tyr Trp Tyr Ala Thr Ser Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Asn Phe Gly Thr Gin Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys 210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 15 <211> 214 <212> PRT <213> Unknown <220> <223> Tanezuab's light chain sequence <400> 15 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Ser lie Ser Asn Asn 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Phe His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Glu His Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 330 <212> PRT <213> Homo sapiens <400> 16 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 17 <211> 993 <212> DNA <213> Homo sapiens <400> 17 gcctctacaa agggcccctc cgtttttcca ctggctccca gcagcaagtc tacctctggt 60 ggaacagccg ctctgggctg cctggtcaag gattactttc ccgagccagt gaccgtgtcc 120 tggaactctg gcgctctgac atctggcgtg cacacatttc cagccgtgct gcagtctagc 180 ggcctgtact ctctgagcag cgtggtcaca gtgcctagct ctagcctggg cacccagacc 240 tacatctgca atgtgaacca caagcctagc aacaccaagg tggacaagaa ggtggaaccc 300 aagagctgcg acaagaccca cacctgtcct ccatgtcctg ctccagaact gctcggcgga 360 ccttccgtgt ttctgttccc tccaaagcct aaggacaccc tgatgatcag cagaacccct 420 gaagtgacct gcgtggtggt ggatgtgtcc cacgaggacc ccgaagtgaa gttcaattgg 480 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagtacaac 540 agcacctaca gagtggtgtc cgtgctgaca gtgctgcatc aggactggct gaacggcaaa 600 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgctc ctatcgagaa aaccatcagc 660 aaggccaagg gccagcctag ggaaccccag gtttacacac tgcctccaag cagggacgag 720 ctgaccaaga atcaggtgtc cctgacctgc ctcgtgaagg gcttctaccc ttccgatatc 780 gccgtggaat gggagagcaa tggccagcct gagaacaact acaagacaac ccctcctgtg 840 ctggacagcg acggctcatt cttcctgtac agcaagctga ccgtggacaa gtccagatgg 900 cagcagggca acgtgttcag ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960 cagaagtccc tgagcctgtc tcctggcaaa taa 993

Claims

1. A recombinant CHO cell, wherein, stably express NGF on the surface of the cell, wherein a nucleic acid sequence of NGF, CD8 hinge region and CD8 transmembrane region in tandem is integrated into the genome of the cell, wherein the NGF is mammalian NGF; the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 7; and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:

10.

2. The recombinant CHO cell of claim 1, wherein, The NGF is human NGF, mouse NGF, rat NGF, bovine NGF, horse NGF or pig NGF.

3. The recombinant CHO cell of claim 1 or 2, wherein, The amino acid sequence of the NGF is shown in any one of SEQ ID NO: 1-3.

4. The recombinant CHO cell of claim 1 or 2, wherein, The nucleic acid sequence of the NGF is shown in any one of SEQ ID NO: 4-6.

5. The recombinant CHO cell of claim 1 or 2, wherein, The nucleic acid sequence encoding the CD8 hinge region comprises the nucleic acid sequence shown in SEQ ID NO: 9 or a nucleic acid sequence having at least 80% sequence identity thereto.

6. The recombinant CHO cell of claim 1 or 2, wherein, The nucleic acid sequence encoding the CD8 transmembrane region comprises the nucleic acid sequence shown in SEQ ID NO: 13 or a nucleic acid sequence having at least 80% sequence identity thereto.

7. A method for constructing the recombinant CHO cell of any one of claims 1-6, comprising: (1) cloning a nucleic acid sequence of NGF, CD8 hinge region and CD8 transmembrane region in tandem into an expression vector to obtain a recombinant expression vector comprising the nucleic acid sequence, wherein the NGF is mammalian NGF; the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 7; and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO: 10; (2) introducing the recombinant expression vector into a CHO cell to obtain a recombinant CHO cell.

8. The method of claim 7, wherein, The NGF is human NGF, mouse NGF, rat NGF, bovine NGF, horse NGF or pig NGF.

9. The method of claim 7 or 8, wherein, The amino acid sequence of the NGF is shown in any one of SEQ ID NO: 1-3.

10. The method of claim 7 or 8, wherein, The nucleic acid sequence of the NGF is shown in any one of SEQ ID NO: 4-6.

11. The method of claim 7 or 8, wherein, The nucleic acid sequence encoding the CD8 hinge region comprises the nucleic acid sequence shown in SEQ ID NO: 9 or a nucleic acid sequence having at least 80% sequence identity thereto.

12. The method of claim 7 or 8, wherein, The nucleic acid sequence encoding the CD8 transmembrane region comprises the nucleic acid sequence shown in SEQ ID NO: 13 or a nucleic acid sequence having at least 80% sequence identity thereto.

13. The method of claim 7 or 8, wherein, The expression vector is a lentivirus expression vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a poxvirus vector or a herpes simplex virus vector.

14. The method of claim 13, wherein, The expression vector is a Lenti-CMV-puro lentivirus expression vector, a pCDH series lentivirus expression vector or a pLenti series lentivirus expression vector.

15. The method of claim 7 or 8, wherein in step (2), the recombinant expression vector and a viral packaging plasmid are co-transfected into packaging cells, and after culturing, a virus containing the nucleic acid sequence is prepared, and then the virus is used to transfect the CHO cells to obtain the recombinant CHO cells.

16. The method of claim 15, wherein, The viral packaging plasmid is pMD2.G, psPAX2, Lenti-packaging Mix, pCMV-dR8.91, pCMV-dR8.74, pLP1, pLP2, or pVSV-G.

17. The method of claim 15, wherein, The packaging cells are 293T cells or 293FT cells.

18. A system for detecting the ADCC effect of an anti-NGF antibody, wherein, The system comprises: The recombinant CHO cell of any one of claims 1-6, and Effector cells.

19. The system of claim 18, wherein, The effector cells are effector T cells, NK cells, phagocytes, eosinophils, or basophils.

20. The system of claim 19, wherein, The effector cells are Jurkat-NFAT-Luciferase-CD16 cells or primary NK cells.

21. The system of any one of claims 18-20, wherein, The effector cells and the recombinant CHO cells have an effector-to-target ratio of 1:1 to 1:20 in number.

22. The system of any one of claims 18-20, wherein, The system further comprises a luciferase substrate.

23. A system for detecting the CDC effect of an anti-NGF antibody, wherein, The system comprises: The recombinant CHO cell of any one of claims 1-6, and Complement.

24. The system of claim 23, wherein, The complement is complement serum containing complement proteins C1-C9.

25. The system of claim 23 or 24, wherein, The system further comprises a luciferase substrate.

26. Use of the recombinant CHO cell of any one of claims 1-6 in detecting ADCC effect or CDC effect of an anti-NGF antibody.

27. A method of detecting the ADCC effect of an anti-NGF antibody, wherein, The method comprises incubating the recombinant CHO cell of any one of claims 1-6 as target cells with an antibody to be tested and effector cells.

28. The method of claim 27, wherein, The effector cells and the target cells have an effector-to-target ratio of 1:1 to 1:20 in number.

29. The method of claim 27 or 28, wherein, The method further comprises adding a luciferase substrate to the system after incubation.

30. A method of detecting the CDC effect of an anti-NGF antibody, wherein, The method comprises incubating the recombinant CHO cell of any one of claims 1-6 as target cells with an antibody to be tested and complement.

31. The method of claim 30, wherein, The complement is complement serum containing complement proteins C1-C9.

32. The method of claim 30 or 31, wherein, The method further comprises adding a luciferase substrate to the system after incubation.

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