Cell culture technology for improving antibody ADCC activity

Abstract

The present invention discloses a cell culture process for effectively improving the ADCC activity of an antibody, comprising: i) adding mammalian cells expressing a monoclonal antibody or a fragment thereof and a basal culture medium to a bioreactor, and setting the culture temperature of the bioreactor to t 1 ii) after a period of cultivation, feeding the feed medium to the bioreactor; and iii) after a period of cultivation, lowering the culture temperature of the bioreactor to t 2 This process can significantly improve the ADCC activity of antibodies while increasing the viability at harvest, and the process has the characteristics of good stability, strong operability, and small batch-to-batch differences. It is easy to be applied to large-scale CHO cell expression of monoclonal antibodies, especially industrial production of monoclonal antibodies.

Description

Technical Field

[0001] The invention discloses a cell culture process, and more specifically relates to a cell culture process for expressing monoclonal antibodies using CHO cells, which can improve the ADCC activity of the monoclonal antibodies and belongs to the field of biological products. Background Art

[0002] Biotechnology drugs, especially monoclonal antibody drugs, have gained high market recognition after listing because of their targeted and tolerable treatment, improving patients' quality of life and prolonging their lives. Since the first monoclonal antibody drug Muromonab CD3 was approved by the FDA for listing in 1986, antibody drugs have made rapid progress in the clinical treatment of malignant tumors, autoimmune diseases, cardiovascular diseases, organ transplant rejection and infections, leading the second wave of biopharmaceutical products. As of May 2018, Europe and the United States have approved more than 80 therapeutic antibody drugs. The average growth rate of global antibody drug sales from 2006 to 2015 was as high as 31.65%. It is predicted that the global share of biological drugs in the total pharmaceutical market will exceed 35% in 2020. The research and development of antibody drugs in my country started late, but in recent years, the antibody field has received more and more attention from the world, and the research and development of antibody drugs has been included in the key research projects.

[0003] Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the important mechanisms of monoclonal antibodies. After the monoclonal antibody targets and binds to the surface molecules of tumor cells, the effector cells bind to the monoclonal antibody Fc fragment through the Fc receptor (FcγR) on their surface, activating a series of signaling pathways, thereby releasing a series of inflammatory or cytotoxic immunoregulatory factors, including cytokines, chemokines, proteases and reactive oxygen molecules, which have a killing effect on tumor cells, thereby exerting the anti-tumor activity of the monoclonal antibody drug.

[0004] As an important indicator of post-translational modification of proteins, the glycosylation of the Fc segment of monoclonal antibodies plays a very important role in the efficacy (such as ADCC) and pharmacokinetics (half-life) of antibodies. At present, some methods have been reported or applied to change the level or type of glycosylation and improve the ADCC activity of antibodies, such as adding mannosidase inhibitors (Kifunensine) to change the glycoform of monoclonal antibodies to high mannose types, or reducing fucose modification through genetic engineering. However, changing the glycosylation type will affect the safety and efficacy of monoclonal antibodies accordingly. For example, high mannose types will accelerate the clearance of antibodies in the body, thereby affecting pharmacokinetics (shortening half-life) and may produce immunogenicity. The genetic engineering method requires the reconstruction of cell lines, which is equivalent to redeveloping monoclonal antibody drugs, extending the drug development cycle and cost.

[0005] Therefore, in the current situation of fierce competition in the development of monoclonal antibody drugs at home and abroad, a simple and easy cell culture process is urgently needed. The inventors accidentally discovered that by lowering the temperature during the cell culture process, the ADCC activity of the produced antibody can be significantly improved without affecting other biological properties, thereby improving the therapeutic effect of the product. Summary of the invention

[0006] The purpose of the present invention is to provide a production process for effectively improving the ADCC activity of antibodies during cell culture, so as to solve the defects existing in the existing cell culture process.

[0007] In one aspect of the present invention, a cell culture process for improving antibody ADCC activity is provided, comprising:

[0008] i) adding mammalian cells expressing a monoclonal antibody or a fragment thereof and a basal culture medium to a bioreactor, and setting the culture temperature of the bioreactor to t1;

[0009] ii) after a period of culturing, adding feed medium to the bioreactor; and

[0010] iii) After continuing the cultivation for a period of time, lowering the cultivation temperature of the bioreactor to t2.

[0011] Preferably, the mammalian cell is selected from Chinese hamster ovary (CHO) cells, NS0 cells and SP2 / 0 cells.

[0012] Preferably, the monoclonal antibody is an OX40 antibody.

[0013] Preferably, the basal culture medium is Actipro medium.

[0014] Preferably, in step i), the parameters of the bioreactor are dissolved oxygen of 40%, stirring speed of 90 rpm, and pH control range of 6.95±0.25.

[0015] Preferably, t1 in step i) is 36° C. to 37° C. More preferably, t1 is 36.5° C.

[0016] Preferably, the cell culture period is 12-16 days. More preferably, the cell culture period is 14 days.

[0017] Preferably, in step ii), the feed medium is fed daily after 2 to 4 days of cell culture. More preferably, the feed medium is fed daily from day 3 to day 13 of cell culture.

[0018] Preferably, in step iii), after 5 to 7 days of cell culture, the culture temperature of the bioreactor is reduced to t2, and the culture temperature is maintained until the end of the entire cell culture cycle.

[0019] Preferably, the fed culture medium in step ii) is Cell boost 7a and Cell boost 7b.

[0020] Preferably, the daily feed volume of Cell boost 7a is 2% of the initial culture volume, and the daily feed volume of Cell boost 7b is 0.15% of the initial culture volume.

[0021] Preferably, t2 is 33°C to 35°C, preferably 33°C.

[0022] Preferably, during the fed-batch culture process, the glucose concentration is maintained above 1 g / L.

[0023] According to any of the above aspects of the present invention, the production process provided by the present invention can simultaneously improve the cell viability at harvest and the ADCC activity of the obtained antibody. In addition, the production process provided by the present invention has strong operability, good stability, and small differences between batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Exemplary embodiments of the present invention will be described based on the following figures, in which:

[0025] Figure 1 The graphs showing the changes in cell density over culture time according to Example 1 of the present invention and Comparative Example 1 are shown.

[0026] Figure 2 The graph shows the change of cell viability with culture time according to Example 1 of the present invention and Comparative Example 1.

[0027] Figure 3 The graph shows the change of lactic acid concentration with culture time according to Example 1 of the present invention and Comparative Example 1.

[0028] Figure 4 The graphs showing the changes in glucose concentration over culture time according to Example 1 of the present invention and Comparative Example 1 are shown.

[0029] Figure 5 The graphs showing the changes in off-line pH with culture time according to Example 1 of the present invention and Comparative Example 1 are shown.

[0030] Figure 6 A graph showing the change of 2pCO2 with culture time according to Example 1 of the present invention and Comparative Example 1 is shown.

[0031] Figure 7The NH4 + Graph showing changes over time.

[0032] Figure 8 The graph shows the change of osmotic pressure (OSM) according to Example 1 of the present invention and Comparative Example 1 with culture time.

[0033] Fig. 9 The dose-dependent response curves of monoclonal antibodies A and B binding to human OX40 expressed in living cells in a FACS assay are shown, wherein HuT78 / OX40 cells were suspended in a 96-well plate with 5x10 4 Monoclonal antibodies A and B bind to the target protein on the cell surface, and FACS detection is performed according to the description of 5) in the experimental discussion of the examples herein.

[0034] Fig.10 The dose-dependent response curves of monoclonal antibodies A and B for promoting IL-2 upregulation are shown, where HuT78 / OX40, 3x10 4 cells, and HEK293 / OS8-FcγRI, 4x10 per well 4 The cells and serially diluted monoclonal antibodies A and B were co-cultured for 16 to 18 hours, and the amount of IL-2 secreted in the conditioned medium was measured using an IL-2 ELISA kit, and the fold increase of IL-2 was calculated.

[0035] Fig.11 The dose-dependent response curve of OX40 antibodies (monoclonal antibodies A and B) binding to human FcγRIIIA expressed by living cells in a FACS assay is shown, wherein NK / CD16 cells were suspended in a 96-well plate with 5×10 cells per well. 4 OX40 antibody binds to FcγRIIIA (CD16a) on the cell surface, and FACS detection is performed according to the description of 7) in the experimental discussion of the example.

[0036] Fig. 12A -C shows the dose-dependent response curve of the cytotoxic activity of OX40 antibody in ADCC assay, in which NK / CD16 cells (3×10 per well) were plated in 96-well U-bottom plates. 4 ) and BW / OX40 (1×10 5 The cells were co-cultured for 5 to 5.5 hours. The release of LDH (lactate dehydrogenase) in the culture supernatant was detected to analyze the cytotoxic activity of monoclonal antibodies A and B. DETAILED DESCRIPTION

[0037] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] For example, the term "bioreactor" refers to a container for culturing cells. The bioreactor can be of any size as long as it can be used to culture cells.

[0039] The term "initial cell culture medium" refers to the cell culture medium at the beginning of the cell culture cycle. The term "initial culture volume" is the culture volume after inoculating cells in the basal medium.

[0040] The term "antibody" refers to any immunoglobulin or fragment thereof, and encompasses any polypeptide comprising an antigen binding site. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, bispecific, trispecific, and in vitro generated antibodies. Antibody fragments include Fab, F(ab')2, Fv, scFv, Fd, dAb, which can retain antigen binding function. Typically, such fragments comprise an antigen binding domain. In a preferred embodiment of the invention, the antibody is selected from: monoclonal and polyclonal antibodies. In a more preferred embodiment, the antibody is a monoclonal antibody.

[0041] The terms "full length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody in substantially intact form, rather than an antibody fragment as defined below. The term specifically refers to an antibody whose heavy chain comprises an Fc region.

[0042] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0043] "Human antibody" refers to an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991)).

[0044] As used herein, the term "titer" refers to the total amount of recombinantly expressed antibody produced by cell culture divided by a given amount of culture medium volume. Titer is usually expressed as units of milligrams of antibody per milliliter of culture medium. Titer can be expressed or evaluated in terms of relative measurements, such as comparing the percentage increase in titer when a protein product is obtained under different culture conditions.

[0045] Monoclonal antibodies can be obtained from the bioreactor using methods commonly used by those skilled in the art, including separation by immunoaffinity or ion exchange columns.

[0046] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0047] Example

[0048] Experimental methods

[0049] NOVA was used to perform biochemical analysis (Gln / Glu / Glucose / Lac / NH4+ / Na+ / K+) on the cell culture medium every day. A blood gas analyzer was used to analyze pH, pCO2, and pO2. The freezing point method was used to measure osmotic pressure. A Vi-cell XR counter was used to count cells. FACS was used to measure the binding activity of monoclonal antibodies to natural OX40 or the binding activity of monoclonal antibodies to Fcγ receptors. ELISA was used to measure IL-2 secretion. A lactate dehydrogenase detection kit was used to detect ADCC activity.

[0050] Experimental instruments

[0051] If no specific conditions are specified below, the normal conditions or the conditions recommended by the manufacturer shall be followed.

[0052] NOVA biochemical analyzer: purchased from Nova Biomedical

[0053] Blood gas analyzer: purchased from Siemens

[0054] Vi-cell XR cell counter: purchased from Beckman Coulter

[0055] FACS flow cytometer: Guava easyCyte 6HT-2L, purchased from Millipore, USA

[0056] ELISA plate reader model SpectraMax Paradigm, purchased from Molecular Devices, USA

[0057] Lactate dehydrogenase detection kit: Promega, G780

[0058] Example 1

[0059] The cells used are CHO cells (constructed by BeiGene), which contain expression vectors and can secrete monoclonal antibodies. Take a cell (constructed by BeiGene) from the cell bank and oscillate rapidly in a 37°C water bath to melt it quickly. The melting time is controlled within 3 minutes. The Actipro basal medium (containing 25nm MSX) purchased from GE is used for recovery. The recovery density is controlled in the range of 0.5±0.1*10^6cells / mL. It is placed in a shaker with a speed of 130rpm, a CO2 concentration of 5% (volume), and 36.5°C for 3 days. When the density reaches 2.5*10^6cells / mL, the passage expansion begins, and the inoculation density is 0.4±0.1*10^6cells / mL. The passage expansion is carried out several times in succession, and then it is inoculated into the XDR10 reactor (purchased from GE) for Fed-batch culture, and the temperature is reduced.

[0060] The parameters of the XDR10 reactor were set as follows: the temperature from the start of culture to the end of the 6th day (i.e., D0-D6) was 36.5°C, the temperature from the 7th day to the 14th day (i.e., D7-D14) was 33.0°C, the rotation speed was 90rpm, the pH range was 6.95±0.25, the DO was 40%, and the inoculation density was 0.5±0.2*10^6cells / mL. When cultured to the 3rd day (D3), feed medium Cell boost 7a (purchased from GE) and Cell boost 7b (purchased from GE) were added. 7a was supplemented with 2% (v / v) of the initial culture volume every day, and 7b was supplemented with 0.15% (v / v) of the initial culture volume every day. Sugar was supplemented according to sugar consumption during the process, and the glucose concentration was maintained above 1g / L during the entire culture process. The culture period was 14 days. After the culture was completed, monoclonal antibody A was obtained by affinity chromatography purification.

[0061] Comparative Example 1

[0062] The cells used are CHO cells (constructed by BeiGene), which contain expression vectors and can secrete monoclonal antibodies. Take a cell (constructed by BeiGene) from the cell bank and oscillate it rapidly in a 37°C water bath to melt it quickly. The melting time must be controlled within 3 minutes. The Actipro basal medium (containing 25um MSX) purchased from GE is used for recovery. The recovery density is controlled in the range of 0.5±0.1*10^6cells / mL. It is placed in a shaker at a speed of 130rpm, a CO2 concentration of 5% (volume), and 36.5°C for 3 days. When the density reaches 2.5*10^6cells / mL, start subculture and amplification, and the inoculation density is 0.4±0.1*10^6cells / mL. Perform several subcultures and amplifications in succession, and then inoculate it into the XDR10 reactor for Fed-batch culture.

[0063] The parameters of the XDR10 reactor were set as follows: the temperature of the entire culture cycle was 36.5°C, the rotation speed was 90rpm, the pH range was 6.95±0.25, the DO was 40%, and the inoculation density was 0.5±0.2*10^6cells / mL. When cultured to D3, feed medium Cell boost 7a (purchased from GE) and Cell boost7b (purchased from GE) were added. 7a was supplemented with 2% (v / v) of the initial culture volume every day, and 7b was supplemented with 0.15% (v / v) of the initial culture volume every day. Sugar was supplemented according to sugar consumption during the process, and the glucose concentration was maintained above 1g / L during the entire culture process. The culture cycle was 14 days. After the culture, monoclonal antibody B was obtained by affinity chromatography purification.

[0064] The above experimental method was used to test Example 1 and Example 2. The experimental results (such as cell growth, cell metabolism, expression level, quality, ADCC activity, etc.) are as follows: Figure 1-1 2 and Table 1-10.

[0065] 1) Evaluation results of cell growth

[0066] Figure 1 and 2 The results of cell growth in Example 1 and Comparative Example 1 are shown, wherein Figure 1 It can be seen that the VCD of Example 1 is slightly lower, which is 29.74*10 6 cells / mL, while the VCD of Comparative Example 1 was relatively high, which was 34.07*10 6 cells / mL; and Figure 2 It can be seen that the cell viability in the later stage of Example 1 is maintained well, with a viability of more than 85%; while the cell viability in the later stage of Comparative Example 1 decreases rapidly, with a viability of 64.5%.

[0067] 2) Evaluation results of cell metabolism

[0068] Figures 3 to 8 The results of cell metabolism in Example 1 and Comparative Example 1 are shown. Figures 2 to 8 It shows that cooling or not has an effect on the glucose concentration and NH4 + The concentration, PCO2, and OSM change trends had basically no significant effect, but had a certain effect on lactic acid metabolism and pH metabolism. Using the non-cooling process of Comparative Example 1, lactic acid would rise in the late stage of cultivation, and pH would drop to below 6.80, while the cooling process in Example 1 would not cause lactic acid to rise in the late stage of cultivation, and pH would be maintained at around 7.0.

[0069] 3) Evaluation results of cell expression

[0070] Table 1: Titer results of Example 1 and Comparative Example 1

[0071]

[0072] Note: D11, D12, D13 and D14 represent the 11th, 12th, 13th and 14th day respectively. The same applies below.

[0073] 4) Evaluation results of monoclonal antibody quality

[0074] Table 2: SEC results of Example 1 and Comparative Example 1

[0075]

[0076]

[0077] Table 3: CE-SDS (NR) results of Example 1 and Comparative Example 1

[0078]

[0079] Table 4: IEC results of Example 1 and Comparative Example 1

[0080]

[0081] Table 5: Glycoform distribution results of Example 1 and Comparative Example 1

[0082]

[0083] 5) Evaluation of the antigen binding activity of monoclonal antibodies A and B

[0084] The binding of monoclonal antibodies to the antigen OX40 is the first step in exercising all functions. Conventional FACS was used to compare the binding activity of monoclonal antibodies A and B to natural OX40. The stable cell line HuT78 / OX40 used in the FACS analysis uses the human leukemia T cell line HuT78 (ATCC) as a host cell to express full-length human OX40. HuT78 / OX40 cells were seeded into 96-well plates and incubated with monoclonal antibodies A and B for 1 hour. A fluorescently labeled anti-human Fab antibody was used to detect the antigen binding activity of monoclonal antibodies A and B on the cell surface. If monoclonal antibody B is used as a reference and monoclonal antibody A is used as a test sample, the relative difference RV% of the peak value is calculated as: (peak value of the test antibody / peak value of the reference sample) x 100%; EC 50 The relative difference RV% was calculated as: (EC 50 / EC of the sample to be tested 50 ) x 100%. In this detection method, when EC 50 When the relative difference RV% is between 70% and 130%, antibodies from different batches are comparable. Fig. 9 The results are shown in Table 6. The results show that the binding of monoclonal antibodies A and B to OX40 is dose-dependent, and the EC 50 All reached nanomolar concentrations. That is, EC 50 (monoclonal antibody B) = 411 ng / mL (2.74 nM), EC 50 (monoclonal antibody A) = 402 ng / mL (2.68 nM), the difference in RV is only 2%. In the case where the peak values ​​are extremely similar (RV difference is 1%), it indicates that the antigen binding activities of monoclonal antibodies A and B are comparable.

[0085] Table 6: Activity analysis results of monoclonal antibodies A and B binding to cell surface OX40

[0086] Antibody Peak value (MFI*) RV%,Peak <![CDATA[EC 50 (μg / mL)]]> <![CDATA[RV%,EC 50 ]]> Monoclonal antibody B 1103 100％ 0A11 100％ Monoclonal Antibody A 1096 99％ 0.402 102％

[0087] *, MFI: mean fluorescence intensity

[0088] 6) Comparison of anti-OX40 antibody DS promoting functional activity (IL-2 detection)

[0089] Anti-OX40 antibodies (monoclonal antibodies A and B) can specifically bind to human OX40, activate downstream OX40 signal transduction, and initiate immune activity of immune cells (Aspeslagh et al., 2016; Voo et al., 2013). This cell-based functional analysis method uses the increase in IL-2 secretion as an indicator of the intensity of the stimulated immune response. In this method, a T cell adapter, i.e., membrane-anchored chimeric antibody OS8 (see US Patent 8735553) and Fc gammareceptor I (FcγRI) were co-transfected into HEK293 cells (ATCC) to establish a stable cell line HEK293 / OS8-FcγRI, which was co-cultured with the aforementioned human T cell line expressing OX40 (HuT78 / OX40) and monoclonal antibodies A and B for 16 to 18 hours, and the supernatant was taken and the IL-2 content was measured using an IL-2 ELISA kit (Invitrogen, Cat. No. 88-7025). This test uses the increase in IL-2 to quantitatively compare the activity of different OX40 antibodies in promoting immune response and leading to upregulation of TCR signal. If monoclonal antibody B is used as the reference and monoclonal antibody A is used as the test sample, the relative difference RV% of the increase in IL-2 is calculated as: (peak value of the test antibody / peak value of the reference sample) x 100%; EC 50 The relative difference RV% was calculated as: (EC 50 / EC of the sample to be tested 50 ) x 100%. In this detection method, when EC 50 When the relative difference RV% is between 70% and 130%, antibodies from different batches are comparable. Fig.10 And as shown in Table 7.

[0090] The results showed that monoclonal antibodies A and B were dose-dependent with IL-2 production. 50 All reached sub-nanomolar concentrations. That is, EC 50 (monoclonal antibody B) = 50.26 ng / mL (0.335 nM), EC 50 (monoclonal antibody A) = 52.76 ng / mL (0.352 nM), the difference in RV is only 5%. In the case of similar fold increase in IL-2 (RV% difference = 6%), it indicates that the binding activities of the two different batches of DS are comparable.

[0091] Table 7: Functional analysis results of monoclonal antibodies A and B promoting IL-2 upregulation

[0092]

[0093] 7) Comparison of the binding activity of anti-OX40 antibodies (i.e., monoclonal antibodies A and B) to Fcγ receptors

[0094] Antibodies bind to cell surface target proteins and then cross-link to Fcγ receptors (FcγRs) expressed by effector cells, which is the basis for some important cellular functions such as ADCC. Monoclonal antibodies A and B are IgG1 wild-type and have significantly higher binding affinity to FcγR, especially to FcγRI and FcγRIIIA. Typical ADCC is initiated by the activation of natural killer (NK) cells by antibodies that bind to FcγRIIIA (also known as CD16a). The binding activity of monoclonal antibodies A and B to native CD16a on the surface of NK cells was compared using conventional FACS. In this method, CD16a (V 158 NK92MI cells (ATCC) were co-transduced with expression plasmids of the 100 allele and FcRγ gene to generate the NK92MI / CD16aV (NK / CD16) cell line. NK / CD16 cells were seeded into 96-well plates and incubated with monoclonal antibodies A and B for 1 hour. Fluorescently labeled anti-human Fab antibodies were used to detect the binding activity of OX40 antibodies to CD16 on the cell surface. If monoclonal antibody B is used as a reference and monoclonal antibody A is used as a test sample, the relative difference RV% of the peak value is calculated as: (peak value of the test antibody / peak value of the reference sample) x 100%; EC 50 The relative difference RV% was calculated as: (EC 50 / EC of the sample to be tested 50 ) x 100%. The result is Fig.11 and as shown in Table 8.

[0095] The results showed that the binding of monoclonal antibodies A and B to FcγRIIIA was dose-dependent. 50 (monoclonal antibody B) = 9.8 μg / mL (65 nM), EC 50 (monoclonal antibody A) = 5.844 μg / mL (39 nM), the difference in RV is 68%, which is obviously significant. When the peak values ​​are similar (RV difference is 4%), the activity of monoclonal antibody A in binding to FcγRIIIA is significantly higher than that of monoclonal antibody B.

[0096] Table 8: Results of analysis of the activity of monoclonal antibodies A and B binding to cell surface FcγRIIIA

[0097] Antibody Peak value*(MFI) RV%,Peak <![CDATA[EC 50 (μg / mL)]]> <![CDATA[RV%,EC 50 ]]> Monoclonal antibody B 2225 100％ 9.8 100％ Monoclonal Antibody A 2311 104％ 5.844 168％

[0098] *, taken from the peak value of the four-parameter fitting curve of Prism software

[0099] 8) Comparison of ADCC activity of anti-OX40 antibodies (i.e., monoclonal antibodies A and B)

[0100] As mentioned above, ADCC (Antibody-dependent cell-mediated cytotoxicity) is initiated when antibodies bind to cell surface target proteins and then cross-link to Fcγ receptors (FcγRs) expressed by effector cells. Typical ADCC is initiated by NK cells activated by antibodies that bind to FcγRIIIA (also known as CD16a), resulting in the release of LDH (lactate dehydrogenase) by the cells. In this method, the aforementioned NK92MI / CD16aV (NK / CD16) cells were used as effector cells, and the mouse T cell line BW5143.7 (ATCC) stably transfected with human OX40 (abbreviated as BW / OX40) was used as the target cell. NK / CD16 cells (3×10 per well) were plated in a 96-well U-bottom plate. 4 ) and BW / OX40 cells (1×10 5 ) were co-cultured for 5 to 5.5 hours. The CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, G780) was used to detect the release of LDH (lactate dehydrogenase) in the culture supernatant to analyze the cytotoxicity of the anti-OX40 antibody and expressed as a percentage. If monoclonal antibody B is used as the reference and monoclonal antibody A is used as the test sample, the relative difference RV% of the ADCC activity peak is calculated as: (ADCC peak% of the test antibody / peak% of the reference sample) x 100%; EC 50 The relative difference RV% was calculated as: (EC 50 / EC of the sample to be tested 50 ) x 100%.

[0101] The results of the three tests are as follows Fig. 12A As shown in Figure 3-C, both monoclonal antibodies A and B showed dose-dependent ADCC activity. The peak values ​​of ADCC and EC 50 The results are listed in Table 9, and the average analysis of the three tests is shown in Table 10. The results show that the EC values ​​of monoclonal antibodies A and B in the three tests were 50 The average difference in RV is as high as 107%. That is, the ADCC activity of monoclonal antibody A is twice as high as that of monoclonal antibody B. It is generally believed that in ADCC analysis, when the ADCC peak values ​​are extremely close (difference of ± 2%), this difference is significant.

[0102] It has been reported that anti-OX40 antibodies promote anti-tumor immune responses that depend on their effector functions, effectively eliminating OX40+Treg cells in tumors through ADCC / ADC, leading to tumor regression and improved patient survival ( Aspeslagh et al., 2016 ; Bulliard et al., 2014 ; Jacquemin et al., 2015 ; Marabelle et al., 2013 ). Antibodies that show high ADCC activity in cell-based assays are obviously more likely to have higher anti-tumor potential.

[0103] Table 9: Results of ADCC activity of monoclonal antibodies A and B in three tests

[0104]

[0105] Table 10: Comparison of the mean relative ADCC activities of monoclonal antibodies A and B in three tests

[0106]

[0107] The present invention has been described in detail above by general description, specific implementation mode and test. Any modification or improvement made without departing from the spirit of the present invention shall fall within the scope of protection claimed by the present invention. Any document cited in this disclosure is incorporated herein by reference.

Claims

1. A method for effectively improving the ADCC activity of OX40 monoclonal antibody or its fragment in a cell culture process, comprising: i) adding CHO cells expressing an OX40 antibody or a fragment thereof and a basal culture medium to a bioreactor, and setting the culture temperature of the bioreactor to t1; ii) after a period of culturing, adding feed medium to the bioreactor; and iii) after continuing the culture for a period of time, lowering the culture temperature of the bioreactor to t2; The basal medium is Actipro medium; wherein t1 in step i) is 36°C to 37°C, and t2 in step iii) is 33°C to 35°C; And in step i), the parameters of the bioreactor are dissolved oxygen of 40%, stirring speed of 90 rpm, and pH control range of 6.95±0.25; The cell culture cycle is 12-16 days; in step ii), after 2 to 4 days of cell culture, feed medium is added every day; in step iii), after 5 to 7 days of cell culture, the culture temperature of the bioreactor is reduced to t2, and the culture temperature is maintained until the end of the entire cell culture cycle; And the feed medium added in step ii) is selected from Cell boost 7a and Cell boost 7b.

2. The method according to claim 1, wherein t1 in step i) is 36.5°C.

3. The method according to claim 1 or 2, wherein the cell culture period is 14 days.

4. The method according to claim 1 or 2, wherein in step ii), feed medium is added every day from day 3 to day 13 of cell culture.

5. The method according to claim 1 or 2, wherein the daily feed volume of Cell boost 7a is 2% of the initial culture volume, and the daily feed volume of Cell boost 7b is 0.15% of the initial culture volume.

6. The method according to claim 1, wherein t2 in step iii) is 33°C.

7. The method according to claim 1, wherein during the fed-batch culture process, the glucose concentration is maintained above 1 g / L.

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