Methods for determining the biological activity of TIGIT antibodies

Abstract

The present invention relates to a method for determining the biological activity of TIGIT antibodies. The method for determining the biological activity of TIGIT antibodies comprises the following steps: constructing NK cells expressing TIGIT in vitro as effector cells; co-culturing TIGIT antibodies of different concentrations with effector cells and tumor cells labeled with a first fluorescent marker at an effector-target ratio (0.2-1.5):1 for 6-24 hours, using another fluorescent dye that can be distinguished from the first fluorescent marker to label apoptotic tumor cells, and calculating the killing rate of effector cells against tumor cells under the action of different concentrations of TIGIT antibodies based on the labeling results, and fitting the relationship curve between TIGIT antibody concentration and killing rate to determine the biological activity of TIGIT antibodies. In the above method, the expression level of TIGIT is stable, and it is easier to detect the biological activity of TIGIT antibodies with higher sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, and in particular to a method for determining the biological activity of TIGIT antibodies. Background Art

[0002] Since its first report in 2009, the T cell immunoreceptor with both Ig and ITIM domains (TIGIT), as an immune checkpoint for T and NK cells, has rapidly become a focus of widespread attention and drug development for major pharmaceutical companies. Genentech's drug is already in Phase III clinical trials, while Bristol-Myers Squibb is also in Phase I clinical trials, with a large number of biopharmaceutical companies both domestically and internationally following suit. Whether in the early screening phase or the later CMC (Chemical, Manufacturing, and Control) phase of antibody drug development targeting this target, we are particularly interested in the in vitro biological activity of these drugs based on MOA.

[0003] In basic research, we know that TIGIT is expressed in lymphocytes, especially in effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells and natural killer (NK) cells. CD155 (also known as PVR, Necl5 or Tage4) is a high-affinity ligand for TIGIT. Once CD155 expressed on the surface of the tumor binds to TIGIT on the surface of NK and T cells, the killing effect of NK and T cells on tumor cells will be inhibited. The purpose of TIGIT antibodies is to block the binding of TIGIT and CD155, thereby relieving the functional inhibitory effect of this signal on T or NK cells and re-killing tumor cells. Therefore, it is particularly important to develop a method that can sensitively detect the biological activity of TIGIT antibodies in vitro.

[0004] Traditional in vitro functional assays for TIGIT antibodies use natural NK cells as target cells and LDH as an indicator to evaluate the biological activity of TIGIT antibodies. However, traditional methods have the following problems: (1) natural NK cells are difficult to obtain from healthy donors, and TIGIT expression levels vary between donors; (2) LDH-based assays often fail to detect the biological activity of TIGIT antibodies. Summary of the Invention

[0005] Based on this, it is necessary to provide a method for determining the biological activity of TIGIT antibodies, which can improve the situation where natural NK cells are difficult to obtain and the expression level of TIGIT is unstable. At the same time, compared with the traditional LDH method, this method is easier to detect the biological activity of TIGIT antibodies and has higher sensitivity.

[0006] A method for determining the biological activity of a TIGIT antibody comprises the following steps:

[0007] In vitro construction of TIGIT-expressing NK cells as effector cells;

[0008] Co-culturing different concentrations of TIGIT antibodies with the effector cells and tumor cells labeled with the first fluorescent marker for 6 hours to 24 hours, respectively, wherein the ratio of the number of the effector cells to the number of the tumor cells labeled with the first fluorescent marker is (0.2-1.5):1;

[0009] The cells after co-culture are labeled with a second fluorescent marker that is distinguishable from the first fluorescent marker; wherein the first fluorescent marker is a fluorescent marker for detecting cell proliferation, and the second fluorescent marker specifically labels apoptotic cells;

[0010] The killing rate of the effector cells on the tumor cells labeled with the first fluorescent marker under the action of different concentrations of TIGIT antibodies is calculated according to the labeling results, and the biological activity of the TIGIT antibody is determined based on a fitting curve of the TIGIT antibody concentration and its corresponding killing rate.

[0011] The above-mentioned method for determining the biological activity of TIGIT antibodies improves the situation where natural NK cells are difficult to obtain and the expression level of TIGIT is unstable by constructing NK cells expressing TIGIT in vitro. At the same time, the effector-target ratio is (0.2-1.5):1 and the co-culture time is 6h-24h, so that the effector cells have a suitable detection window for killing tumors and can be detected. Compared with the traditional method of detecting LDH, it is easier to determine the biological activity of TIGIT antibodies and has higher sensitivity.

[0012] In one embodiment, the step of calculating, based on the labeling results, the killing rate of the effector cells against the tumor cells labeled with the first fluorescent marker under the action of different concentrations of the TIGIT antibody comprises:

[0013] Using flow cytometry to count the number of tumor cells labeled with the first fluorescent marker and the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker under the action of different concentrations of TIGIT antibodies; and

[0014] The killing rate of the effector cells against the tumor cells labeled with the first fluorescent marker under the action of different concentrations of TIGIT antibodies is calculated, wherein the killing rate is the ratio of the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker to the number of tumor cells labeled with the first fluorescent marker.

[0015] In one embodiment, the tumor cell is the osteosarcoma cell line U2OS.

[0016] In one embodiment, the ratio of the number of the effector cells to the number of the tumor cells labeled with the first fluorescent marker is (1-1.5):1; and the co-culture time is 12h-24h.

[0017] In one embodiment, the ratio of the number of the effector cells to the number of the tumor cells labeled with the first fluorescent marker is 1.5:1, and the co-culture time is 12 hours;

[0018] Alternatively, the ratio of the number of the effector cells to the number of the tumor cells labeled with the first fluorescent marker is 1:1, and the co-culture time is 16 hours;

[0019] Alternatively, the ratio of the number of the effector cells to the number of the tumor cells labeled with the first fluorescent marker is 1:2, and the co-culture time is 8 hours or 24 hours;

[0020] Alternatively, the ratio of the number of the effector cells to the number of the tumor cells labeled with the first fluorescent marker is 1:5, and the co-culture time is 24 hours.

[0021] In one embodiment, the concentration of the TIGIT antibody solution is 1×10 -6 μg / mL~10μg / mL.

[0022] In one embodiment, the first fluorescent marker is 5(6)-carboxyfluorescein diacetate succinimidyl ester, and the second fluorescent marker is propidium iodide.

[0023] In one embodiment, in the co-culture system, the density of the tumor cells labeled with the first fluorescent marker is 1×10 5 / mL~2×10 5 pieces / mL.

[0024] In one embodiment, the NK cells are NK92 cells.

[0025] In one embodiment, the step of constructing NK cells expressing TIGIT in vitro comprises:

[0026] The nucleic acid fragment for expressing TIGIT is transfected into NK cells via lentivirus and expressed to prepare NK cells expressing TIGIT. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the vector map of the human TIGIT gene in Example 1;

[0028] Figure 2 The expression of TIGIT in NK92-TIGIT cells in Example 1;

[0029] Figure 3 The PVR expression of U2OS cells in Example 1;

[0030] Figure 4 The LDH method in Example 2 was used to detect the killing of U2OS cells by NK92-TIGIT cells mediated by TIGIT antibodies;

[0031] Figures 5 to 12 The killing of U2OS cells by NK92-TIGIT cells mediated by TIGIT antibodies at different effector-target ratios and different co-culture times in Example 3;

[0032] Figure 13 This is the relationship curve between TIGIT antibody and killing rate when the effector-target ratio is 1:1 and the co-culture time is 16 hours in Example 4. DETAILED DESCRIPTION

[0033] For ease of understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive. The terms "first", "second", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0034] One embodiment of the present application provides a method for evaluating the biological activity of a TIGIT antibody, comprising steps S100, S200, S300, and S400. Specifically:

[0035] Step S100: Constructing NK cells expressing TIGIT in vitro.

[0036] Specifically, genetic engineering technology is used to integrate a nucleic acid fragment for expressing TIGIT into NK cells, causing them to express TIGIT. Alternatively, the nucleic acid fragment for expressing TIGIT is transfected into NK cells via a lentivirus and expressed to prepare NK cells expressing human TIGIT. More specifically, the packaging vector is co-transfected with a transfer vector carrying a nucleic acid fragment for expressing TIGIT into packaging cells to prepare a lentivirus carrying a nucleic acid fragment for expressing TIGIT, and then the lentivirus carrying a nucleic acid fragment for expressing TIGIT is used to transfect NK cells to prepare NK cells expressing TIGIT.

[0037] In this embodiment, the NK cell is an NK92 cell. TIGIT is human TIGIT. Correspondingly, the TIGIT antibody is a human TIGIT antibody.

[0038] Step S200: co-culture TIGIT antibodies of different concentrations with effector cells and tumor cells labeled with a first fluorescent marker.

[0039] Tumor cells labeled with a first fluorescent marker are target cells. When TIGIT antibodies, effector cells, and tumor cells labeled with a first fluorescent marker are co-cultured, the TIGIT antibodies and TIGIT on the surface of the effector cells will block the binding of PVR on the surface of the tumor cells with TIGIT on the surface of the effector cells, thereby releasing the effector cells to kill tumor cells. Therefore, the half-maximal effect concentration (EC) of the TIGIT antibody can be calculated by the killing rate of the effector cells against the tumor cells labeled with the first fluorescent marker after the addition of the TIGIT antibody. 50 ), thereby determining the biological activity of TIGIT antibodies.

[0040] Specifically, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is (0.2-1.5):1, and the co-culture time is 8-24 hours. By setting the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker to (0.2-1.5):1 and the co-culture time to 8-24 hours, the above method has a suitable detection window.

[0041] Furthermore, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is (1-1.5):1; and the co-culture time is 12 hours to 24 hours.

[0042] In one embodiment, the ratio of the effector cells to the tumor cells labeled with the first fluorescent marker is 1.5:1, and the co-culture time is 12 hours.

[0043] In one embodiment, the ratio of the effector cells to the tumor cells labeled with the first fluorescent marker is 1:1, and the co-culture time is 12 hours or 16 hours.

[0044] In one embodiment, the ratio of the effector cells to the tumor cells labeled with the first fluorescent marker is 1:2, and the co-culture time is 8 hours.

[0045] In one embodiment, the ratio of the effector cells to the tumor cells labeled with the first fluorescent marker is 1:2, and the co-culture time is 24 hours.

[0046] In one embodiment, the ratio of the effector cells to the tumor cells labeled with the first fluorescent marker is 1:5, and the co-culture time is 24 hours.

[0047] Optionally, the TIGIT antibody at different concentrations is a TIGIT antibody at gradient concentrations. For example, in some embodiments, the TIGIT antibody at different concentrations is a TIGIT antibody solution diluted 5-fold or 10-fold gradient.

[0048] Alternatively, different concentrations of TIGIT antibodies were added at a concentration of 1 × 10 -6 The range is μg / mL~10μg / mL.

[0049] Optionally, in the co-culture system, the density of tumor cells labeled with the first fluorescent marker is 1×10 5 / mL~2×10 5 pieces / mL.

[0050] Step S300: labeling the cells after the co-culture with a second fluorescent marker that can be distinguished from the first fluorescent marker.

[0051] Specifically, the first fluorescent marker is a fluorescent marker for cell proliferation detection, used to mark living cells; the second fluorescent marker specifically marks apoptotic cells.

[0052] In this embodiment, the first fluorescent marker is 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), and the second fluorescent marker is propidium iodide (PI). CFSE can easily penetrate the cell membrane, covalently bind to intracellular proteins in living cells, and release green fluorescence after hydrolysis, which is used to label living cells; PI is a nuclear staining reagent that can stain DNA. It is an analog of ethidium bromide and releases red fluorescence after intercalation into double-stranded DNA, which is used to label apoptotic cells. Therefore, the different fluorescent signals on tumor cells can be used to distinguish whether the tumor is apoptotic and count the cells.

[0053] It is understandable that, in other embodiments, the combination of the first fluorescent marker and the second fluorescent marker is not limited to the above-mentioned CFSE and PI, and may be other combinations as long as they can distinguish between apoptotic tumor cells and living tumor cells.

[0054] Optionally, the tumor cell is an osteosarcoma cell line U2OS. The osteosarcoma cell line U2OS highly expresses PVR, which is more conducive to the release of the effector cell killing effect of the TIGIT antibody on tumor cells.

[0055] In some embodiments, the method further includes the step of preparing tumor cells labeled with a first fluorescent marker, comprising: mixing the tumor cells with the first fluorescent marker; and removing unreacted first fluorescent marker to prepare tumor cells labeled with the first fluorescent marker. It is understood that in other embodiments, the step of preparing tumor cells labeled with the first fluorescent marker may be omitted; in such cases, commercially available tumor cells labeled with the first fluorescent marker may be purchased directly.

[0056] Step S400: Counting the killing rate of effector cells against target cells under the action of different concentrations of TIGIT antibodies, and determining the biological activity of the TIGIT antibody based on a fitting curve of the TIGIT antibody concentration and its corresponding killing rate.

[0057] Specifically, the killing rate is the ratio of the number of tumor cells marked with the first fluorescent marker and the second fluorescent marker (i.e., tumor cells that have undergone apoptosis) to the number of tumor cells marked with the first fluorescent marker (i.e., total tumor cells), that is, the proportion of tumor cells that have undergone apoptosis in the total tumor cells. Therefore, by counting the number of tumor cells marked with the first fluorescent marker and the number of tumor cells marked with the first fluorescent marker and the second fluorescent marker under the action of different concentrations of TIGIT antibodies, the killing rate of effector cells against target cells under the action of different concentrations of TIGIT antibodies can be calculated. EC can be determined based on the TIGIT antibody concentration and its corresponding killing rate fitting curve. 50 , thereby determining the biological activity of TIGIT antibodies.

[0058] Optionally, flow cytometry is used to count the number of tumor cells labeled with the first fluorescent marker and the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker. It is understood that in other embodiments, other methods can be used to count the number of tumor cells labeled with the first fluorescent marker and the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker, for example, using a fluorescence microscope.

[0059] The above method for determining the biological activity of TIGIT antibodies improves the defects of natural NK cells, which are difficult to obtain and the unstable expression level of TIGIT, by constructing NK cells expressing TIGIT in vitro. At the same time, the effector-target ratio is (0.2-1.5):1 and the co-culture time is 6h-24h, so that the effector cells have a suitable detection window for the tumor-killing effect and can be detected. It has been verified that compared with the traditional detection method of LDH, the above method is easier to determine the biological activity of TIGIT antibodies and has higher sensitivity. Specific embodiments

[0061] The following is a detailed description with reference to specific examples. Unless otherwise specified in the following examples, other components except unavoidable impurities are not included. The reagents and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or methods recommended by the manufacturer. In the accompanying drawings, group "T" represents a group with only target cells, that is, an experimental group with only U2OS cells; group "T+E" refers to a group with effector cells (NK92-TIGIT cells) and target cells (U2OS cells) but no TIGIT antibodies. The TIGIT antibody can be any TIGIT antibody, and the TIGIT antibodies selected in the examples herein are only exemplary and cannot be a limitation of this application.

[0062] Example 1

[0063] Construction of NK92-TIGIT

[0064] Experimental materials include: NK92 cell line (Beina Biotechnology; BNCC236019); osteosarcoma cell line U2OS (Shanghai Cell Bank SCSP-5030, referred to as U2OS cells or U2OS); Dynamis medium (Gibco; A2617502); double antibody (Gibco; 15140122); anticoagulant (Gibco; 0010057); pcDNA3. TM3.4TOPOTMTA Cloning Kit (Invitrogen A14697); 5-alpha Competent E. coli (NEB C2987I); DL2000 DNA Marker (TAKARA 3427A); Q5 High-Fidelity DNA Polymerase (NEB M0491L); AxyPrep DNA Gel Extraction Kit (AxyGEN AP-GX-50); Hind III (NEB); EcoRI (NEB); T4 DNA Ligase (TAKARA 2011A); Endotoxin-Free Plasmid Extraction Kit (TIANGEN DP117); PE-anti-human TIGIT antibody (Biolegend 372704); anti-PVR antibody (Biolegend 337602).

[0065] The instruments and equipment included: PCR instrument (Tprofessional TR20); electrophoresis apparatus (DYY-TC); gel imager (Smart Gel N); centrifuge (H1650-W); micro-vacuum thermostat (HW-8C); clean bench (SDJ series); constant temperature oscillator (H2-9211K); constant temperature water bath (HH-4A); cell culture shaker (Adolf Kuhner; ISF4-XC); high-speed centrifuge (Xiangyi, H2050R); biological safety cabinet (ESCO AC2-4S1); CO2 incubator (Sanyo MCO-18AC, Japan); cell viability analyzer (Shanghai Yiheng DK-8AX); and flow cytometer (Beckman cytoflex).

[0066] The experimental steps include:

[0067] (1) Construction of NK92 cell line stably expressing TIGIT: Construct a lentivirus containing the full-length sequence of human TIGIT (Gene ID: 201633) (the vector map containing the human TIGIT gene is as follows Figure 1 After infection, single clones were picked to obtain NK92 cell lines stably expressing TIGIT (referred to as NK92-TIGIT cells).

[0068] (2) Detection of TIGIT expression in NK92-TIGIT cells: The NK92 cell line stably expressing TIGIT obtained in step (1) was labeled with a flow cytometric fluorescent antibody and incubated at 4°C for 30 minutes to detect TIGIT expression using a flow cytometer. The results were as follows: Figure 2 shown. Figure 2 “BLANK” refers to the blank control, i.e., unmodified NK92 cells.

[0069] (3) Detection of PVR expression in U2OS cells. The results are as follows Figure 3 shown. Figure 3 “BLANK” refers to the blank control, which is a control group in which no antibody is added and only the same volume of culture medium is used instead.

[0070] Depend on Figure 2 It can be seen that according to the above experimental method, a NK92 cell line that stably and highly expresses human TIGIT was constructed, which can be used as an effector cell to detect the biological function of TIGIT antibodies. Figure 3 It can be seen that U2OS cells also stably and normally express PVR and can be used as target cells to detect the biological functions of TIGIT antibodies.

[0071] Example 2

[0072] LDH detection of TIGIT antibody-mediated NK92-TIGIT cell killing effect on U2OS cells

[0073] Experimental materials include: NK92-TIGIT cells constructed in Example 1; osteosarcoma cell line U2OS (Shanghai Cell Bank SCSP-5030); 1640 culture medium (Gibco 11875085); LDH detection kit (Biyuntian C0017); TIGIT antibody (TIGIT monoclonal antibody 22G2 in WO2016106302A1, referred to as R0223).

[0074] Instruments and equipment include: biological safety cabinet (ESCO AC2-4S1); CO2 incubator (Japan Sanyo MCO-18AC); cell viability analyzer (Shanghai Yiheng DK-8AX); multifunctional microplate reader (Molecular Devices Spectramax i3x).

[0075] The experimental steps include:

[0076] (1) Count the cultured U2OS cells, centrifuge them, and adjust the cell number to 5×10 cells using serum-free 1640 medium. 5 The cells were plated evenly on a 96-well plate at 100 μL / well and cultured overnight.

[0077] (2) After the overnight culture was stable, NK92-TIGIT cells in good culture condition (“good culture condition” in this article refers to the cells being in a state of uniform aggregation, translucent cells, clear culture medium, and viability of more than 90% by cell counter) were taken and the cell number was adjusted to 1×10 6 50 μL of the TIGIT antibody was added to the 96-well plate containing U2OS cells in step (1). -6 μg / mL, 10-fold gradient dilution), and the release of LDH was detected after 4 hours. Figure 4 shown.

[0078] Depend on Figure 4 It can be seen that the biological activity window of TIGIT antibody detected by LDH detection is low (the background is high and cannot be distinguished), and the biological activity of the antibody (EC 50 ).

[0079] Example 3

[0080] Dual fluorescence flow cytometry was used to detect the cell-killing effect of NK92-TIGIT mediated by TIGIT antibody on U2OS cells

[0081] The experimental materials included: NK92-TIGIT cells constructed in Example 1; osteosarcoma cell line U2OS (Shanghai Cell Bank SCSP-5030); 1640 culture medium (Gibco 11875085); CFSE (eBioscience 85-65-0850-84); PI (Sigma P4170-25MG); TIGIT antibody 1 (TIGIT monoclonal antibody 22G2 in WO2016106302A1, abbreviated as R0223); TIGIT antibody 2 (tiragolumab in WO2017053748A2, abbreviated as R0300); TIGIT antibody 3 (TIGIT antibody obtained by replacing the Fc region of tiragolumab in WO2017053748A2 with the Fc region of mIgG1, abbreviated as R0300-CH1, as isotype (isotype control)); mIgG1 (Biolegend / 401411).

[0082] Instruments and equipment include: biological safety cabinet (ESCO AC2-4S1), CO2 incubator (Japan Sanyo MCO-18AC), cell viability analyzer (Shanghai Yiheng DK-8AX), and flow cytometer (Beckman cytoflex).

[0083] The experimental steps include:

[0084] (1) U2OS cells were labeled with CFSE and the cells were plated at 2×10 5 The cells were resuspended at a density of 1 μg / mL and inoculated into 96-well plates at a volume of 100 μL and cultured overnight.

[0085] (2) After overnight culture, NK92-TIGIT cells with good growth status were taken and divided into four groups. The cell density was adjusted. The first group was 6×10 5 / mL (corresponding to an effector-target ratio of 1.5:1), and the second group was 4×10 5 / mL (corresponding to the effector-target ratio of 1:1), and the third group was 2×10 5 / mL (corresponding to the effector-target ratio of 1:2), and the fourth group was 8×10 4 / mL (corresponding to an effector-target ratio of 1:5), and inoculated into different wells of a 96-well plate containing U2OS cells in a volume of 50 μL. At the same time, three TIGIT antibodies (R0300, R0223 and R0300-CH1) and mIgG1 were diluted to the required concentrations (final concentration of 0.1 μg / mL, 10-fold gradient dilution, a total of 3 gradients), and the diluted antibodies were added to the wells of the 96-well plate with different effector-target ratios at 50 μL, and continued to be cultured. It should be noted that the effector-target ratio refers to the ratio of the number of effector cells to target cells. In this embodiment, the effector-target ratio refers to the ratio of the number of NK92-TIGIT cells to U2OS cells.

[0086] (3) After 6 h, 8 h, 12 h, and 24 h, the cells of different groups were collected and resuspended into single cell suspension. PI was added to each well for staining before flow cytometry detection. After flow cytometry detection, CFSE-positive cells were regarded as the total number of cells, CFSE and PI double-positive cells were regarded as apoptotic cells, and (PI + +CFSE + ) / CFSE + Calculate the antibody-mediated killing rate, some of the results are as follows Figures 5 to 12 shown.

[0087] Figures 5 to 12 In the figure, "cytotoxicity" in the vertical axis refers to the killing rate, in %; group "0" represents the group with only NK92-TIGIT cells and U2OS cells and no added TIGIT antibodies; group "U2OS only" represents the group with only U2OS cells. Figure 5 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1.5:1 and the culture time was 12 hours; Figure 6 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1.5:1 and the culture time was 24 hours; Figure 7 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1:1 and the culture time was 12 h. Figure 8 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1:2 and the culture time was 6 hours; Figure 9 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1:2 and the culture time was 12 h. Figure 10 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1:2 and the culture time was 24 hours; Figure 11 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1:5 and the culture time was 12 h. Figure 12 The killing of target cells by effector cells mediated by TIGIT antibodies was observed when the effector-target ratio was 1:5 and the culture time was 24 hours.

[0088] Example 4

[0089] Experimental materials include: NK92-TIGIT cells constructed in Example 1; osteosarcoma cell line U2OS (Shanghai Cell Bank SCSP-5030); 1640 culture medium (Gibco 11875085); CFSE (eBioscience 85-65-0850-84); PI (Sigma P4170-25MG); TIGIT antibody (tiragolumab in WO2017053748A2, referred to as R0300), hIgG1 (Biolegend / 403502, as an isotype, that is, a control antibody in the experiment that is similar in structure to the drug but has no function).

[0090] Instruments and equipment include: biological safety cabinet (ESCO AC2-4S1), CO2 incubator (Japan Sanyo MCO-18AC), cell viability analyzer (Shanghai Yiheng DK-8AX), and flow cytometer (Beckman cytoflex).

[0091] The experimental steps include:

[0092] (1) U2OS cells were labeled with CFSE and the cells were plated at 2×10 5 The cells were resuspended at a density of 1 μg / mL and inoculated into 96-well plates at a volume of 100 μL and cultured overnight.

[0093] (2) After overnight culture, NK92-TIGIT cells with good growth status were taken and the cell density was adjusted to 4×10 5 The cells were plated at a volume of 50 μL into the wells of a 96-well plate containing U2OS cells.

[0094] (3) Dilute the TIGIT antibody (R0300) to the desired concentration (final concentration of 66 nM, 5-fold gradient dilution, a total of 9 gradients). Add 50 μL of the diluted antibody to the wells containing target cells and effector cells in a 96-well plate and continue culturing. hIgG1 was used as a control.

[0095] (4) After culturing for 16 h, the cells were collected and resuspended into single cell suspension. PI was added to each well for staining before flow cytometry detection. After flow cytometry, CFSE-positive cells were counted as the total number of cells, CFSE and PI double-positive cells were counted as apoptotic cells, and (PI + +CFSE + ) / CFSE + The killing rate mediated by TIGIT antibodies was calculated, and the results are shown in Figure 13.

[0096] Depend on Figure 13 It can be seen that when the effector-target ratio is 1:1 and the reaction time is 16 hours, the killing effect of effector cells on target cells in the presence of TIGIT antibodies can be detected by flow cytometry. The TIGIT antibody concentration and killing rate can be fitted into a better killing curve, which can be used to evaluate the biological activity of TIGIT antibodies.

[0097] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for determining the biological activity of a TIGIT antibody, characterized in that: The following steps are involved: In vitro construction of TIGIT-expressing NK cells as effector cells; Co-culturing different concentrations of TIGIT antibodies with the effector cells and tumor cells labeled with the first fluorescent marker for 16 hours, wherein the ratio of the effector cells to the tumor cells labeled with the first fluorescent marker is 1:1; The cells after co-culture are labeled with a second fluorescent marker that is distinguishable from the first fluorescent marker; wherein the first fluorescent marker is a fluorescent marker for detecting cell proliferation, and the second fluorescent marker specifically labels apoptotic cells; Calculating the killing rate of the effector cells on the tumor cells labeled with the first fluorescent marker under the action of different concentrations of the TIGIT antibody according to the labeling results, and determining the biological activity of the TIGIT antibody according to a fitting curve of the killing rate corresponding to the TIGIT antibody concentration; The step of calculating the killing rate of the effector cells against the tumor cells marked with the first fluorescent marker under the action of different concentrations of TIGIT antibodies according to the labeling results comprises: Using flow cytometry to count the number of tumor cells labeled with the first fluorescent marker and the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker under the action of different concentrations of TIGIT antibodies; and The killing rate of the effector cells against the tumor cells labeled with the first fluorescent marker under the action of different concentrations of TIGIT antibodies is calculated, wherein the killing rate is the ratio of the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker to the number of tumor cells labeled with the first fluorescent marker.

2. The method according to claim 1, characterized in that The tumor cells are osteosarcoma cell line U2OS.

3. The method according to claim 1, characterized in that The concentration of the TIGIT antibody solution was 1×10 -6 μg / mL~10μg / mL.

4. The method according to claim 1, wherein The first fluorescent marker is 5(6)-carboxyfluorescein diacetate succinimidyl ester, and the second fluorescent marker is propidium iodide.

5. The method according to claim 1, wherein In the co-culture system, the density of the tumor cells labeled with the first fluorescent marker is 1×10 5 / mL~2×10 5 pieces / mL.

6. The method according to claim 1, characterized in that The NK cells are NK92 cells.

7. The method according to any one of claims 1 to 6, characterized in that The step of constructing NK cells expressing TIGIT in vitro comprises: The nucleic acid fragment for expressing TIGIT is transfected into NK cells via lentivirus and expressed to prepare NK cells expressing TIGIT.

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