CN115704822A - Method for determining biological activity of TIGIT antibody - Google Patents
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Abstract
The invention relates to a method for determining the biological activity of a TIGIT antibody. The method for determining the biological activity of the TIGIT antibody comprises the following steps: constructing NK cells expressing TIGIT in vitro as effector cells; respectively mixing TIGIT antibodies with different concentrations with effector cells and tumor cells marked with a first fluorescent marker according to an effective target ratio (0.2-1.5): 1 co-culturing for 6-24 h, marking the tumor cells which are subjected to apoptosis by adopting another fluorescent dye which can be distinguished from the first fluorescent marker, calculating the killing rate of the effector cells to the tumor cells under the action of the TIGIT antibodies with different concentrations according to a marking result, and fitting a relation curve between the TIGIT antibody concentration and the killing rate to determine the biological activity of the TIGIT antibody. The TIGIT expression level in the method is stable, the biological activity of the TIGIT antibody is easier to detect, and the sensitivity is higher.
Description
Technical Field
The invention relates to the technical field of immunization, in particular to a method for determining the biological activity of a TIGIT antibody.
Background
T cell immune receptor (TIGIT) with Ig and ITIM domains was first reported since 2009, as an immune checkpoint for T cells and NK cells, it has rapidly become the direction of extensive attention and drug development for each large drug enterprise. Among them Genentech, pharmaceutical research has been in phase iii of clinical practice, BMS is also in phase i of clinical practice, and a large number of biopharmaceutical companies compete at home and abroad. We are particularly concerned with the MOA-based in vitro biological activity of these drugs, whether in the early screening phase of the target antibody drug or in the following CMC (Chemical, manufacturing and Control) phase.
In basic studies, we know that TIGIT is expressed in lymphocytes, particularly 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 Tage 4) is a high affinity ligand for TIGIT. Once CD155 expressed on the surface of tumors binds to TIGIT on the surface of NK and T cells, the killing of tumor cells by NK and T cells is inhibited. The TIGIT antibody has the significance of blocking the combination of TIGIT and CD155, releasing the function inhibition of the signal on T or NK cells and killing tumor cells again. Therefore, it is important to develop a method for sensitively detecting the biological activity of the TIGIT antibody in vitro.
In a traditional in vitro functional experiment for detecting the TIGIT antibody, natural NK cells are used as target cells, and LDH (layered double hydroxide) is used as an index to evaluate the biological activity of the TIGIT antibody. However, the conventional method has the following problems: (1) Natural NK cells are troublesome to obtain from healthy donors, and TIGIT expression levels of different donors are different; (2) Detection methods based on detection of LDH often fail to detect the biological activity of TIGIT antibodies.
Disclosure of Invention
Based on this, it is necessary to provide a method for measuring the biological activity of a TIGIT antibody, which is capable of improving the situation in which natural NK cells are difficult to obtain and the TIGIT expression level is unstable, and which is capable of detecting the biological activity of the TIGIT antibody more easily and with higher sensitivity than the conventional LDH method.
A method of determining the biological activity of a TIGIT antibody, comprising the steps of:
constructing NK cells expressing TIGIT in vitro as effector cells;
co-culturing TIGIT antibodies with different concentrations with the effector cells and the tumor cells marked with the first fluorescent markers for 6-24 h respectively, wherein the number ratio of the effector cells to the tumor cells marked with the first fluorescent markers is (0.2-1.5): 1;
labeling the cells after the end of co-culture with a second fluorescent label distinguishable from the first fluorescent label; wherein the first fluorescent marker is a fluorescent marker for detecting cell proliferation, and the second fluorescent marker specifically marks apoptotic cells;
and calculating the killing rate of the effector cells to the tumor cells marked with the first fluorescent marker under the action of the TIGIT antibodies with different concentrations according to the marking result, and determining the biological activity of the TIGIT antibodies according to a curve fitted by the TIGIT antibody concentration and the corresponding killing rate.
According to the method for determining the biological activity of the TIGIT antibody, the situation that natural NK cells are difficult to obtain and the TIGIT expression level is unstable is improved by constructing TIGIT-expressing NK cells in vitro, and meanwhile, the effective target ratio is (0.2-1.5): 1 and the co-culture time is 6-24 h, so that the effector cells have a proper detection window for detecting the tumor killing effect, and compared with the traditional detection method for detecting LDH, the biological activity of the TIGIT antibody is more easily determined, and the sensitivity is higher.
In one embodiment, the step of calculating the killing rate of the effector cells to the tumor cells labeled with the first fluorescent marker under the action of different concentrations of TIGIT antibody according to the labeling result comprises:
counting the number of the tumor cells marked with the first fluorescent marker and the second fluorescent marker under the action of TIGIT antibodies with different concentrations by using a flow cytometer; and
and calculating the killing rate of the effector cells to the tumor cells marked with the first fluorescent marker under the action of TIGIT antibodies with different concentrations, wherein the killing rate is the ratio of the number of the tumor cells marked with the first fluorescent marker and the second fluorescent marker to the number of the tumor cells marked with the first fluorescent marker.
In one embodiment, the tumor cell is osteosarcoma cell line U2OS.
In one embodiment, 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; the co-culture time is 12-24 h.
In one embodiment, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1.5:1, the co-culture time is 12h;
or, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is 1:1, the co-culture time is 16h;
or, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is 1:2, the co-culture time is 8h or 24h;
or, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is 1:5, the co-culture time is 24h.
In one embodiment, the TIGIT antibody solution has a concentration of 1 × 10 -6 μg/mL～10μg/mL。
In one embodiment, the first fluorescent label is fluorescein-5 (6) -carboxydiacetate succinimidyl ester and the second fluorescent label is propidium iodide.
In one embodiment, the density of the tumor cells labeled with the first fluorescent marker in the co-cultured system is 1 × 10 5 2X 10 per mL 5 one/mL.
In one embodiment, the NK cell is an NK92 cell.
In one embodiment, the step of constructing in vitro TIGIT-expressing NK cells comprises:
a nucleic acid fragment for expressing TIGIT was transfected into NK cells by lentivirus and expressed, and TIGIT-expressing NK cells were prepared.
Drawings
FIG. 1 is a vector map of the human TIGIT gene in example 1;
FIG. 2 shows TIGIT expression of NK92-TIGIT cells in example 1;
FIG. 3 shows PVR expression of U2OS cells in example 1;
FIG. 4 shows the LDH method in example 2 to detect TIGIT antibody-mediated killing of NK92-TIGIT cells to U2OS cells;
FIGS. 5 to 12 are the killing of U2OS cells by NK92-TIGIT cells mediated by TIGIT antibody at different effective target ratios and different co-culture times in example 3;
FIG. 13 shows the effective target ratio of 1: 1. the co-incubation time was the curve of the relationship between TIGIT antibody and killing rate after 16 hours.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. The terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. Unless defined otherwise, all 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
An embodiment of the present application provides a method for evaluating a biological activity of a TIGIT antibody, which includes step S100, step S200, step S300, and step S400. Specifically, the method comprises the following steps:
step S100: constructing NK cells expressing TIGIT in vitro.
Specifically, a nucleic acid fragment for expressing TIGIT is integrated into NK cells using genetic engineering techniques and allowed to express TIGIT. Alternatively, a nucleic acid fragment for expressing TIGIT is transfected into NK cells by lentivirus and expressed to prepare NK cells expressing human TIGIT. More specifically, a packaging vector and a transfer vector carrying a nucleic acid segment for expressing TIGIT are co-transfected into a packaging cell to prepare lentivirus carrying the nucleic acid segment for expressing TIGIT, and NK cells are transfected by the lentivirus carrying the nucleic acid segment for expressing TIGIT to prepare the NK cells for expressing TIGIT.
In this embodiment, the NK cells are NK92 cells. TIGIT is human TIGIT. Correspondingly, the TIGIT antibody is a human TIGIT antibody.
Step S200: TIGIT antibody at different concentrations was co-cultured with effector cells and tumor cells labeled with a first fluorescent marker, respectively.
The tumor cells labeled with the first fluorescent marker are target cells. And (3) co-culturing the TIGIT antibody, the effector cell and the tumor cell marked with the first fluorescent marker, wherein the TIGIT antibody and the TIGIT on the surface of the effector cell can block the combination of PVR on the surface of the tumor cell and the TIGIT on the surface of the effector cell, so that the killing effect of the effector cell on the tumor cell is released. Therefore, by the killing rate of the effector cells after addition of the TIGIT antibody to the tumor cells labeled with the first fluorescent marker, the half maximum effect concentration of the TIGIT antibody (concentration for 50% of maximum effect,EC 50 ) Thereby determining the biological activity of the TIGIT antibody.
Specifically, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is (0.2-1.5): 1; the co-culture time is 8-24 h. By setting the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker to (0.2 to 1.5): 1. the co-culture time is set to be 8-24 h, so that the method has a proper detection window.
Further, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is (1-1.5): 1; the co-culture time is 12-24 h.
In one embodiment, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1.5:1, the co-culture time is 12h.
In one embodiment, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1:1, the co-culture time is 12h or 16h.
In one embodiment, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1:2, the co-culture time is 8h.
In one embodiment, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1:2, the co-culture time is 24h.
In one embodiment, the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1:5, the co-culture time is 24h.
Optionally, the different concentrations of TIGIT antibody are gradient concentrations of TIGIT antibody. For example, in some embodiments, the different concentrations of TIGIT antibody are 5-fold or 10-fold gradient diluted TIGIT antibody solutions.
Optionally, the TIGIT antibody concentrations at different concentrations are 1 × 10 -6 Mu g/mL to 10 mu g/mL.
Alternatively, in a co-cultured system, the tumor cells labeled with the first fluorescent marker have a density of 1X 10 5 2X 10 per mL 5 one/mL.
Step S300: and (3) carrying out cell labeling on the cells after the co-culture is finished by adopting a second fluorescent marker which can be distinguished from the first fluorescent marker.
Specifically, the first fluorescent marker is a fluorescent marker for detecting cell proliferation and is used for marking living cells; the second fluorescent label specifically labels the cells undergoing apoptosis.
In this embodiment, the first fluorescent marker is 5 (6) -carboxyfluorescein diacetate succinimidyl ester (CFSE) and the second fluorescent marker is Propidium Iodide (PI). The CFSE can easily penetrate through cell membranes, is covalently combined with intracellular proteins in living cells, and releases green fluorescence after hydrolysis for marking the living cells; PI is a nuclear staining reagent that stains DNA, an analog of ethidium bromide, which releases red fluorescence after intercalating into double-stranded DNA, and is used to label apoptotic cells. Thus, tumor apoptosis can be distinguished and counted based on the different fluorescent signals on the tumor cells.
It is understood that in other embodiments, the combination of the first fluorescent marker and the second fluorescent marker is not limited to the CFSE and PI described above, but may be other combinations as long as it can distinguish between apoptotic tumor cells and live tumor cells.
Alternatively, the tumor cell is osteosarcoma cell line U2OS. The osteosarcoma cell line U2OS has high expression PVR, and is more favorable for releasing the effect of a response TIGIT antibody on killing tumor cells by effector cells.
In some embodiments, further comprising the step of preparing tumor cells labeled with a first fluorescent marker, the preparing step comprising: mixing the tumor cells with a first fluorescent marker; and removing the unreacted first fluorescent marker to prepare the tumor cells 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 which case commercial tumor cells labeled with the first fluorescent marker may be purchased directly.
Step S400: and counting the killing rate of the effector cells to the target cells under the action of the TIGIT antibodies with different concentrations, and determining the biological activity of the TIGIT antibodies according to a fitting curve of the TIGIT antibody concentrations and the killing rates corresponding to the TIGIT antibodies.
Specifically, the killing rate is the ratio of the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker (i.e., tumor cells undergoing apoptosis) to the number of tumor cells labeled with the first fluorescent marker (i.e., total tumor cells), i.e., the ratio of tumor cells undergoing apoptosis to total tumor cells. Therefore, the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker under the action of different concentrations of the TIGIT antibody are counted, and the killing rate of effector cells to target cells under the action of different concentrations of the TIGIT antibody can be calculated. EC can be determined according to a fit curve of the TIGIT antibody concentration and the killing rate corresponding to the TIGIT antibody concentration 50 Thereby determining the biological activity of the TIGIT antibody.
Optionally, counting the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker using flow cytometry. It is understood that in other embodiments, other means may be used to count the number of tumor cells labeled with the first fluorescent marker and the second fluorescent marker. For example, using fluorescence microscopy.
According to the method for determining the TIGIT antibody biological activity, the defects that natural NK cells are difficult to obtain and the TIGIT expression level is unstable are overcome by constructing TIGIT-expressing NK cells in vitro. Meanwhile, the effective target ratio is (0.2-1.5): 1 and the co-culture time is set to be 6-24 h, so that effector cells have a proper detection window for tumor killing and can be detected. Proved by verification, compared with the traditional detection method for detecting LDH, the method is easier to determine the biological activity of the TIGIT antibody and has higher sensitivity.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except for inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. In the figure, the group "T" indicates a group having only target cells, that is, an experimental group having only U2OS cells; group "T + E" refers to the group with effector cells (NK 92-TIGIT cells) and target cells (U2 OS cells) but without TIGIT antibody. The TIGIT antibody can be any TIGIT antibody, and the TIGIT antibody selected in the examples herein is merely exemplary and should not be a limitation of the present application.
Example 1
Construction of NK92-TIGIT
The experimental materials included: NK92 cell line (Beinai organism; BNCC 236019); osteosarcoma cell line U2OS (Shanghai cell bank SCSP-5030, abbreviated as U2OS cell or U2 OS) Dynamis medium (Gibco; A2617502); double antibody (Gibco; 15140122); anticoagulants (Gibco; 0010057); pcdDNA TM 3.4TOPOTMTA Cloning Kit (Invitrogen A14697); 5-alpha Competent E.coli (complete E.coli) (NEB C2987I); DL2000 DNA Marker (TAKARA 3427A); q5 High Fidelity DHA Polymerase (High-Fidelity DNA Polymerase) (NEB M0491L); axyPrep DNA Gel Extraction Kit (Gel Extraction Kit) (AxyGEN AP-GX-50); hind III (NEB); ecoRI (NEB); t4 DNA ligase (TAKARA 2011A); endotoxin-free plasmid great extract kit (TIANGEN DP 117); PE-anti-human TIGIT antibody (Biolegend 372704); anti-PVR antibody (Biolegend 337602).
The instrument device includes: PCR instrument (Tprofessional TR 20); an electrophoresis apparatus; (DYY-TC); gel imager (Smart Gel N); a centrifuge (H1650-W); micro thermostats (HW-8C); clean bench (SDJ series); a constant temperature oscillator (H2-9211K); a constant temperature water bath (HH-4A); cell culture shaking tables (Adolf Kuhner; ISF 4-XC); high speed centrifugation (xiang apparatus, H2050R); a biological safety cabinet (ESCO AC2-4S 1); CO 2 2 Incubators (MCO-18 AC, sanyo, japan); cell viability analyzer (Shanghai-Heng DK-8 AX); flow cytometer (Beckman cytoflex).
The experimental steps include:
(1) Constructing an NK92 cell strain for stably expressing TIGIT: constructing lentivirus (vector map containing human TIGIT Gene is shown in figure 1) containing human TIGIT full-length sequence (Gene ID: 201633) and infecting NK92 cells, and selecting monoclone after infection to obtain NK92 cell strain (NK 92-TIGIT cell for short) for stably expressing TIGIT.
(2) Detecting the expression of TIGIT of NK92-TIGIT cells: the flow fluorescent antibody was used to label the NK92 cell line capable of stably expressing TIGIT obtained in step (1), and the TIGIT expression was detected by a flow cytometer after incubation for 30min at 4 ℃, with the results shown in FIG. 2. "BLANK" in FIG. 2 refers to the BLANK control, i.e., unmodified NK92 cells.
(3) PVR expression was measured in U2OS cells and the results are shown in FIG. 3. "BLANK" in FIG. 3 refers to a BLANK control, i.e., a control group that was replaced with an equivalent volume of medium without antibody.
As can be seen from FIG. 2, a NK92 cell line stably and highly expressing human TIGIT was constructed according to the experimental method, and the cell line can be used as an effector cell for detecting the biological function of the TIGIT antibody. As is clear from fig. 3, U2OS cells stably and normally express PVR, and can be used as target cells for detecting the physical function of TIGIT antibody.
Example 2
LDH (LDH-detection) TIGIT (tungsten inert gas) mediated cell killing effect of NK92-TIGIT on U2OS
The experimental materials included: NK92-TIGIT cells constructed in example 1; osteosarcoma cell line U2OS (Shanghai cell Bank SCSP-5030); 1640 medium (Gibco 11875085); LDH detection kit (bi yun day C0017); TIGIT antibody (TIGIT monoclonal antibody 22G2, abbreviated as R0223 in WO2016106302 A1).
The instrument device includes: a biological safety cabinet (ESCO AC2-4S 1); CO 2 2 Incubators (MCO-18 AC, sanyo, japan); cell viability analyzer (Shanghai-Heng DK-8 AX); multifunctional microplate reader (Molecular devices spectramax i3 ×).
The experimental steps include:
(1) The cultured U2OS cells were counted, centrifuged and the cell number was adjusted to 5X 10 using serum-free 1640 medium 5 one/mL, and spread evenly on a 96-well plate at 100. Mu.L/well, and thenAnd (5) culturing overnight.
(2) After the overnight culture is stable, taking NK92-TIGIT cells with good culture state (the "good culture state" in the description of the cell state in the present document refers to the culture state that the cells are uniformly agglomerated, the cells are bright, the culture medium is clear, and the cell counter counts the activity by more than 90%), and adjusting the cell number to 1 × 10 6 one/mL of the cells was added to the U2OS cell-plated 96-well plate of step (1) in an amount of 50. Mu.L. Then 50 mu.L of TIGIT antibody (final concentration is 1-10) diluted in a gradient way is added -6 μ g/mL, 10-fold gradient dilution), LDH release was detected after 4h, and the results are shown in fig. 4.
As shown in FIG. 4, the window of biological activity for detecting TIGIT antibody by the LDH detection method is low (higher background and indistinguishable), and the biological activity (EC) of the antibody cannot be fitted 50 )。
Example 3
Double-fluorescence flow method for detecting cell killing effect of TIGIT antibody mediated NK92-TIGIT on U2OS
The experimental materials included: NK92-TIGIT cells constructed in example 1; osteosarcoma cell line U2OS (Shanghai cell Bank SCSP-5030); 1640 medium (Gibco 11875085); CFSE (eBioscience 85-65-0850-84); PI (Sigma P4170-25 MG); TIGIT antibody 1 (TIGIT monoclonal antibody 22G2, abbreviated as R0223 in WO2016106302 A1); TIGIT antibody 2 (tiragolumab in WO2017053748A2, R0300 for short); TIGIT antibody 3 (TIGIT antibody obtained by replacing the Fc region of tiragolumab in WO2017053748A2 with the Fc region of mIgG1, R0300-CH1 for short, as isotype (isotype control)); mIgG1 (Biolegend/401411).
The instrument device includes: biological safety cabinet (ESCO AC2-4S 1), CO 2 Incubator (MCO-18 AC, sanyo, japan), cell viability analyzer (constant DK-8AX, shanghai), and flow cytometer (Beckman cytoflex).
The experimental steps include:
(1) U2OS cells were labeled with CFSE and the cells were plated at 2X 10 5 The cells/mL were resuspended at density and seeded into 96-well plates at a volume of 100 μ L and incubated overnight.
(2) After overnight culture, the growth state was goodThe NK92-TIGIT cells were divided into four groups, and the cell density was adjusted, wherein the first group was 6X 10 5 counts/mL (corresponding to an effective target ratio of 1.5: 1), and a second set of 4 × 10 5 one/mL (corresponding to an effective target ratio of 1), and a third set of 2 × 10 5 counts/mL (corresponding to an effective target ratio of 1 to 2), fourth group 8 × 10 4 one/mL (corresponding to an effective target ratio of 1 to 5) and seeded in a volume of 50 μ Ι _ into different wells of a 96-well plate containing U2OS cells. Meanwhile, three TIGIT antibodies (R0300, R0223 and R0300-CH 1) and mIgG1 are respectively diluted to required concentrations (final concentration is 0.1 mu g/mL,10 times of gradient dilution, 3 gradients in total), the diluted antibodies are added into wells with different effective target ratios of a 96-well plate in 50 mu L, and the culture is continued. The effective target ratio is the ratio of the number of effector cells to target cells. In this example, the effective target ratio is the ratio of the number of NK92-TIGIT cells to U2OS cells.
(3) Collecting cells of different groups after 6h, 8h, 12h and 24h respectively, re-suspending the collected cells to a single cell suspension state respectively, and adding PI into each hole for staining before detection by a flow cytometer; after flow detection, CFSE positive cells are taken as the total number of cells, and CFSE and PI double positive cells are taken as apoptotic cells to obtain (PI) + +CFSE + )/CFSE + The antibody-mediated killing rate was calculated and part of the results are shown in fig. 5-12.
In fig. 5 to 12, "cytoxicity" in the ordinate indicates the killing rate in units; group "0" represents the group with NK92-TIGIT cells and U2OS cells only and with no added TIGIT antibody; the group "U2OS only" represents the group with only U2OS cells. Fig. 5 shows the effective target ratio of 1.5: 1. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 12h; fig. 6 shows the effective target ratio of 1.5: 1. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 24h; fig. 7 shows the effective target ratio is 1: 1. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 12h; fig. 8 shows the effective target ratio is 1: 2. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 6h; fig. 9 shows the effective target ratio of 1: 2. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 12h; fig. 10 shows the effective target ratio is 1: 2. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 24h; fig. 11 shows the effective target ratio of 1: 5. killing the target cells by the TIGIT antibody mediated effector cells when the culture time is 12h; fig. 12 shows the effective target ratio is 1: 5. and (3) killing of target cells by the TIGIT antibody mediated effector cells at the culture time of 24h.
Example 4
The experimental materials included: NK92-TIGIT cells constructed in example 1; osteosarcoma cell line U2OS (Shanghai cell Bank SCSP-5030); 1640 medium (Gibco 11875085); CFSE (eBioscience 85-65-0850-84); PI (Sigma P4170-25 MG); TIGIT antibody (tiragolumab in WO2017053748A2, R0300 for short), hIgG1 (Biolegend/403502 as isotype, a control antibody with a structure similar to a drug but not functional in the experiment).
The instrument device includes: biological safety cabinet (ESCO AC2-4S 1), CO 2 Incubator (MCO-18 AC, sanyo, japan), cell viability analyzer (constant DK-8AX, shanghai), and flow cytometer (Beckman cytoflex).
The experimental steps include:
(1) U2OS cells were labeled with CFSE and the cells were plated at 2X 10 5 The cells/mL were resuspended at density and seeded into 96-well plates at a volume of 100. Mu.L and incubated overnight.
(2) After overnight culture, NK92-TIGIT cells in good growth state were collected and cell density was adjusted to 4X 10 5 one/mL (corresponding to an effective target ratio of 1), and seeded in a volume of 50 μ L into wells of a 96-well plate containing U2OS cells.
(3) TIGIT antibody (R0300) was diluted to the desired concentration (final concentration 66nm, 5-fold gradient dilution, total 9 gradients), and the diluted antibody was added to wells containing target cells and effector cells in a 96-well plate at 50 μ L, and culture was continued. hIgG1 was also used as a control.
(4) Collecting cells after culturing for 16h, respectively re-suspending the collected cells to a single cell suspension state, and adding PI into each hole for staining before detection by a flow cytometer; after flow detection, CFSE positive cells are taken as the total number of cells, CFSE and PI double positive cells are apoptotic cells, with (PI) + +CFSE + )/CFSE + The TIGIT antibody mediated killing rate was calculated and the results are shown in 13.
As can be seen from fig. 13, the effective target ratio was 1:1, when the reaction time is 16h, the killing effect of effector cells on target cells can be detected by a flow method in the presence of a TIGIT antibody, the concentration and the killing rate of the TIGIT antibody can be fitted into a better killing curve, and the biological activity of the TIGIT antibody can be evaluated.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, which is convenient for specific and detailed understanding of the technical solutions of the present invention, but the present invention should not be construed as being limited to the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present patent shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.
Claims (10)
1. A method of determining the biological activity of a TIGIT antibody, comprising the steps of:
constructing NK cells expressing TIGIT in vitro as effector cells;
and respectively co-culturing TIGIT antibodies with different concentrations with the effector cells and the tumor cells marked with the first fluorescent markers for 6-24 h, wherein the number ratio of the effector cells to the tumor cells marked with the first fluorescent markers is (0.2-1.5): 1;
labeling the cells after the co-culture is finished with a second fluorescent marker 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 marks apoptotic cells;
and calculating the killing rate of the effector cells to the tumor cells marked with the first fluorescent marker under the action of the TIGIT antibodies with different concentrations according to the marking result, and determining the biological activity of the TIGIT antibodies according to a curve fitted by the TIGIT antibody concentration and the corresponding killing rate.
2. The method according to claim 1, wherein the step of calculating from the labeling results the killing rate of the effector cells against the tumor cells labeled with the first fluorescent label by different concentrations of TIGIT antibody comprises:
counting the number of the tumor cells marked with the first fluorescent marker and the second fluorescent marker under the action of TIGIT antibodies with different concentrations by using a flow cytometer; and
and calculating the killing rate of the effector cells to the tumor cells marked with the first fluorescent marker under the action of different concentrations of TIGIT antibodies, wherein the killing rate is the ratio of the number of the tumor cells marked with the first fluorescent marker and the second fluorescent marker to the number of the tumor cells marked with the first fluorescent marker.
3. The method of claim 1, wherein the tumor cell is osteosarcoma cell line U2OS.
4. The method of claim 1, wherein 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; the co-culture time is 12-24 h.
5. The method of claim 1, wherein the ratio of the number of effector cells to tumor cells labeled with the first fluorescent marker is 1.5:1, the co-culture time is 12h;
or, 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, the co-culture time is 16h;
or, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is 1:2, the co-culture time is 8h or 24h;
or, the ratio of the number of effector cells to the number of tumor cells labeled with the first fluorescent marker is 1:5, the co-culture time is 24h.
6. The method according to claim 1, characterized in that the TIGIT antibody solution has a concentration of 1 x 10 -6 μg/mL～10μg/mL。
7. The method of claim 1, wherein the first fluorescent label is fluorescein 5 (6) -carboxydiacetate succinimidyl ester and the second fluorescent label is propidium iodide.
8. The method of claim 1, wherein the tumor cells labeled with the first fluorescent marker have a density of 1 x 10 in the co-cultured system 5 2X 10 units/mL 5 One per mL.
9. The method of claim 1, wherein said NK cells are NK92 cells.
10. The method according to any one of claims 1 to 9, wherein the step of constructing in vitro NK cells expressing TIGIT comprises:
a nucleic acid fragment for expressing TIGIT was transfected into NK cells by lentivirus and expressed, and TIGIT-expressing NK cells were prepared.
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