ADCP biological activity detection method based on single luciferase

Abstract

The invention discloses a monoluciferase-based ADCP biological activity detection method, which comprises the following steps: separating a mononuclear cell from a human peripheral blood mononuclear cell as an effector cell, phagocytizing a Raji cell transfected by Luciferase as a target cell by the mononuclear cell, acidifying and degrading the Raji cell in a phagocytic body, and degrading the Luciferase so as to attenuate a light-emitting signal, so that the ADCP biological activity is detected. The ADCP biological activity of the antibody therapeutic drug is accurately quantified by detecting the weakening of the light-emitting signal. According to the present invention, the method only needs to separate the mononuclear cells and perform Luciferase infection on the target cells, and only needs to add the luciferase substrate after the phagocytosis process, such that the luminescence value can be read by using the microplate reader, the operation process is simple, the steps are less, the time consumption is short, the high-throughput screening is achieved, and the new direction is provided for the ADCP detection.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a method for detecting ADCP biological activity based on single luciferase. Background Art

[0002] Therapeutic antibodies have been used to treat a variety of diseases, including cancer, autoimmune diseases, and other metabolic-related diseases. The mechanism of action (MOA) of therapeutic antibodies is mainly binding to specific targets through the Fab region of the antibody, while the induction of effector functions through the Fc region of the antibody is equally important for the therapeutic effect of the antibody. Through the binding of the Fc region to Fcγ receptors (FcγRs) or complement factors on the surface of immune cells, antibody therapy exerts effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and contribute to the therapeutic effect. Another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the induction of phagocytosis by antibody-activated target cells activating FcγRs on the surface of macrophages, which leads to target cell degradation through target cell internalization and phagosome acidification. ADCP is considered to be the main MOA of several therapeutic antibodies.

[0003] At present, the main detection method of ADCP is flow cytometry. Most of them use flow cytometry to detect double-positive cells by simultaneously labeling macrophages and target cells to determine the phagocytosis of target cells. However, the production of macrophages in flow cytometry requires seven days of induction with stimulating factors, and the fluid needs to be changed in the middle; when collecting macrophages, it takes a long time to digest; at the same time, both cells need to be labeled, and the macrophages take a long time to adhere to the wall and digest, resulting in more experimental steps, longer cycles, and difficulty in achieving high-throughput detection. At the same time, ADCP detection is mainly used in the drug development stage. Therefore, it is urgent to develop a detection method for ADCP biological activity in the treatment stage to achieve fewer operating steps, shorter time consumption and high-throughput screening. Summary of the invention

[0004] The purpose of the present invention is to provide a single luciferase-based ADCP biological activity detection method, which uses Luciferase-transfected target cells, which are engulfed by monocytes (CD16 is not expressed to avoid ADCC interference), enter the phagosome for acidification and degradation, and Luciferase is subsequently degraded, thereby causing the luminescence signal to attenuate. The ADCP activity of the antibody therapeutic drug is accurately quantified by detecting the attenuation of the luminescence signal with an ELISA instrument, solving the problems existing in the prior art.

[0005] In order to solve the above technical problems, the present invention adopts the following solutions:

[0006] A method for detecting ADCP biological activity based on single luciferase comprises the following steps:

[0007] Step S1, plate the target cells transfected with Luciferase, add pre-diluted Rituximab, and incubate once;

[0008] Step S2, after the first incubation is completed, monocytes separated from human peripheral blood mononuclear cells are added and then incubated for the second time;

[0009] Step S3, adding luciferase substrate and detecting the luminescent signal;

[0010] The monocytes serve as effector cells.

[0011] The present application uses monocytes as effector cells, Luciferase-transfected Raji cells (Raji-luciferase) as target cells, and adds gradiently diluted antibody Rituximab for co-culture. There is no need to label macrophages and target cells, thus reducing experimental steps and cycles. After the phagocytosis process is completed, only luciferase substrate needs to be added to detect the phagocytosis rate of target cells, making the detection operation process of ADCP biological activity simple.

[0012] More preferably, the target cells are Raji cells transfected with Luciferase.

[0013] Further preferably, in step S1, the number of target cells per well is 1×10 4 ~10×10 4 The preferred value is 2.5×10 4 ~5×10 4 More preferably, 2.5×10 4 or 5×10 4 indivual.

[0014] Further preferably, in step S1, the ratio of target cells to effector cells is 2:1.

[0015] More preferably, the temperature of the primary incubation and the secondary incubation are both 35-38°C, preferably 37°C.

[0016] More preferably, the first incubation time is 60 min, and the second incubation time is 4 h to 5 h.

[0017] More preferably, in step S2, the number of mononuclear cells per well is 5×10 4 indivual.

[0018] More preferably, in the step S3, after adding the luciferase substrate, the cleavage is carried out for 3 to 5 minutes.

[0019] Further preferably, the luciferase substrate is luciferin, including but not limited to Promega's ONE-Glo TM Luciferase Assay System and Bright-Lumi TM .

[0020] More preferably, the luminescent signal is detected using an enzyme reader.

[0021] The beneficial effects of the present invention are as follows: the present invention is a method for detecting the biological activity of ADCP based on single luciferase, wherein the separated monocytes are used as effector cells, and the Raji cells transfected with Luciferase are used as target cells. After being engulfed by the monocytes, the cells enter the phagosomes for acidification and degradation, and the Luciferase is subsequently degraded, thereby causing the attenuation of the luminescent signal, and the ADCP biological activity of the antibody therapeutic drug is accurately quantified by detecting the attenuation of the luminescent signal. It is only necessary to separate the monocytes and infect the target cells with Luciferase, and after the engulfment process is completed, it is only necessary to add the luciferase substrate, and the luminescent value can be read with an enzyme reader, the operation process is simple, the steps are few, the time consumption is short, and high-throughput screening is achieved, providing a new direction for the detection of ADCP. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a data diagram of the phagocytic rate in monocytes in Example 2 of the present invention;

[0023] Figure 2 This is a diagram for identifying the expression level of CD16a in monocytes in Example 3 of the present invention;

[0024] Figure 3 This is a data graph showing the phagocytic rate of monocytes when the blocking antibody 3G8 is added in Example 3 of the present invention;

[0025] Figure 4 This is a data diagram of the phagocytic rate in monocytes in Comparative Example 1 of the present invention;

[0026] Figure 5 This is a data graph of the phagocytic rate in monocytes in Comparative Example 2 of the present invention;

[0027] Figure 6 This is a data graph of the phagocytic rate in monocytes in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The relative arrangement of components and steps, the numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

[0030] At the same time, it should be understood that for the convenience of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship.

[0031] Additionally, descriptions of well-known structures, functions, and configurations may be omitted for clarity and conciseness.One of ordinary skill in the art will recognize that various changes and modifications may be made to the examples described herein without departing from the spirit and scope of the present disclosure.

[0032] Technologies, methods, and apparatus known to ordinary technicians in the relevant field may not be discussed in detail, but where appropriate, such technologies, methods, and apparatus should be considered part of the authorization specification.

[0033] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0034] Example 1

[0035] Example 1 of the present invention is the preparation of a tumor cell line transfected with luciferase, which specifically includes the following steps:

[0036] (1) Collection and storage of viral supernatant

[0037] Add 9.5mL Opti-MEM serum-free medium to the HEK293T cell culture dish, and evenly and slowly drop the PEI-Luciferase gene-containing plasmid complex. After 2 hours of transfection, supplement the culture medium with FBS to a volume concentration of 10% (1mL). After 6 hours, replace the culture medium in the culture dish with 10mL complete medium (DMEM + 10% FBS) and continue to culture in a carbon dioxide incubator. After 48 hours of culture, collect the virus in the supernatant for use.

[0038] (2) Infection

[0039] One day before infection, target cells (Raji, Daudi, SKOV-3) were seeded in 6-well plates and cultured in a carbon dioxide incubator.

[0040] On the day of infection, the virus supernatant was diluted to 30-50% of the initial concentration with complete medium (without antibiotics); and the transduction aid Polybrene was added at a final concentration of 10 μg / mL, and centrifuged at 1000g for one hour at room temperature; after centrifugation, the cells were placed in a carbon dioxide incubator for culture; after 6 hours or overnight, the supernatant was discarded and replaced with complete medium (basal medium + 10% FBS) for continued culture. After 48-72 hours of infection, the expression of the target gene and its positive rate were verified, and antibiotics were added for pressure screening. The positive rate of the screened cells was greater than or equal to 95%. At this time, the cells obtained were target cells transfected with Luciferase, and monoclonal screening could be performed as needed.

[0041] Example 2

[0042] Example 2 of the present invention is the establishment of a single luciferase-based ADCP biological activity detection method.

[0043] (1) Isolation of monocytes

[0044] Monocytes were isolated from human peripheral blood mononuclear cells (PBMC) (Mitenyi Biotec, 130-118-906) and used as is.

[0045] (2) Target cells (Raji-Luc) were plated and added to a 96-well cell culture plate. The number of target cells per well was 2.5×10 4 pcs, 50μL / well.

[0046] (3) Add pre-diluted Rituximab to a 96-well cell culture plate and incubate at 37°C for 60 min.

[0047] (4) Add monocytes to a 96-well cell culture plate, with 5×10 monocytes per well. 4 , 50 μL / well, the monocytes are used as effector cells, and the ratio of effector cells to target cells is 2:1;

[0048] (5) After a second incubation at 37°C for 4-5 hours, add Bright-Lumi TM Luciferase substrate (Biyuntian RG052M) was lysed for 3-5 min, transferred to a white 96-well plate, and the luminescent signal was read using an ELISA reader (see Figure 4 ).

[0049] Since the acidification and degradation of phagosomes leads to the degradation of Luciferase, which in turn leads to the attenuation of the light signal, the weakening of the luminescent signal is detected by an ELISA instrument to calculate the ADCP activity of the antibody therapeutic drug.

[0050] Experimental results: The ADCP effect of antibody Rituximab in monocytes was detected, and the phagocytic rate in monocytes was Figure 1 .according to Figure 1 It can be seen that monocytes were used as effector cells, Luciferase-transfected Raji cells (Raji-luciferase) were used as target cells, and gradiently diluted antibody Rituximab (rituximab, bridging target cells and monocytes) was added for co-culture. The ADCP effect of the antibody was detected, and it was found that the phagocytic rate of monocytes increased with the increase of antibody concentration within a certain antibody concentration range, and then reached an upper platform.

[0051] Example 3

[0052] Example 3 of the present invention identifies whether there is a CD16a-mediated ADCC effect in monocytes in human peripheral blood.

[0053] (1) Identification of CD16a expression in monocytes

[0054] This example verifies whether CD16a is expressed in monocytes by flow cytometry. Since CD16a can mediate antibody-dependent cellular cytotoxicity (ADCC), killing target cells will also lead to a decrease in Luciferase activity, so it is necessary to first eliminate the interference of CD16a. More than 85% of monocytes in the blood are classical monocytes, and CD16a expression is negative. This application separates monocytes from human PBMCs, and then adds fluorescently labeled CD16a antibodies (Biolegend, 360716) for flow cytometry to confirm the expression of CD16a on monocytes. The expression level of CD16a is shown in the result graph. Figure 2 .according to Figure 2 It can be seen that monocytes hardly express CD16a, so the influence of ADCC effect on this experimental system can be ruled out.

[0055] (2) Further eliminating the influence of ADCC effect by adding CD16a blocking antibody

[0056] To further determine whether monocytes have CD16a-mediated ADCC effect, the experimental system of Example 2 was used, the concentration of Rituximab was 5 or 0.05 ug / mL, and CD16a blocking antibody 3G8 (5 ug / mL) was added to some control wells of the 96-well cell culture plate and incubated at 37°C for 30 min.

[0057] Raji cells transfected with Luciferase were added as target cells and pre-diluted Rituximab, and incubated for 60 minutes; the effector-target ratio was 2:1. After adding monocytes, they were incubated for 4 to 5 hours in a 37°C incubator, and then the luciferase substrate Bright-Lumi TM (Biyuntian RG052M), lyse for 3-5 minutes, transfer to a white 96-well plate and read the luminescent signal using an ELISA reader.

[0058] Experimental results: In order to further exclude the influence of a small amount of non-classical monocytes expressing CD16a, CD16a blocking antibody 3G8 was added in Example 2 to detect ADCP. CD16a blocking antibody 3G8 can bind to CD16a and block the binding of CD16a to antibody Fc, thus blocking the ADCC effect mediated by CD16a; compared with the group without CD16a blocking antibody, CD16a blocking antibody 3G8 had no effect on the phagocytosis of monocytes (see Figure 3 ). Therefore, it can be determined that there is no interference from ADCC in the ADCP experimental system.

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 2 is mainly in the target cells. The target cells used are Daudi-Luc cells transfected with Luciferase, and specifically include the following steps:

[0061] (1) Isolation of monocytes

[0062] Monocytes were isolated from human peripheral blood mononuclear cells (PBMC) (Mitenyi Biotec, 130-118-906) and used as is.

[0063] (2) Target cells (Daudi-Luc) were plated and added to a 96-well cell culture plate. The number of target cells per well was 5×10 4 pcs, 50μL / well.

[0064] (3) Add pre-diluted Rituximab to a 96-well cell culture plate and incubate at 37°C for 60 min.

[0065] (4) Add monocytes to a 96-well cell culture plate, with the number of monocytes in each well being 5×10 4 pcs, 50μL / well.

[0066] (5) After a second incubation at 37°C for 4-5 hours, add Bright-Lumi TM Luciferase substrate (Biyuntian RG052M) was lysed for 3-5 min, transferred to a white 96-well plate, and the luminescent signal was read using an ELISA reader (see Figure 4 ).according to Figure 4 It can be seen that monocytes were used as effector cells, Luciferase-transfected Daudi cells (Daudi-luc) were used as target cells, and gradiently diluted antibody Rituximab (rituximab, bridging target cells and monocytes) was added for co-culture. The ADCP effect of the antibody was detected, and it was found that the phagocytic rate of monocytes increased with the increase of antibody concentration within a certain antibody concentration range, and then reached an upper platform.

[0067] Comparative Example 2

[0068] This comparative example is different from Example 2 mainly in the difference between the monoclonal antibody and the target cell. The antibody used is the HER2 monoclonal antibody Trastuzumab (Biointron B7432), and the target cell is the HER2 positive tumor cell SKOV-3 (SKOV-3-Luc) transfected with Luciferase. This comparative example changes both the antibody and the target cell to verify the versatility of the experimental system of the present invention. Specifically, the following steps are included:

[0069] (1) Isolation of monocytes

[0070] Monocytes were isolated from human peripheral blood mononuclear cells (PBMC) (Mitenyi Biotec, 130-118-906) and used as is.

[0071] (2) Target cells (SKOV-3-Luc) were plated and added to a 96-well cell culture plate. The number of target cells per well was 2.5×10 4 pcs, 50μL / well.

[0072] (3) Add pre-diluted Trastuzumab to a 96-well cell culture plate and incubate at 37°C for 60 min.

[0073] (4) Add monocytes to a 96-well cell culture plate, with 5×10 monocytes per well. 4 pcs, 50μL / well.

[0074] (5) After a second incubation at 37°C for 4-5 hours, add Bright-Lumi TM Luciferase substrate (Biyuntian RG052M) was lysed for 3-5 min, transferred to a white 96-well plate, and the luminescent signal was read using an ELISA reader (see Figure 5 ).according to Figure 5It can be seen that monocytes were used as effector cells, Luciferase-transfected SKOV3 cells (SKOV3-luc) were used as target cells, and gradiently diluted antibody Trastuzumab (trastuzumab, bridging target cells and monocytes) was added for co-culture. The ADCP effect of the antibody was detected, and it was found that the phagocytic rate of monocytes increased with the increase of antibody concentration within a certain antibody concentration range, and then reached an upper platform.

[0075] Comparative Example 3

[0076] The difference between this comparative example and Example 2 is mainly that the method used is different. The conventional dual fluorescence staining flow method is used to detect the biological activity of ADCP, which specifically includes the following steps:

[0077] (1) Target cell labeling: Daudi target cells were labeled with pHrodo green AM dye and resuspended in PBS at 1×10 6 Cells were added with 1 μM dye and incubated at 37°C for 30 min;

[0078] (2) Add 5 volumes of culture medium (1640 + 10% FBS), incubate at 37°C for 5 min, and terminate labeling;

[0079] (3) Target cell plating: Add target cells to a 96-well cell culture plate, with 5×10 target cells per well. 4 ;

[0080] (4) Add pre-diluted Rituximab to the 96-well cell culture plate and incubate at 37°C for 60 min;

[0081] (5) Monocytes were isolated from human peripheral blood mononuclear cells (Mitenyi Biotec, 130-118-906);

[0082] (6) Monocyte labeling: Resuspend the cells in PBS, 1×10 6 The cells were added with 1 μM Cell Trace Far red (Invitrogen, C34572) (1 μL Cell Trace was added to 1 μL of cells suspended in PBS, the final concentration was 1 μM) and incubated at 37°C for 20 min;

[0083] (7) Add 5 volumes of culture medium (1640 + 10% FBS), incubate at 37°C for 5 min, and terminate labeling;

[0084] (8) Wash cells twice with flow cytometry buffer;

[0085] (9) Monocyte plating: add monocytes to a 96-well cell culture plate containing target cells, and 5×104 ;

[0086] (10) After incubation at 37°C for 4–5 h, centrifuge, wash cells three times with flow cytometry buffer, and resuspend in flow cytometry buffer;

[0087] (11) Flow cytometry was used to detect the proportion of double-positive cells, i.e., monocytes that engulfed target cells. Figure 6 .

[0088] Experimental results: Comparative Examples 1 and 3 were detected using the Luciferase labeling method and the dual fluorescence staining flow cytometry method, respectively, and the detection results were relatively consistent. Figure 1 The Luciferase method of the invention of this patent has a smoother curve, simpler operation steps, shorter time consumption, and does not require fluorescent dyes to label target cells. Figure 6 The ADCP effect of antibodies was detected by flow cytometry. It was found that the phagocytic rate increased with the increase of antibody concentration within a certain antibody concentration range and then reached an upper platform. However, the linear range of the curve was Figure 1 Narrower.

[0089] At present, the main detection method for ADCP is flow cytometry, which requires two fluorescent labels for both effector cells and target cells. In addition, flow cytometry requires repeated washing of cells, which will cause a decrease in cell viability. In addition, the experimental steps are many and the cycle is long, making it difficult to achieve rapid high-throughput detection. In the early stages of antibody drug discovery, a large number of antibodies need to be screened, and a convenient high-throughput ADCP detection method is urgently needed.

[0090] The present invention creatively uses luciferase to transfect target cells. After the target cells are engulfed by macrophages, they enter the lysosomes and are degraded, so that the luciferase can no longer catalyze the substrate to produce fluorescence. The ADCP activity of macrophages can be analyzed by adding luciferase substrate to a 96-well plate and detecting the attenuation of the fluorescence signal using an enzyme marker. And the detection speed is faster, usually reading an enzyme-labeled plate in less than one minute, while a flow cytometer takes 40 to 60 minutes to read a 96-well plate. At the same time, there is no need to use a large amount of fluorescent dyes, only one Luciferase substrate needs to be prepared to transfect all target cells. Furthermore, since different types of high-sensitivity luciferases, such as NanoLuc, have been developed in the art, this method has more room for development.

[0091] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for detecting ADCP biological activity based on single luciferase, characterized in that: The following steps are involved: Step S1, plate the target cells transfected with Luciferase, add pre-diluted Rituximab, and incubate once; Step S2, after the first incubation is completed, monocytes separated from human peripheral blood mononuclear cells are added and then incubated for a second time; Step S3, adding luciferase substrate and detecting the luminescent signal; The monocytes serve as effector cells.

2. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: The target cells are Raji cells transfected with Luciferase.

3. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: In step S1, the number of target cells per well is 1×10 4 ~10×10 4 indivual.

4. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: In step S1, the ratio of target cells to effector cells is 2:

1.

5. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: The temperatures of the first incubation and the second incubation are both 35-38°C.

6. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: The first incubation time is 60 minutes, and the second incubation time is 4 to 5 hours.

7. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: In step S2, the number of mononuclear cells per well was 5×10 4 indivual.

8. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: In the step S3, after adding the luciferase substrate, the cleavage is carried out for 3 to 5 minutes.

9. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: The luciferase substrate is luciferin.

10. The method for detecting ADCP biological activity based on single luciferase according to claim 1, characterized in that: The luminescent signal was detected using a microplate reader.

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