CN114807299B

CN114807299B - Dual-luciferase method, cell and kit for detecting ADC drug activity

Info

Publication number: CN114807299B
Application number: CN202210732636.XA
Authority: CN
Inventors: 徐涵文; 范贝贝; 秦刚
Original assignee: Qiguang Dejian Pharmaceutical Technology Suzhou Co ltd; Genequantum Healthcare Suzhou Co Ltd
Current assignee: Qiguang Dejian Pharmaceutical Technology Suzhou Co ltd; Genequantum Healthcare Suzhou Co Ltd
Priority date: 2022-06-27
Filing date: 2022-06-27
Publication date: 2022-11-18
Anticipated expiration: 2042-06-27
Also published as: CN114807299A

Abstract

The invention provides a dual-luciferase method, cells and a kit for detecting ADC drug activity. The invention provides a novel dual-luciferase method for detecting ADC drug activity based on dual-luciferase activity detection. The detection method of the invention has the same result with the existing flow detection method, and has the advantages of simple operation, high accuracy, high flux, good repeatability, wide applicability and the like.

Description

Dual-luciferase method, cell and kit for detecting ADC drug activity

Technical Field

The invention particularly relates to a dual-luciferase method, cells and a kit for detecting ADC drug activity.

Background

The antibody-conjugated drug (ADC) is formed by connecting a monoclonal antibody and small molecular cytotoxin by a linker, so that the high specificity of the monoclonal antibody and the cell killing activity of the small molecular toxin are combined, the targeting property of the tumor drug is improved, and toxic and side effects are reduced. The general pharmacological mechanism of killing tumor cells by the ADC medicament is that an antibody part in the ADC medicament specifically recognizes and binds to an antigen on the surface of the tumor cells, the ADC medicament is endocytosed into the cells by the tumor cells with positive antigen and degraded in the cells to release micromolecular toxin, and the micromolecular toxin kills the tumor cells with positive antigen. After the antigen positive tumor cells are killed, the micromolecule toxin is released to the periphery of the tumor cells, and the micromolecule toxin with strong membrane permeability can permeate cell membranes of other peripheral cells to further kill the other peripheral cells. ADC drugs are degraded in antigen-positive tumor cells to release small molecule drugs, or release small molecule drugs in extracellular spaces, and the action of killing other cells around the antigen-positive tumor cells is called bystander killing or bystander killing effect.

The parakilling effect of ADC drugs is very important for the killing of solid tumors. The reasons mainly include the following two points: 1. rapid proliferation and multiple gene mutation of tumors, heterogeneous antigen expression or cell subsets with different biological characteristics in the tumors; the parakilling effect can kill tumor cells which do not express or express a low-expression target antigen at the same time, and the action range of the medicine is enlarged; 2. the interior of the tumor contains a large number of mesenchymal cells (fibroblasts, immune and inflammatory cells, glial cells and the like), and the cells provide environments of cytokines, chemokines and immunosuppression for the growth of the tumor and promote the growth of the tumor; the parakilling effect can also kill mesenchymal cells in the tumor, thereby inhibiting the growth of the tumor. Evaluating the parakilling effect of ADC drugs is also one of the important indexes for evaluating ADC drugs at present.

At present, a relatively classic in vitro detection method is, for example, the detection method of trastuzumab conjugates of the first three-co-company Kyoto (Enhertu DS8201 a): inoculating KPL-4 with HER2 positive and MDA-MB-468 cells with HER2 negative into a 6-well plate for co-culture, adding ADC drug after adherence overnight, continuing to culture for 5 days, digesting and collecting cells after the culture is finished, counting the cells and calculating the total number of the cells in each well; staining cells with FITC-labeled anti-HER 2 antibody, washing, and analyzing the proportion of FITC positive cells and FITC negative cells by using a flow cytometer; finally, calculating the absolute values of FITC positive and negative cells according to the total number of the cells; the magnitude of the parakilling effect is mainly determined by the reduction rate of MDA-MB-468 cells. Another method is CN114045325B, which is a method of fluorescence cell counting based on cell imaging to determine the number of the survival negative cells, thereby calculating the proportion of the parakilling.

The existing detection method for the side-killing effect has the following defects:

1. the flow detection method has complicated steps, cannot process a large number of samples at the same time, and is difficult to realize high throughput; cell digestion, counting, staining and flow detection may cause large errors, resulting in inaccurate experimental results and poor repeatability among batches;

2. the fluorescence cell counting method based on cell imaging has the advantages that the flux is improved, but special fluorescence photographing and imaging equipment is needed, and counting deviation can occur due to inaccurate focusing; cells grow in the plate and are not completely uniform, in order to ensure accurate reading, a plurality of or even dozens of areas need to be shot, the reading time is long, the difference between the holes is large due to the change of the cell state sometimes, and the fluorescence counting method has poor applicability to cells growing in clusters;

3. the prior method needs to screen positive cells and negative cells for co-culture according to targets, and different cells selected according to different targets have different required complete culture media and inconsistent growth speeds, so the prior method has no broad-spectrum applicability.

Disclosure of Invention

The invention aims to solve the technical problem of providing a dual-luciferase method which has high flux, good stability, high repeatability and easy operation and is used for detecting the activity of ADC drugs, and the detection method can simultaneously detect the killing effect of the ADC drugs on antigen positive cells and the killing effect (namely the side killing effect) on antigen negative cells.

The invention also aims to provide an antigen negative cell and an antigen positive cell for detecting the ADC pharmaceutical activity, and develop a kit for detecting the ADC pharmaceutical activity, which has the advantages of good stability, wide applicability, simple operation and low cost by using the antigen negative cell and the antigen positive cell.

In order to achieve the purpose, the invention adopts the technical scheme that:

a dual luciferase assay for detecting ADC drug activity:

co-culturing antigen-negative cells and antigen-positive cells in the presence of an ADC drug, and detecting the bystander rate of the ADC drug to the antigen-negative cells and the killer rate of the ADC drug to the antigen-positive cells, wherein the antigen-negative cells carry a first luciferase gene for detecting the antigen-negative cells, the antigen-positive cells carry a second luciferase gene for detecting the antigen-positive cells, and an antigen gene of interest, the antigen gene of interest encodes a target antigen which is specifically bound with an antibody of the ADC drug, and the luciferase encoded by the first luciferase gene and the luciferase encoded by the second luciferase gene are different.

Preferably, the first luciferase gene and the second luciferase gene are independently selected from the group consisting of a firefly luciferase gene, a click beetle luciferase gene, a renilla luciferase gene, or a gauss (Gaussia) luciferase gene.

Preferably, the target antigen comprises a tumor therapy target protein or an immune checkpoint protein.

Preferably, the antibody to the ADC drug comprises a tumor therapeutic or an immune checkpoint inhibitor.

Further preferably, the tumor therapeutic agent comprises a monoclonal antibody, a diabody, an scFv fragment, or an antibody fusion protein.

In the invention, the tumor therapy target protein is preferably a protein overexpressed in tumor cells.

In the present invention, the tumor includes, but is not limited to nasopharyngeal carcinoma, intestinal cancer, liver cancer, gastric cancer, breast cancer, lung cancer, non-small cell lung cancer, melanoma, glioma, gastrointestinal stromal tumor, cervical cancer, ovarian cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, or cholangiocarcinoma.

Specifically, the target antigen includes, but is not limited to, HER2, HER3, EGFR, TROP2, CEACEM5, CD22, SIRPa, PD-L2, PD-L1, TIM3, CTLA4, CD103, LAG3, TIGIT, CD47, B7H3, B7H4, OX40, or VISTA.

Preferably, the antigen-positive cells and the antigen-negative cells are immortalized murine, human or primate cells as host cells.

Further preferably, the host cell is a human a549 cell, heLa cell, 293 cell or primate Vero cell (Vero cell). More preferably, the host cell is a HEK293 cell or a HEK293T cell.

Further preferably, the antigen-positive cells and the antigen-negative cells use the same host cells.

According to some preferred embodiments, the antigen positive cells and the antigen negative cells are HEK293 cells as host cells. Human embryonic kidney HEK293 rarely expresses endogenous receptors required by extracellular ligands, is sensitive to toxins commonly used by ADC drugs, is easy to transfect or infect and has universality.

Preferably, the method for preparing the antigen-negative cell comprises the following steps:

a1 Constructing a first luciferase reporter gene vector comprising a first luciferase gene and a first resistance gene;

a2 B) infecting cells by means of viral transfection using the first luciferase reporter gene vector constructed in step a 1), and obtaining the antigen negative cells by resistance screening using an antibiotic corresponding to the first resistance gene;

and/or, the preparation method of the antigen positive cell comprises the following steps:

b1 Constructing a second luciferase reporter vector comprising a second luciferase gene and a second resistance gene;

b2 Constructing an expression vector of an antigen gene of interest, which comprises the antigen gene of interest and a third resistance gene;

b3 B) infecting cells by means of virus transfection using the second luciferase reporter gene vector constructed in the step b 1) and the target antigen gene expression vector constructed in the step b 2), and obtaining antigen positive cells carrying the second luciferase gene and the target antigen gene simultaneously by resistance screening using an antibiotic corresponding to the second resistance gene and an antibiotic corresponding to the third resistance gene, wherein the antibiotic corresponding to the second resistance gene is different from the antibiotic corresponding to the third resistance gene.

Further preferably, the first resistance gene, the second resistance gene, and the third resistance gene are independently selected from puromycin resistance gene, hygromycin resistance gene (e.g., hygromycin B resistance gene), blasticidin resistance gene (e.g., blistic idin S resistance gene), and bleomycin (Zeocin) resistance gene.

Preferably, the first luciferase reporter gene vector further comprises a first fluorescent protein gene, the second luciferase reporter gene vector further comprises a second fluorescent protein gene, and fluorescent proteins encoded by the first fluorescent protein gene and the second fluorescent protein gene show different fluorescence, so that the fluorescent protein vector can be used for fluorescent cell observation confirmation, photographing and counting, or for confirming the concentration of ADC drugs, and can also be used as quality control or control in detection to verify the accuracy of the dual-luciferase method.

Further preferably, the first fluorescent protein gene and the second fluorescent protein gene are independently selected from a green fluorescent protein gene, a red fluorescent protein gene, and a blue fluorescent protein gene.

According to some preferred embodiments, the first luciferase reporter vector is constructed by: a first luciferase gene, a linker sequence, a first fluorescent protein gene and a first resistance gene are inserted in this order downstream of a promoter (e.g., CMV promoter).

According to some preferred embodiments, the first luciferase reporter gene vector comprises the following nucleic acid sequence in the direction of transcription: a promoter (such as a CMV promoter), a first luciferase gene, a linker sequence, a first fluorescent protein gene, a promoter (such as a PGK promoter) and a first resistance gene, wherein the linker sequence in the first luciferase reporter gene vector is a 2A connecting peptide coding sequence.

Further, the linker sequence in the first luciferase reporter vector is a P2A linker peptide coding sequence, an E2A linker peptide coding sequence, an F2A linker peptide coding sequence, or a T2A linker peptide coding sequence.

Further, in the first luciferase reporter gene vector, the first luciferase gene is a firefly luciferase gene, the linker sequence is a 2A linker peptide coding sequence (e.g., a T2A linker peptide coding sequence), the first fluorescent protein gene is a green fluorescent protein gene, and the first resistance gene is a puromycin resistance gene.

According to some preferred embodiments, the second luciferase reporter vector is constructed by: a second fluorescent protein gene, a linker sequence, a second luciferase gene and a second resistance gene are inserted in this order downstream of a promoter (e.g., CMV promoter).

According to some preferred embodiments, the second luciferase reporter vector comprises the following nucleic acid sequences in order along the direction of transcription: a promoter (e.g., CMV promoter), a second fluorescent protein gene, an IRES linker sequence, a second luciferase gene, a promoter (e.g., PGK promoter), and a second resistance gene.

Further, in the second luciferase reporter gene vector, the second fluorescent protein gene is a red fluorescent protein gene, the linker sequence is an IRES linker sequence, the second luciferase gene is a renilla luciferase gene, and the second resistance gene is a puromycin resistance gene.

According to some preferred embodiments, the method for constructing the target antigen gene expression vector comprises: the target antigen gene, the linker sequence and the third resistance gene are inserted in sequence at the downstream of the promoter (such as CMV promoter).

According to some preferred embodiments, the target antigenic gene expression vector comprises, in order along the direction of transcription, the following nucleic acid sequences: a promoter (such as a CMV promoter), a target antigen gene, a linker sequence and a third resistance gene, wherein the linker sequence in the target antigen gene expression vector is a 2A connecting peptide coding sequence or an IRES linker sequence. In some embodiments, the 2A linker peptide coding sequence in the antigenic gene expression vector of interest is a P2A linker peptide coding sequence, an E2A linker peptide coding sequence, an F2A linker peptide coding sequence, or a T2A linker peptide coding sequence.

Further, the third resistance gene in the target antigen gene expression vector is a hygromycin resistance gene.

Further, the target antigen gene in the target antigen gene expression vector is a HER2 gene, a HER3 gene, an EGFR gene, a TROP2 gene, a CEACEM5 gene, a CD22 gene, a sirpa gene, a PD-L2 gene, a PD-L1 gene, a TIM3 gene, a CTLA4 gene, a CD103 gene, a LAG3 gene, a TIGIT gene, a CD47 gene, a B7H3 gene, a B7H4 gene, an OX40 gene, or a VISTA gene.

According to some specific and preferred embodiments, the screening fluorescence intensity is 10 ⁵ ~10 ⁶ The positive tool cell of (1) (a cell containing the second luciferase gene to be transfected with the antigen gene of interest) is used to prepare the antigen-positive cell.

According to some specific and preferred embodiments, the screening fluorescence intensity is 10 ⁶ ~10 ⁷ The antigen-negative cells of (4) are used for co-culture.

According to some embodiments, the antigen-negative cells and the antigen-positive cells are co-cultured in the presence of the ADC drug, after co-culturing, the first luciferase substrate is added and the first luciferin signal value is detected, then the second luciferase substrate is added and the second luciferin signal value is detected, the parasubstitution rate of the ADC drug to the antigen-negative cells is calculated from the first luciferin signal value, and the killing rate of the ADC drug to the antigen-positive cells is calculated from the second luciferin signal value.

Specifically, the dual-luciferase method for detecting the ADC drug activity specifically comprises the following steps:

1) Setting an experimental group and a control group: the experimental group is characterized in that antigen negative cells and antigen positive cells are co-cultured in the presence of ADC (azodicarbonamide) drugs, the control group is characterized in that the antigen negative cells and the antigen positive cells are co-cultured, and a culture system does not contain the ADC drugs;

2) After the co-culture is finished, using cell lysate to lyse cells of an experimental group and a control group, simultaneously setting a background group, only adding the cell lysate with the same volume to the background group, then respectively adding a first luciferase substrate to the experimental group, the control group and the background group, respectively detecting first luciferin signal values in the experimental group, the control group and the background group, then respectively adding a second luciferase substrate to the experimental group, the control group and the background group, and respectively detecting second luciferin signal values in the experimental group, the control group and the background group;

wherein the first fluorescein signal value in the experimental group is RLU1, the first fluorescein signal value in the control group is RLU1', the first fluorescein signal value in the background group is RLU3, and the parakilling rate of the ADC medicament on antigen-negative cells = (1- (RLU 1-RLU 3)/(RLU 1' -RLU 3)) × 100%;

the second fluorescein signal value in the experimental group was RLU2, the second fluorescein signal value in the control group was RLU2', and the second fluorescein signal value in the background group was RLU3', and the killing rate of the ADC drug against the antigen positive cells = (1- (RLU 2-RLU3 ')/(RLU 2' -RLU3 ')) × 100%.

Further preferably, the method further comprises the step of determining the added concentration of the ADC drug during co-culture, wherein the added concentration of the ADC drug is the concentration of the ADC drug that achieves the maximum killing value for the antigen-positive cells and has no growth inhibition for the antigen-negative cells.

Further preferably, the final concentration of the ADC drug is controlled to be 0-10nM and is not 0; for example, 0.0001 nM,0.001 nM,0.002 nM,0.008 nM,0.01 nM,0.03 nM,0.07 nM,0.1 nM,0.5 nM,1nM, 2 nM,5 nM,6 nM,7 nM,8 nM,10 nM.

Further preferably, the method further comprises the step of determining the ratio of the inoculated number of said antigen positive cells and said antigen negative cells in co-culture.

According to some embodiments, the ratio of the number of the antigen-positive cells to the number of the antigen-negative cells at the time of inoculation is 0.5.

Further preferably, the ratio of the number of the antigen-positive cells to the number of the antigen-negative cells at the time of inoculation is 1 to 1, more preferably 1 to 2.5.

Preferably, the detection method further comprises the step of determining the total number of vaccinations of said antigen positive cells and said antigen negative cells in co-culture.

According to some embodiments, the total number of vaccinations of the antigen-positive cells and the antigen-negative cells is controlled to be 1000 to 2000 per well, such as 1000, 1200, 1400, 1500, 1600, 1800, 2000 per well.

Preferably, the co-culturing is performed using a cell culture plate.

According to some embodiments, the co-cultivation is controlled for a period of 4~7 days.

According to some embodiments, the co-culturing is performed by incubating the antigen positive cells and the antigen negative cells overnight, then adding the ADC drug, and continuing the incubation.

According to some embodiments, the dual luciferase method for detecting ADC drug activity is:

(1) Selecting or constructing antigen negative cells carrying a first luciferase gene according to an ADC medicament to be detected, and selecting or constructing antigen positive cells carrying a target antigen gene and a second luciferase gene simultaneously;

(2) Taking antigen positive cells and antigen negative cells with cell fusion degree of 80-90% and good growth state to determine the addition concentration of the ADC medicament;

(3) Taking the antigen positive cells and the antigen negative cells with the cell fusion degree of 80-90% and good growth state to determine the inoculation number ratio and the total inoculation number of the antigen positive cells and the antigen negative cells;

(4) Paving the antigen positive cells and the antigen negative cells according to the inoculation number ratio and the total inoculation number determined in the step (3) or paving the antigen positive cells and the antigen negative cells directly according to the inoculation number ratio of 0.5 to 1 and the total inoculation number of 1000 to 2000/hole in the step (3), after incubating overnight, adding the ADC drug into culture holes of an experimental group to the final concentration determined in the step (2) or directly adding the ADC drug to the final concentration of 0 to 10nM and not 0 in the step (2), wherein the control group and the experimental group are only different from the control group in that the ADC drug is not added; the experimental group and the control group are incubated for 4~7 days;

(5) Cracking the cells in the experimental group and the control group after the co-culture in the step (4) by using a cell lysate, and setting a background group, wherein the cell lysate with the same volume is only added into the background group;

(6) And detecting a first fluorescein signal value and a second fluorescein signal value in the experimental group, the control group and the background group by using a dual-luciferase activity detection method, calculating the bykilling rate of the ADC medicament to the antigen negative cells according to the first fluorescein signal value, and calculating the killing rate of the ADC medicament to the antigen positive cells according to the second fluorescein signal value.

The invention also provides an antigen-negative cell and an antigen-positive cell for detecting the ADC pharmaceutical activity, wherein the antigen-negative cell and the antigen-positive cell are the antigen-negative cell and the antigen-positive cell in the dual-luciferase method for detecting the ADC pharmaceutical activity.

Preferably, the antigen-negative cell contains a first luciferase reporter gene vector comprising, in order along the direction of transcription, a promoter, a first luciferase gene, a linker sequence, a first fluorescent protein gene, a promoter, and a first resistance gene.

The antigen positive cell contains a second luciferase reporter gene vector and a target antigen gene expression vector.

The second luciferase gene reporter gene vector sequentially comprises a promoter, a second fluorescent protein gene, a connector sequence, a second luciferase gene, a promoter and a second resistance gene along the transcription direction.

The target antigen gene expression vector sequentially comprises a promoter, a target antigen gene, a linker sequence and a third resistance gene along the transcription direction.

According to some preferred embodiments, the antigen-negative cell contains a first luciferase reporter vector comprising, in order in the direction of transcription, the following nucleic acid sequences: the gene comprises a promoter, a firefly luciferase gene, a T2A connecting peptide coding sequence, a green fluorescent protein gene, a promoter and a puromycin resistance gene.

A second luciferase reporter vector in the antigen positive cells comprising the following nucleic acid sequences in order along the direction of transcription: a promoter, a red fluorescent protein gene, an IRES linker sequence, a renilla luciferase gene, a promoter, and a puromycin resistance gene.

The target antigen gene expression vector in the antigen positive cell sequentially comprises the following nucleic acid sequences along the transcription direction: a promoter, a target antigen gene, an IRES linker sequence and a hygromycin resistance gene.

The present invention also provides a kit for detecting ADC pharmaceutical activity, which includes the antigen-negative cells and the antigen-positive cells in the dual-luciferase method for detecting ADC pharmaceutical activity.

Preferably, the kit also comprises reagents required for the dual luciferase activity detection method.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

the invention can simultaneously detect the side killing rate of the ADC medicine to the antigen negative cells and the killing rate to the antigen positive cells, and has the advantages of simple operation process, high sensitivity, high flux, good consistency and broad-spectrum applicability.

Drawings

FIG. 1 is a schematic structural diagram of a firefly luciferase reporter gene vector pLVX-Luc-EGFP;

FIG. 2 is a diagram of the flow analysis of the HEK293-Luc-EGFP cell line (firefly luciferase tool cell) as a negative tool cell;

FIG. 3 is a schematic structural diagram of Renilla luciferase reporter vector pLVX-RFP-Rluc;

FIG. 4 is a diagram of flow analysis of the HEK293-RFP-Rluc cell line (Renilla luciferase tool cells) as a positive tool cell;

FIG. 5 is a schematic structural diagram of a human TROP2 gene overexpression lentiviral vector pLVX-TROP 2-Hyg;

FIG. 6 is a graph showing the cell viability effect of the DS-1062 drug on antigen positive cells (HEK 293-RFP-Rluc-TROP 2-Hyg) and antigen negative cells (HEK 293-Luc-EGFP);

FIG. 7 is a graph of the parakilling efficiency of drug DS-1062 against antigen negative cells (graph A in FIG. 7) and the killing efficiency against antigen positive cells (graph B in FIG. 7) at different cell densities and different cell ratios;

FIG. 8 shows the parakilling efficiency of the drug DS-1062 against antigen-negative cells (graph A in FIG. 8) and the killing efficiency against antigen-positive cells (graph B in FIG. 8) measured by the dual-luciferase method;

FIG. 9 shows the detection of the parakilling efficiency of drug DS-1062 on IRES-Rluc negative cells (Panel A in FIG. 9) and the killing efficiency on Luc-Trop2 positive cells (Panel B in FIG. 9) by the dual luciferase method.

Detailed Description

All of the features disclosed in this specification, or all of the steps of a method or process so disclosed, may be combined in any combination, except combinations where mutually exclusive features or steps are present. The invention will now be further described with reference to specific examples, but the invention should not be limited to these examples, but may be substituted by other equivalent or similarly purposed alternative features unless specifically stated. Unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features. Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The current flow detection method for detecting the activity of ADC drugs has the problems of complex steps, difficulty in realizing high flux, large detection error, low repeatability among batches and the like. In order to solve the problems of the prior art, the inventors of the present application have conducted extensive research and experimental verification to develop a dual-luciferase assay for detecting ADC drug activity, in which antigen-negative cells and antigen-positive cells are co-cultured in the presence of an ADC drug, the antigen-negative cells carry a first luciferase gene, the antigen-positive cells carry a second luciferase gene and an antigen gene of interest, the first luciferase gene is used to detect the antigen-negative cells, the second luciferase gene is used to detect the antigen-positive cells, and the antigen gene of interest encodes a target antigen that specifically binds to an antibody of the ADC drug.

Specifically, the dual-luciferase assay method comprises the following steps:

1) Setting an experimental group and a control group: the experimental group is characterized in that antigen negative cells and antigen positive cells are co-cultured in the presence of ADC drugs, the control group and the experimental group are only different in that the ADC drugs are not used, the antigen negative cells and the antigen positive cells are directly co-cultured, the first luciferase gene and the second luciferase gene are used for distinguishing and detecting the antigen negative cells and the antigen positive cells, and the antigen positive cells and the antigen negative cells are the same in used host cells.

In a specific and preferred embodiment, the methods for preparing antigen negative and positive cells and the dual luciferase assay for the parakilling rate of the ADC drugs on antigen negative cells and the killing rate on antigen positive cells are as follows:

1. vector construction

1) Constructing a firefly luciferase reporter gene vector pLVX-Luc-EGFP, wherein the pLVX-Luc-EGFP sequentially comprises a CMV promoter, a firefly luciferase gene (Luc), a T2A connecting peptide coding sequence, a green fluorescent protein (EGFP) gene, a PGK promoter and a puromycin (puromycin) resistance gene along a transcription direction;

2) Constructing a Renilla luciferase reporter gene vector pLVX-RFP-Rluc, wherein the pLVX-RFP-Rluc sequentially comprises a CMV promoter, a Red Fluorescent Protein (RFP) gene, an IRES linker sequence (internal ribosome entry site sequence), a Renilla luciferase gene (RLuc), a PGK promoter and a puromycin resistance gene along the transcription direction;

3) Constructing a target antigen gene expression vector: taking the TROP2 target as an example, the TROP2 gene expression vector pLVX-TROP2-Hyg sequentially comprises a CMV promoter, a human TROP2 gene, an IRES linker sequence and a hygromycin resistance gene (a hygromycin B resistance gene is selected) along the transcription direction.

The resistance genes carried by the pLVX-Luc-EGFP and the pLVX-RFP-Rluc can be replaced by other resistance genes, and the resistance genes can be the same or different, so that cells successfully transfected with luciferase genes can be screened in the later period.

The resistance gene carried by the target antigen gene expression vector can also be replaced by other resistance genes, but the resistance gene carried by the target antigen gene expression vector is different from the resistance gene carried by pLVX-RFP-Rluc, so that antigen positive cells which are simultaneously and successfully transfected with the luciferase gene and the target antigen gene (namely target gene) can be screened out by different antibiotics in the later period.

Fluorescent protein genes carried by pLVX-Luc-EGFP and pLVX-RFP-Rluc can be replaced by other fluorescent protein genes, but fluorescent proteins coded by the fluorescent protein genes in the two reporter gene vectors show different fluorescence and are used for fluorescent cell observation confirmation, photographing and counting, and the fluorescent protein genes can play a role in quality control or contrast.

Since the focus of the parakilling is to detect antigen-negative cells, it is necessary to reduce the interference of fluorescence displayed by antigen-positive cells with fluorescence displayed by antigen-negative cells without affecting the sensitivity. In the above embodiment, the gene expression downstream of the IRES linker sequence is generally weaker than that of the gene immediately downstream of the promoter, and the gene expression efficiency can be regulated by using the IRES linker, and the renilla luciferase gene in pLVX-RFP-Rluc for displaying antigen-positive cells is located downstream of the IRES sequence, and the firefly luciferase gene in pLVX-Luc-EGFP for displaying antigen-negative cells is located downstream of the CMV promoter, thereby expanding the range of detection; in addition, since the green fluorescent protein EGFP has higher fluorescence intensity and sensitivity than the red fluorescent protein RFP, the EGFP gene can be placed downstream of the T2A linker coding sequence, while the RFP gene is placed directly downstream of the CMV promoter. The inventive combination of the plasmid of the present application uses a T2A linker peptide (or other linker peptide) with an IRES linker to achieve simultaneous compromise: 1) Reducing background noise of renilla luciferase on firefly luciferase; 2) Reduce the background noise of the red fluorescent protein to the green fluorescent protein.

2. Virus package

And respectively co-transferring the constructed plasmids and auxiliary packaging plasmids into HEK293T cells to obtain lentiviruses pLVX-Luc-EGFP, lentiviruses pLVX-RFP-Rluc and lentiviruses pLVX-TROP2-Hyg.

3. Establishment of stably expressing cell lines

Infecting HEK293 cells by using lentivirus pLVX-Luc-EGFP to obtain HEK293-Luc-EGFP cell strains (antigen negative cells) integrated with firefly luciferase genes and EGFP genes; HEK293 cells were infected with lentivirus pLVX-RFP-Rluc to obtain HEK293-RFP-Rluc cell lines having integrated renilla luciferase gene and RFP gene, and then HEK293-RFP-Rluc cell lines were infected with lentivirus pLVX-TROP2-Hyg to obtain HEK293-RFP-Rluc-TROP2-Hyg cell lines (antigen positive cells) having integrated TROP2 gene.

4. Determination of ADC drug concentration by CTG assay

Respectively testing the cell viability of the antigen positive cells and the antigen negative cells under different ADC drug concentrations, and selecting the ADC drug concentration which achieves the maximum killing value on the antigen positive cells and has no growth inhibition on the antigen negative cells.

Respectively taking antigen positive cells and antigen negative cells with fusion degree of 80-90% and good growth state, digesting and counting the cells, adding 100 μ L cell suspension containing 1500 cells into each well of 96-well plate with black wall bottom, placing the cell plate at 37 deg.C and 5% CO ₂ Culturing overnight in a cell culture box; diluting the drug to 400 nM by DMEM complete cell culture medium, sequentially diluting the drug by 5 times of gradient to obtain 10 concentrations, adding 100 mu L of ADC drug with different concentrations into cell wells to obtain an experimental group, and setting a control group (the difference from the experimental group is only that the ADC drug is not added), wherein each experimental group and the control group have 3 wells; standing at 37 deg.C for 5% CO ₂ The cell culture box is continuously cultured for 5 days; taking out the 96-well plate from the incubator, balancing to room temperature, and standing for about 20 min; the medium in the culture well was aspirated, 100. Mu.L of DMEM complete medium at room temperature was added, and the background group was set (only addition ofInto an equal volume of DMEM complete medium); equal volume of CellTiter-Glo was added to the experimental, control and background groups ^TM The reagent is placed in a track shaking table, and the cell is shaken for 20 min at the room temperature at the frequency of 200 revolutions per minute, so as to achieve the purpose of fully cracking the cell; incubating for 10 min at room temperature to stabilize luminescent signals; reading the signal values of the experimental group, the control group and the background group by using an MD enzyme reader, and recording the signal values in relative luminescence units RLU (radio link unit), wherein the experimental group is marked as RLU ₁ The control group was designated as RLU ₂ And the background group is RLU ₃ (ii) a And calculating the survival rate of the cells in the experimental group according to the RLU signal value by the following method: cell viability (%) = (RLU) ₁ -RLU ₃ ）/（RLU ₂ -RLU ₃ ) X 100%; the mean and standard deviation error bars (error bar) were plotted using a Prism scientific plotting tool (Graphpad Prism).

5. Co-incubation of antigen-positive and antigen-negative cells

Respectively taking antigen-positive cells and antigen-negative cells with fusion degree of 80% -90% and good growth state, digesting, counting and diluting the cells to the same concentration, and mixing according to a certain cell ratio (such as antigen-positive cells: antigen-negative cells = 1,1 or 0.5, preferably 2:1; taking a certain amount of mixed cells and adding the mixed cells into a 96-well plate with a black wall bottom (such as 1000, 1500 or 2000 cells per well, and preferably 1500 cells per well); then placed at 37 ℃ and 5% CO ₂ The incubator of (2) for overnight culture; adding 100 μ L ADC drug with final concentration of 1-80 nM, preferably 10nM, setting control group without ADC drug, and continuously placing at 37 deg.C and 5% CO ₂ Cultured in an incubator for 5 days.

6. Detecting the activities of firefly luciferase and renilla luciferase by using a dual-luciferase detection kit

The test can be carried out by using the existing dual-luciferase detection kit, including the dual-luciferase detection kit of manufacturers such as Promega (Promega), kookay Bessel Gene technology (Genecopoeia), bosey Biotechnology Limited (BioAssay Systems) in the United states, and the like, and the operation can be carried out according to the manufacturer's instructions. The invention uses the commonDual-Luciferase reporters (DLR) of Lomega corporation (Promega) ^TM ) Assay System (Dual Luciferase. RTM. Double Luciferase reporter gene detection System) kits were tested.

The cell plate is placed at room temperature for balancing and is placed for about 30 min; the medium in the cell wells was aspirated, 20 μ L of cell lysate was added, and blank background wells were set (only equal volume of cell lysate added); shaking on a track shaking table at 200 rpm for 15 min to fully lyse cells; simultaneously setting parameters of the microplate reader, adopting a chemiluminescence mode, oscillating for 5 seconds, and switching an end point detection mode; adding 100 mu L of Luciferase Assay Reagent II (LAR II) into a cell hole, reading a relative fluorescence value of firefly luciferin by using an enzyme labeling instrument, namely a signal value of an antigen negative cell in the cell hole, and recording the signal value by using a relative luminescence unit RLU; adding 100 mu L of renilla luciferase detection reagent Stop & Glo reagent into the cell wells, reading the relative fluorescence value of renilla luciferin by a microplate reader, namely the signal value of the antigen positive cells in the cell wells, and recording the signal value by relative luminescence unit RLU.

7. Calculating the mortality of antigen-positive and antigen-negative cells (i.e., the killing efficiency of the ADC drug to antigen-positive and antigen-positive cells)

And respectively calculating the cell death rate of the antigen positive cells and the antigen negative cells in each group of experiments according to a formula:

cell death rate (%) = (1- (RLU) ₁ -RLU ₃ ）/（RLU ₂ -RLU ₃ ））×100%，RLU ₁ Represents the experimental group, RLU ₂ Denotes control group, RLU ₃ Representing a background group; the mean and standard deviation error bars (error bars) were plotted using a Prism scientific plotting tool (Graphpad Prism).

The above exemplified dual luciferase assay has the following advantages over the prior art:

1. the method comprises the steps of (1) using a dual-luciferase labeling design, labeling target antigen positive cells with renilla luciferase (renilla luciferase), labeling target antigen negative cells with firefly luciferase (firefly luciferase), co-culturing the antigen positive cells and the antigen negative cells, incubating the antigen positive cells and the antigen negative cells with an ADC (azodicarbonamide) drug, detecting reading values of two kinds of fluorescence simultaneously, and calculating the killing rate of the ADC drug on the antigen positive cells and the side killing rate of the ADC drug on the antigen negative cells;

2. compared with a flow cytometry detection method and a fluorescence cell counting detection method, the dual-luciferase method is based on mutual reaction luminescence of enzyme and substrate, has strong specificity and high sensitivity, and has higher signal-to-noise ratio due to no non-specific interference of exciting light;

3. compared with a flow cytometry method and a fluorescent cell counting method, the dual-luciferase method has the advantages of simple operation process, high flux and better consistency;

4. through molecular cloning design, the expression quantities of renilla luciferase and firefly luciferase are adjusted to a reasonable range, and the method is suitable for simultaneous detection; meanwhile, antigen positive cells expressing renilla luciferase are also marked by Red Fluorescent Protein (RFP), and antigen negative cells expressing firefly luciferase are also marked by green fluorescent protein (EGFP), so that the method can be used for observing, confirming, photographing and counting fluorescent cells; as a control, there were two sets of detection methods;

5. the universality is improved by selecting the human embryonic kidney cell HEK293, the cell rarely expresses an endogenous receptor required by an extracellular ligand, is sensitive to a toxin commonly used by ADC and is easy to transfect or infect; the HEK293 cell can stably express firefly luciferase and can be widely applied to antigen negative cells; the HEK293 cell can stably express renilla luciferase and target antigen and can be constructed into an antigen positive cell;

6. because the antigen negative cells and the antigen positive cells are constructed from the HEK293 cells, the growth rate of the cells is relatively consistent with the sensitivity of the cells to the same small molecule drugs, and although some adjustment may be needed according to the influence of targets, the proportion of the total antigen negative cells and the antigen positive cells is relatively fixed, and the subsequent experiment searching process is relatively simple.

The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry, for example, the cell culture conditions, culture media and the like which are not noted are conventional conditions and conventional culture media. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

Example 1: establishment of HEK293-Luc-EGFP cell line

1) The firefly luciferase reporter gene vector pLVX-Luc-EGFP (the plasmid map of which is shown in figure 1) containing the firefly luciferase gene and the green fluorescent protein gene is constructed, and comprises the firefly luciferase gene (firefly luciferase), a T2A connecting peptide coding gene, the green fluorescent protein (EGFP) gene and a Puromycin (Puromycin) resistance gene. Co-transferring a firefly luciferase reporter gene vector pLVX-Luc-EGFP and an auxiliary plasmid into HEK293T cells, collecting virus supernatant (containing lentivirus pLVX-Luc-EGFP) after 72h, filtering with a 0.45 mu m filter membrane, subpackaging, and storing at-80 ℃ for later use.

2) Reviving HEK293 cells: the cryovial with the HEK293 cells frozen therein was removed from the liquid nitrogen, immediately placed in a 37 ℃ water bath and gently shaken. After complete thawing (about 1 min), the cell suspension was transferred to a centrifuge tube of 15 mL containing 5 mL complete medium and centrifuged at 1500 rpm for 5 min; pouring out the supernatant, suspending the cells by using 1 mL complete culture medium, transferring the cells to a T75 culture bottle filled with 10 mL complete culture medium, and slightly shaking front and back and left and right to uniformly distribute the cells in the culture bottle; put at 37 ℃ and contain 5% of CO ₂ The cells were cultured in an incubator (preparation of complete medium: DMEM (high sugar) +10% Fetal Bovine Serum (FBS)).

3) Digesting HEK293 cells in logarithmic growth phase by trypsin, counting the cells, inoculating the cells into a T25 cell culture flask, and adjusting the cell number to be 1.0 multiplied by 10 ⁶ Cells/vial; according to the result of the measurement of the lentivirus titer, an appropriate amount of lentivirus pLVX-Luc-EGFP is added to make the multiplicity of infection (MOI) 1, and polybrene (polybrene) with a final concentration of 4 mug/mL is added; after infection with lentivirus 8 h, virus-containing cells are removedAdding 6 mL fresh complete culture medium again; continuously culturing 48 h, then transferring to a T75 cell culture bottle, adding antibiotic puromycin with the final concentration of 2 mug/mL for screening; after further culturing for 5 days, the cells expressing the green fluorescent protein were sorted by flow cytometry, and the green fluorescence intensity was collected at 10 ⁶ The HEK293-Luc-EGFP cell line, which is a negative tool cell and into which a firefly luciferase gene and a green fluorescent protein gene were integrated, was obtained from the left and right cells (FIG. 2).

Example 2: establishment of HEK293-RFP-Rluc cell strain

1) The Renilla luciferase reporter vector pLVX-RFP-Rluc (plasmid map, see FIG. 3) containing the Renilla luciferase gene and the red fluorescent protein gene, which comprises the Red Fluorescent Protein (RFP) gene, the IRES linker sequence, the Renilla luciferase gene (renilla luciferase) and the Puromycin (Puromycin) resistance gene, was constructed. Co-transferring a Renilla luciferase reporter gene vector pLVX-RFP-Rluc and an auxiliary plasmid into HEK293T cells, collecting virus supernatant after 72h, filtering by a 0.45 mu m filter membrane (containing lentivirus pLVX-RFP-Rluc), subpackaging, and storing at-80 ℃ for later use.

2) Reviving HEK293 cells: the cryovial with the HEK293 cells frozen therein was removed from the liquid nitrogen, immediately placed in a 37 ℃ water bath and gently shaken. After complete thawing (about 1 min), the cell suspension was transferred to a centrifuge tube of 15 mL containing 5 mL complete medium and centrifuged at 1500 rpm for 5 min; pouring out the supernatant, suspending the cells by using a1 mL complete culture medium, transferring the cells into a T75 culture bottle filled with a 10 mL complete culture medium, and slightly shaking the cells back and forth and left and right to uniformly distribute the cells in the culture bottle; at 37 deg.C, with 5% CO ₂ Culturing in an incubator.

3) Trypsinizing HEK293 cells in logarithmic growth phase, counting the cells, inoculating the cells into a T25 cell culture flask, and adjusting the number of the cells to 1.0X 10 ⁶ Cells/vial; according to the result of the lentivirus titer determination, adding an appropriate amount of lentivirus pLVX-RFP-Rluc to make the multiplicity of infection (MOI) be 1, and simultaneously adding polybrene (polybrene) with the final concentration of 4 mug/mL; after infection with lentivirus 8 h,removing the cell culture medium containing the virus, and adding fresh complete culture medium of 6 mL again; continuously culturing 48 h, then transferring to a T75 cell culture bottle, adding antibiotic puromycin with the final concentration of 2 mug/mL for screening; after further culturing for 5 days, the cells expressing the red fluorescent protein were sorted by flow cytometry, and the red fluorescence intensity was collected at 10 ⁵ Left and right cells, a positive tool cell line HEK293-RFP-Rluc cell line integrated with a red fluorescent protein gene and a Renilla luciferase gene was obtained (FIG. 4).

Example 3: establishment of HEK293-RFP-Rluc-TROP2-Hyg cell strain

1) A human TROP2 gene-overexpressing lentiviral vector pLVX-TROP2-Hyg (plasmid map, see FIG. 5) containing a human TROP2 gene, an IRES linker sequence and a hygromycin B resistance gene was constructed. Co-transferring the pLVX-TROP2-Hyg overexpression lentivirus vector and the helper plasmid into HEK293T cells, collecting virus supernatant (containing the lentivirus pLVX-TROP 2-Hyg) after 72 hours, filtering by a 0.45 mu m filter membrane, subpackaging and storing at-80 ℃ for later use.

2) Digesting HEK293-RFP-Rluc cells in logarithmic growth phase by trypsin, counting the cells, inoculating the cells into a T25 cell culture flask, and adjusting the cell number to be 1.0 multiplied by 10 ⁶ Cells/vial; according to the result of the measurement of the lentivirus titer, an appropriate amount of lentivirus pLVX-TROP2-Hyg was added to make the multiplicity of infection (MOI) 1, and polybrene (polybrene) was added to a final concentration of 4. Mu.g/mL; after infecting lentivirus 8 h, removing the cell culture medium containing virus, and adding fresh complete culture medium of 6 mL again; continuously culturing 48 h, then transferring to a T75 cell culture bottle, adding antibiotic puromycin with the final concentration of 5 mug/mL and antibiotic hygromycin B with the final concentration of 5 mug/mL for screening; meanwhile, adding the antibiotic puromycin with the final concentration of 5 mug/mL and the antibiotic hygromycin B with the final concentration of 5 mug/mL into the positive tool cell HEK293-RFP-Rluc which is not infected with the virus, and taking the antibiotic puromycin and the antibiotic hygromycin B as a control group for drug screening; culturing for 10 days, and after the cells of the control group are completely killed by the antibiotics, obtaining the HEK293-RFP-Rluc-TROP2-Hyg cell strain integrated with the red fluorescent protein, the renilla luciferase gene and the human TROP2 gene.

Example 4: in vitro cell viability assay

The in vitro killing effect of the ADC medicament DS-1062 on antigen positive cells and antigen negative cells is respectively measured by a cell viability test of the following scheme, so that the appropriate ADC medicament concentration is selected to enable the medicament to have the maximum killing effect on the antigen positive cells, and not to have the killing effect on the antigen negative cells.

1) Add 100. Mu.L of cell culture medium containing 1500 antigen positive cells (HEK 293-RFP-Rluc-TROP 2-Hyg) or antigen negative cells (HEK 293-Luc-EGFP) per well of a 96-well black-walled bottom-permeable plate containing the corresponding selection antibiotics at 37 ℃ with 5% CO ₂ Was incubated overnight in the incubator of (1).

2) The treatment group and the control group (the control group is different from the experimental group only in that the ADC drug DS-1062 is not added) were set using the ADC drug DS-1062 (synthesized by wuxi drug minkangde new drug development gmbh). Experimental group settings were: diluting ADC drug DS-1062 with complete culture medium, wherein the highest drug concentration is 400 nM, diluting in 5-fold gradient, and adding 100 μ L of drugs with different concentrations into the cell hole to make the highest final concentration of the drug 200 nM; place the cell plate at 37 ℃ in 5% CO ₂ Was incubated in the incubator of (1) for 5 days.

3）CellTiter-Glo ^TM And (3) cell viability detection: in the experiment, cellTiter-Glo luminescence method cell activity detection kits (CellTiter-Glo) of Promega is adopted ^TM Luminecent Cell Viability Assay kit) according to the following steps in the specification: the cell plates were removed from the incubator, equilibrated to room temperature, and the (serum-free) DMEM medium and CellTiter-Glo were simultaneously incubated ^TM Taking out the reagent, and balancing to room temperature for about 30 min; the cell supernatant was removed, 100. Mu.L of DMEM medium was added, and an equal volume of CellTiter-Glo was added ^TM Reagents, together with a blank background set (adding only equal volumes of DMEM medium and CellTiter-Glo) ^TM Reagents); shaking and mixing the contents on a track shaker at a speed of 200 rpm for 20 min to induce cell lysis, and incubating at room temperature for 10 min to stabilize luminescent signals; collecting luminescence signal value on MD enzyme-labeling instrument as RLU relative luminescence unit record, where the experimental group is RLU ₁ The control group was designated as RLU ₂ And the background group is RLU ₃ (ii) a Calculating the survival rate of the cells in the experimental group according to the RLU signal value, and calculating the survival rate of the cells in each experimental hole according to the luminescence signal value of the control hole, wherein the calculation method comprises the following steps: cell viability (%) = (RLU) ₁ -RLU ₃ ）/（RLU ₂ -RLU ₃ ) X 100%, the survival rates of antigen positive cells and antigen negative cells under different concentrations of ADC drugs were calculated, and plotted with a Prism scientific drawing tool (Graphpad Prism) using a mean and standard deviation error bar (error bar) (fig. 6). As shown in fig. 6, the ADC drug DS-1062 reached maximum lethality against antigen positive cells at concentrations above 1nM; only at the highest concentration point of 200 nM showed lethality against antigen negative cells, and DS-1062 concentration below 100 nM showed no significant inhibition against antigen negative cells. In general, for cell lines sensitive to a certain ADC drug, the concentration of the drug reaching the maximum cell killing is generally not more than 10nM, and the concentration point of 10nM is selected as the drug concentration for cell killing.

Example 5: determination of the Total number and proportion of cells inoculated during Co-culture of antigen-Positive cells and antigen-negative cells

1) To each well of a flat-bottom transparent 96-well plate, 100. Mu.L of a suspension containing 1000 to 2000 cells (sum of the number of antigen-positive cells and antigen-negative cells) was added, and in this example, 1000, 1500, 2000 cells were set per well, and the ratio of antigen-positive cells to antigen-negative cells was 4: 1.2: 1. 1: 1. 0.5:1; cell plates at 37 ℃ 5% CO ₂ Was incubated overnight in the incubator.

2) Diluting the drug with complete culture medium, adding 100 μ L of ADC drug DS-1062 into the cell well of the experimental group, and setting the control group; at 37 deg.C, 5% CO ₂ The final concentration of the drug in the experimental group of the present example was 10nM, and the control group and the experimental group were different only in that no ADC drug was added to the control group.

3) Flow cytometry analysis of lethality of ADC drug DS-1062: removing culture medium from the cell plate, adding 50 μ L of 0.25% trypsin per well, and digesting for 5 min;adding 100 μ L complete culture medium to stop digestion, and blowing to obtain single cell; transferring the cells to a V-bottom 96-well plate, and centrifuging at 1500 rpm for 5 min; removing the supernatant, and adding 50 μ L PBS to each well to resuspend the cells; analyzing and recording the number of antigen positive cells expressing red fluorescent protein and antigen negative cells expressing green fluorescent protein in the experimental group and the control group by a flow cytometer (the experimental group is C) ₁ Labeled, control group with C ₂ Marker), the cell death rates of the antigen-positive cells and the antigen-negative cells (namely the killing rate of DS-1062 to the antigen-positive cells and the killing rate of DS-1062 to the antigen-negative cells) are respectively calculated by the following formula: cell death rate (%) = (1-C) ₁ /C ₂ ) X is 100%; the mean and standard deviation error bars (error bar) were plotted using a Prism scientific plotting tool (Graphpad Prism) (fig. 7). As shown in panel B of FIG. 7, DS-1062 achieved the highest lethality of antigen-positive cells in all experimental groups at the drug concentration of 10 nM; as shown in a diagram in fig. 7, in the lethality analysis of the antigen-negative cells, it was found that the ratio of the antigen-positive cells to the antigen-negative cells was 1:1 or 0.5:1, the side killing effect of the medicine on antigen negative cells is weaker than that of other experimental groups, and the optimal side killing effect is not achieved; at a cell ratio of 4:1 and 2:1, the parakilling effect is better, because the cell ratio is 2:1, more antigen-negative cells exist, and the parakilling effect has higher accuracy and sensitivity, so that the ratio of the selected antigen-positive cells to the selected antigen-negative cells is 2:1; in comparison of the total number of different cell inoculations, 1500 or 2000 cells per well achieved higher and similar lethality, and 1500 cells per well were selected in experiments to verify ADC drug lethality, with a ratio of antigen positive cells to antigen negative cells of 2:1.

because the invention uses the tool cells HEK293-luc-EGFP and HEK293-RFP-Rluc and is established by expressing the target to be detected, the basic genetic phenotype of the cells is the same, the growth speed is similar, the ratio of antigen positive cells to antigen negative cells is 2:1 has certain versatility.

In addition, the target of the ADC drug targeting, such as HER2 or EGFR, often has the effect of promoting tumor, the growth rate of the antigen positive cells may increase, which results in that the total number of cells at the end of the experiment and the ratio of the antigen positive cells to the antigen negative cells are higher than those of the currently set experimental conditions, but the results show that a good experimental result can be obtained even when the ratio of 2000 cells per well or the ratio of the antigen positive cells to the antigen negative cells is 4:1, so that the increase in the growth rate of the antigen positive cells has little influence on the experimental result under the appropriate initial number of cells per well and the ratio of the antigen positive cells to the antigen negative cells, and thus the scheme has strong robustness and at the same time has universality.

Example 6: double-fluorescein method for detecting ADC (azodicarbonamide) medicament side-killing effect

At 1500 cells per well, the ratio of antigen positive to antigen negative cells was 2:1, the killing efficiency of the ADC drug DS-1062 to antigen positive cells and antigen negative cells at drug concentrations of 0.1nM, 1nM and 10nM was tested by the dual luciferase method.

1) Adding 100 mu L of suspension containing 1500 cells (the sum of the number of antigen positive cells and antigen negative cells) into each well of a 96-well black-wall bottom-penetrating plate, wherein the number ratio of the antigen positive cells to the antigen negative cells is 2:1; cell plates at 37 ℃ 5% CO ₂ Was incubated overnight in the incubator of (1).

2) Drug was diluted with complete medium, 100. Mu.L of drug was added to the cell wells, and a control group containing no drug in medium was set up at 37 ℃ with 5% CO ₂ Incubating for 5 days in the incubator; the final concentration of drug in this example was selected to be 0.1nM, 1nM and 10nM for comparison.

3) The double luciferase method is adopted to detect the lethality of the medicine: operating according to the experimental method of the Promega dual-luciferase detection kit, completely sucking the culture medium in the cell plate, adding a cell solution to lyse cells in the control holes and the experimental holes, and lysing; adding a firefly luciferase detection reagent LAR II, collecting a luminescence signal value of an antigen-negative cell, recording the luminescence signal value by relative luminescence units of RLU, recording the test group as RLU1, recording the control group as RLU1', recording the background group as RLU3, recording the antigen-negative cell death rate (namely the bykilling rate of the ADC medicament to the antigen-negative cell) = (1- (RLU 1-RLU 3)/(RLU 1' -RLU 3)). Times.100%, and drawing a graph by using a Prism scientific research and drawing tool (Graphpad Prism) according to a mean value and a standard deviation error bar (error bar) (A picture in figure 8); adding a Renilla luciferase detection reagent Stop & Glo reagent, collecting a luminescence signal value of antigen-positive cells, recording by relative luminescence units of RLU, recording the test group as RLU2, the control group as RLU2', the background group as RLU3', and the antigen-positive cell death rate (namely the killing rate of the ADC drug on the antigen-positive cells) = (1- (RLU 2-RLU3 ')/(RLU 2' -RLU3 ')) × 100%, and drawing by using a Prism scientific research drawing tool (Graphpad Prism) and standard deviation error bars (error bars) (B picture in FIG. 8). As shown in FIG. 8, the dual luciferase method is used for detecting the lethality of DS-1062 to antigen positive cells and antigen negative cells at different concentration points, and when the lethality is 0.1nM, the medicament does not reach the maximum killing effect on antigen positive cells, so that the parakilling effect on antigen negative cells is weakest; when the drug concentration is 1nM and 10nM, the lethality to antigen positive cells reaches the maximum value, and the death rate of the antigen positive cells is about 95 percent; the lethality to the antigen negative cells is in direct proportion to the concentration of the medicament, and the bykilling rate to the antigen negative cells is about 40% at the concentration of 10 nM; the above results also reflect that the killing of antigen negative cells by ADC drugs is caused by the collateral killing effect of the drugs after killing antigen positive cells.

In this example, the killing efficiency of the drug was measured using 3 drug concentrations, and the results show that: in 3 experimental groups with different concentrations, the killing of the antigen positive cells and the antigen negative cells by the medicament is positively correlated, because the antigen positive cells and the antigen negative cells in the same cell hole are detected, the side killing effect caused by the killing of the antigen positive cells by the medicament can be better explained.

Comparative example 1

In order to verify the accuracy, sensitivity and universality of the detection of the killing effect of the ADC drug by using the method for expressing the tool cells and the target points, the comparative example adopts a classical flow cytometry method for detection and compares the detection result with a dual-luciferase method. In this comparative example, reference is made to a method for detecting a killing effect by a first three-Co-Ltd trastuzumab conjugate (Enhertu, DS8201 a): the method comprises the steps of inoculating TROP2 positive tumor cells BxPC-3 and TROP2 negative tumor cells HEPG2 into a 6-well plate for co-culture, adding ADC medicine DS-1062 after the cells are attached to the wall overnight, continuing to culture for 5 days, digesting and collecting the cells after the culture is finished, counting the cells and calculating the total number of the cells in each well, staining the cells by using FITC marked anti-TROP 2 antibodies, washing, analyzing the proportion of FITC positive and negative cells by using a flow cytometer, calculating the absolute value of the FITC positive and negative cells according to the total number of the cells, and detecting the killing effect of the ADC medicine on the tumor cells and the parakilling effect on the negative cells by using antigen positive tumor cells BxPC-3 and antigen negative tumor cells HEPG2 which endogenously express TROP2, wherein the results are shown in Table 1.

As shown in Table 1, antigen-positive tumor cells BxPC-3 and antigen-negative tumor cells HEPG2 were 15X 10 cells per well ⁴ Culturing the cells in a 6-well plate, wherein the ratio of antigen positive tumor cells BxPC-3 to antigen negative tumor cells HEPG2 is 2:1, the treatment concentration of the ADC drug DS-1062 is 10nM, and the result shows that the killing rate of DS-1062 on positive tumor cells BxPC-3 is 93.1%; the bykilling rate to the antigen-negative tumor cell HEPG2 is 34.6%; the result is more consistent with the result detected by a dual-luciferase method, and the killing effect of the ADC medicament on tumor antigen positive and antigen negative cells can be further proved by comparing the killing power of the tumor antigen positive and negative tumor cells with the killing power of the medicament antigen negative tumor cells.

Classical flow cytometry requires digestion of cells, counting and counting the total number of cells; then, incubating and rinsing the antigen positive cells in the mixed cell suspension by the antibody; then, incubating and rinsing by using a fluorescence-labeled secondary antibody; performing flow analysis after re-suspending, and finally calculating the number of cells according to the proportion of the antigen positive cells and the antigen negative cells so as to calculate the killing power of the medicament on the antigen positive cells and the antigen negative cells; the flow method has complicated steps and long time consumption, and is difficult to realize high flux and high repeatability. By adopting the dual-luciferase detection system, the lethality of the medicament to the antigen positive cells and the antigen negative cells can be simultaneously detected in the same cell hole, the cell treatment steps are few, the operation is simple, the experimental error can be reduced, the repeatability is high, and high flux can be realized.

The detection method provided by the invention has the same detection result as that of the first three-common corporation, and has the advantages of good accuracy, high repeatability, good stability and broad-spectrum applicability.

Comparative example 2

Infecting the HEK293-Luc-EGFP tool cells established in example 1 with the lentivirus pLVX-TROP2-Hyg established in example 3 to construct antigen positive cells (Luc-TROP 2 positive cells) highly expressing firefly luciferase, while using the HEK293-RFP-Rluc cells linked to the Renilla luciferase gene by an IRES linker established in example 2 as negative cells (IRES-Rluc negative cells); luc-Trop2 positive cells and IRES-Rluc negative cells were plated at a rate of 2:1, 1500 cells per well, incubated for 5 days in the presence of ADC drug DS1062, with DS1062 concentrations set at 10nM,1nM and 0.1nM; referring to example 6, the dual luciferase assay kit was used to detect the killing power of the drug on Luc-Trop 2-positive cells and IRES-Rluc-negative cells at different concentrations.

The results are shown in FIG. 9: when the concentration of the drug is 0.1nM, the drug does not reach the maximum killing effect on Luc-Trop2 positive cells, and the side killing effect on IRES-Rluc negative cells is weaker; when the drug concentration is 1nM and 10nM, the lethality to Luc-Trop2 positive cells reaches the maximum value, and the death rate of the positive cells is about 95 percent; the side-killing effect of the drug on IRES-Rluc negative cells is proportional to the drug concentration. However, the result of the combination mode of the antigen positive cells of the Luc-Trop2 highly expressing the firefly luciferase and the antigen negative cells of the IRES-Rluc lowly expressing the Renilla luciferase gene shows that the bykilling rate of the DS1062 to the antigen negative cells is about 25 percent and is obviously lower than the detection results of the classical methods of the example 6 and the comparative example 1, which shows that the detection result of the bykilling rate of the antigen negative cells is lower in the combination mode of the Luc-Trop2 positive cells and the IRES-Rluc negative cells. The reason for the analysis may be: IRES-Rluc negative cells are connected with renilla luciferase through IRES connectors, the expression level of the renilla luciferase is low, the number of the negative cells in an experiment is small, the total signal value of the renilla luciferase is small due to the synthesis, the change of a signal value corresponding to the renilla luciferase is small under the partial killing of a drug on the negative cells, and the parakilling rate calculated according to the signal value is low. In addition, the use of a tool cell that highly expresses firefly luciferase to construct antigen-positive cells leads to a detection result that exceeds the upper limit of detection of firefly luciferase due to the high expression of firefly luciferase and the large number of positive cells, resulting in a limitation of the degree of freedom of the total number of cells in the experiment.

In conclusion, the HEK293-Luc-GFP highly expressing firefly luciferase is used as an antigen negative cell, the HEK293-RFP-Rluc lowly expressing renilla luciferase is used as an antigen positive tool cell, the antigen positive tool cell is used as an antigen positive cell after expressing a target antigen gene, and the antigen negative cell and the antigen positive cell are co-cultured in the presence of an ADC medicament, so that the lethality and the parakilling effect of the ADC medicament can be accurately and efficiently reflected, and the stability and the repeatability are good.

Claims

1. A dual-luciferase method for detecting ADC drug activity is characterized in that antigen-negative cells and antigen-positive cells are co-cultured in the presence of an ADC drug, and the bystander rate of the ADC drug to the antigen-negative cells and the killer rate of the ADC drug to the antigen-positive cells are detected, wherein the antigen-negative cells carry a first luciferase gene, the antigen-positive cells carry a second luciferase gene and an antigen gene of interest, the first luciferase gene is used for detecting the antigen-negative cells, the second luciferase gene is used for detecting the antigen-positive cells, the antigen gene of interest encodes a target antigen which is specifically combined with an antibody of the ADC drug, the first luciferase gene is a firefly luciferase gene, the second luciferase gene is a renilla luciferase gene, and host cells used by the antigen-positive cells and the antigen-positive cells are HEK293 cells;

the preparation method of the antigen negative cell comprises the following steps:

a1 Constructing a first luciferase reporter vector comprising in the direction of transcription the following nucleic acid sequences: a promoter, a first luciferase gene, a 2A linker peptide coding sequence, a first fluorescent protein gene, a promoter, and a first resistance gene;

a2 Using the first luciferase reporter gene vector constructed in the step a 1) to infect cells by means of viral transfection; obtaining the antigen negative cell by resistance screening by adopting an antibiotic corresponding to the first resistance gene;

the preparation method of the antigen positive cell comprises the following steps:

b1 Constructing a second luciferase reporter vector comprising the following nucleic acid sequences in order along the direction of transcription: a promoter, a second fluorescent protein gene, an IRES linker sequence, a second luciferase gene, a promoter, and a second resistance gene;

b3 B) infecting cells by means of viral transfection using the second luciferase reporter gene vector constructed in step b 1) and the target antigen gene expression vector constructed in step b 2), and obtaining antigen-positive cells carrying both the second luciferase gene and the target antigen gene by resistance screening using an antibiotic corresponding to the second resistance gene and an antibiotic corresponding to the third resistance gene,

wherein the antibiotic corresponding to the second resistance gene is different from the antibiotic corresponding to the third resistance gene, and the fluorescent proteins encoded by the first fluorescent protein gene and the second fluorescent protein gene show different fluorescence.

2. The dual luciferase method for detecting ADC pharmaceutical activity of claim 1, wherein the target antigen comprises a tumor therapy target protein or an immune checkpoint protein.

3. The dual luciferase method for detecting ADC drug activity of claim 1, wherein the target antigen comprises HER2, HER3, EGFR, TROP2, CEACEM5, CD22, SIRPa, PD-L2, PD-L1, TIM3, CTLA4, CD103, LAG3, TIGIT, CD47, B7H3, B7H4, OX40 or VISTA.

4. The dual luciferase method for detecting ADC drug activity of claim 1, wherein the first resistance gene, the second resistance gene, the third resistance gene are each independently selected from puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, or bleomycin resistance gene;

and/or the first fluorescent protein gene and the second fluorescent protein gene are respectively and independently selected from a green fluorescent protein gene, a red fluorescent protein gene or a blue fluorescent protein gene.

5. The dual luciferase method for detecting ADC drug activity according to claim 1, wherein the target antigen gene expression vector comprises the following nucleic acid sequences in sequence along the transcription direction: the gene comprises a promoter, a target antigen gene, a linker sequence and a third resistance gene, wherein the linker sequence in the target antigen gene expression vector is a 2A connecting peptide coding sequence or an IRES linker sequence.

6. The dual luciferase method for detecting ADC pharmaceutical activity according to claim 1, wherein the first fluorescent protein gene is a green fluorescent protein gene, and the first resistance gene is a puromycin resistance gene;

the second fluorescent protein gene is a red fluorescent protein gene, and the second resistance gene is a puromycin resistance gene; the third resistance gene is a hygromycin resistance gene, and the linker sequence in the target antigen gene expression vector is an IRES linker sequence.

7. The dual luciferase method for detecting ADC drug activity according to any one of claims 1 to 6, wherein the antigen negative cells and the antigen positive cells are co-cultured in the presence of ADC drug, after co-culture is finished, a first luciferase substrate is added and a first luciferin signal value is detected, then a second luciferase substrate is added and a second luciferin signal value is detected, the parakilling rate of the ADC drug on the antigen negative cells is calculated according to the first luciferin signal value, and the killing rate of the ADC drug on the antigen positive cells is calculated according to the second luciferin signal value.

8. The dual luciferase method for detecting ADC drug activity according to claim 7, comprising the steps of:

1) Setting an experimental group and a control group: the experimental group is characterized in that antigen negative cells and antigen positive cells are co-cultured in the presence of ADC (azodicarbonamide) drugs, the control group is characterized in that the antigen negative cells and the antigen positive cells are co-cultured, and the culture system does not contain the ADC drugs;

9. The dual luciferase method for detecting ADC drug activity according to claim 8, wherein the final concentration of the ADC drug is controlled to be 0 to 10nm and is not 0;

and/or, controlling the number ratio of the antigen positive cells to the antigen negative cells in inoculation to be 0.5;

and/or controlling the total inoculation number of the antigen positive cells and the antigen negative cells to be 1000-2000 cells/hole.

10. Antigen-negative cells and antigen-positive cells for detecting ADC pharmaceutical activity, wherein the antigen-negative cells and the antigen-positive cells are the antigen-negative cells and the antigen-positive cells in the dual-luciferase method for detecting ADC pharmaceutical activity according to any one of claims 1 to 9.

11. A kit for detecting ADC pharmaceutical activity, characterized in that it comprises the antigen-negative cells and the antigen-positive cells in the dual-luciferase method for detecting ADC pharmaceutical activity according to any one of claims 1 to 9.