CN113009157B - RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application thereof - Google Patents

RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application thereof Download PDF

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CN113009157B
CN113009157B CN202110308027.7A CN202110308027A CN113009157B CN 113009157 B CN113009157 B CN 113009157B CN 202110308027 A CN202110308027 A CN 202110308027A CN 113009157 B CN113009157 B CN 113009157B
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ctla
monoclonal antibody
biological activity
reporter gene
cells
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CN113009157A (en
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王兰
刘春雨
于传飞
杨雅岚
崔永霏
段茂芹
俞小娟
徐苗
王军志
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National Institutes for Food and Drug Control
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Abstract

The invention discloses an RGA method for detecting the biological activity of an anti-CTLA-4 monoclonal antibody and application thereof. The method comprises the steps of constructing and obtaining target cells for stably expressing the CD3scFv and effector cells for stably expressing the CTLA-4 gene and the reporter gene, mixing the target cells and the effector cells according to a certain proportion, adding a drug sample of the anti-CTLA-4 monoclonal antibody and a reference substance to activate a signal path, and fitting a four-parameter curve according to a measured signal value of the reporter gene to determine the biological activity of the antibody. The invention establishes a quantitative detection method with accuracy, sensitivity, rapidness, strong specificity and high durability aiming at the biological activity detection of the anti-CTLA-4 monoclonal antibody, and has important significance on the quality control and clinical application of the anti-CTLA-4 monoclonal antibody.

Description

RGA method for detecting biological activity of anti-CTLA-4 monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, relates to a detection method of biological activity, and particularly relates to an RGA method for detecting the biological activity of an anti-CTLA-4 monoclonal antibody and application thereof.
Background
Cytotoxic T Lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, is located in 3-band (2q33) of long arm 3 of chromosome 2, encodes 233 amino acids, and is one member of immunoglobulin-associated receptor family. CTLA-4 is expressed on the surface of activated T cells, and the high expression of the CTLA-4 in the T cells can obviously inhibit the activity of the T cells, further weaken the capability of the T cells in killing cancer cells, is an important negative regulatory factor of T cell immune response, and meanwhile, the CTLA-4 is the first discovered immune checkpoint with treatment potential in tumor immunotherapy. T cell activation is dependent on both signals: one is an antigenic signal and the other is a costimulatory signal. The antigen signal is mainly generated by the recognition of T Cell Receptor (TCR) on the surface of T cell and the binding of Major Histocompatibility Complex (MHC) molecules on the surface of Antigen Presenting Cell (APC), and the costimulatory signal is mainly generated by the binding of the costimulatory molecule CD28 on the surface of T cell and the B7 molecule on the surface of antigen presenting cell.
In the past, the immunotherapy for tumors mainly comprises means of developing small molecular target drugs, anti-tumor vaccines, adoptive cell immunotherapy and the like, and in recent years, the development of inhibitors targeting tumor immune check points becomes a novel immunotherapy. For example, by designing a blocking agent targeting CTLA-4, the inhibition effect of the blocking agent on the activation of T cells is reduced, thereby achieving the aim of resisting tumors. The development of antibody drugs targeting CTLA-4 for immunotherapy of tumors is one of the current research hotspots. Currently, representative CTLA-4 single-resistant drugs in the market mainly comprise Ipiimumab (Ipilimumab) and Tremelimumab, are mainly used for treating solid tumors such as melanoma and the like clinically, and achieve better curative effect compared with the traditional chemotherapy.
At present, there is still a lack of a cell-based assay method for biological activity of anti-CTLA-4 monoclonal antibodies, and the present invention has developed and verified a reporter assay (RGA) method for determining biological activity of CTLA-4 monoclonal antibodies by constructing two cell lines, a target cell stably expressing anti-CD 3 single-chain antibody fragment (scFv), referred to as Raji-CD3scFv cell line, and an effector cell stably expressing CTLA-4 gene and a luciferase reporter gene expressed by a NFAT response element driven by NFAT response element, referred to as Jurkat-CTLA-4-NFAT-luc cell line, respectively, which shows good specificity, accuracy, durability, and at the same time, which is sensitive to structural changes of anti-CTLA-4 monoclonal antibody drugs, and can be used not only for batch quality release and stability detection, it can also be used in the development and characterization of novel anti-CTLA-4 monoclonal antibodies.
Disclosure of Invention
Aiming at the defects of the existing biological activity detection method of the anti-CTLA-4 monoclonal antibody, the invention aims to provide an RGA method for detecting the biological activity of the anti-CTLA-4 monoclonal antibody, which comprises the steps of constructing Raji target cells for stably expressing CD3scFv and Jurkat effector cells for stably expressing a CTLA-4 gene and a luciferase reporter gene, mixing the target cells and the effector cells according to a certain proportion, and adding a drug sample of the anti-CTLA-4 monoclonal antibody for detection. The optimization, verification and practical application of the method prove that the method can be used for the stability test of the anti-CTLA-4 monoclonal antibody, the activity evaluation of the anti-CTLA-4 monoclonal antibody medicament and the batch quality release.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect, the invention provides an RGA method for the detection of biological activity of anti-CTLA-4 monoclonal antibodies.
The invention constructs a transgenic cell Jurkat-CTLA-4-NFAT-luc (anti-CTLA-4 monoclonal antibody transgene activity-measuring effect cell) by co-transfecting and expressing CTLA-4 and NFAT-luc reporter gene in Jurkat cell, and transfects and expresses CD3scFv (CD3 single chain antibody) in Raji cell, constructs Raji-CD3scFv (anti-CTLA-4 monoclonal antibody transgene activity-measuring target cell) as transgenic cell, when the CD3scFv expressed by Raji cell is combined with CD3 molecule in TCR compound on Jurkat cell, the signal path is activated, and when the CTLA-4 molecule expressed on Jurkat cell is combined with CD80/86 molecule constitutively expressed on the surface of Raji cell, the signal path is inhibited, when the anti-CTLA-4 monoclonal antibody is combined with CTLA-4, the combination of CTLA-4 and CD80/86 can be inhibited, thereby the inhibition effect is achieved, the expression of transcription factor NFAT reporter gene is restored, after adding the substrate, chemiluminescence values are generated, and a dose-effect curve is drawn to determine the biological activity of the anti-CTLA-4 monoclonal antibody.
Further, the method comprises the steps of:
(1) constructing target cells and effector cells;
(2) mixing the target cell suspension and the effector cell suspension according to a certain proportion to obtain a mixed solution;
(3) diluting the anti-CTLA-4 monoclonal antibody drug sample and a reference substance in equal proportion, respectively transferring the diluted antibodies to the mixed solution in the step (2), and incubating and culturing;
(4) adding a luciferase substrate to step (3), and fitting a four-parameter curve according to the signal value to determine the biological activity of the antibody.
Further, the constructed target cells are Raji cells stably expressing CD3 scFv.
Furthermore, the constructed effector cells are Jurkat cells which stably express CTLA-4 genes and enzyme reporter genes.
Further, the reporter gene is a luciferase reporter gene.
Further, the luciferase reporter gene is a luciferase reporter gene whose expression is driven by an NFAT responsive element;
further, the cell density of the prepared target cell suspension was 6X 105Per mL, the cell density of the prepared effector cell suspension is 6X 106one/mL.
Furthermore, the anti-CTLA-4 monoclonal antibody is diluted in equal proportion at a ratio of 1:3-1:5, preferably 1: 4.
Further, the incubation time is 4-7h, preferably 6 h.
Further, the conditions for incubation culture are preferably 37 ℃ and 5% CO2。
Further, the ratio of effector cells to target cells is 2:1-25:1, preferably 10: 1.
In the examples of the present invention, Raji cells stably expressing CD3scFv, i.e., target cells Raji-CD3scFv, are prepared by transfecting Raji cells with a vector containing the sequence of CD3 scFv; jurkat cells, i.e., Effector cells, that stably express both a CTLA-4 gene and a luciferase reporter gene whose expression is driven by an NFAT responsive element, Jurkat-CTLA-4-NFAT-luc, are prepared by transfecting Jurkat cells with a luciferase reporter vector comprising a CTLA-4-NFAT sequence.
In the embodiment of the invention, chemiluminescence is used for reading relative chemiluminescence unit value (RLU) on a microplate reader, and a four-parameter curve is fitted through data processing, so that the four-parameter curve can reflect upper and lower asymptotes and half effective concentration value (EC)50Value) and slope.
The second aspect of the invention provides a system for evaluating the pharmaceutical biological activity of the anti-CTLA-4 monoclonal antibody.
Further, the system comprises the following components:
(1) raji target cells stably expressing CD3scFv, Jurkat effector cells stably expressing CTLA-4 gene and reporter gene;
(2) anti-CTLA-4 monoclonal antibody drug samples and reference samples;
(3) a luciferase substrate.
Further, the reporter gene is a luciferase reporter gene.
Further, the luciferase reporter gene is a luciferase reporter gene whose expression is driven by an NFAT responsive element.
Furthermore, the biological activity evaluation index of the system is relative titer.
Further, the relative titer is obtained by comparing the half effective concentration value of the four-parameter curve of the sample and the reference product.
Further, the median effective concentration value is obtained by fitting a four-parameter curve to the relative chemiluminescence unit value through data processing.
In the examples of the present invention, Raji cells stably expressing CD3scFv, i.e., target cells Raji-CD3scFv, are prepared by transfecting Raji cells with a vector containing the sequence of CD3 scFv; jurkat cells, i.e., Effector cells, that stably express both a CTLA-4 gene and a luciferase reporter gene whose expression is driven by an NFAT responsive element, Jurkat-CTLA-4-NFAT-luc, are prepared by transfecting Jurkat cells with a luciferase reporter vector comprising a CTLA-4-NFAT sequence.
Furthermore, the CTLA-4 is CTLA-4-Y165F obtained by mutating CTLA-4.
In the embodiment of the invention, chemiluminescence is used for reading relative chemiluminescence unit value (RLU) on a microplate reader, and a four-parameter curve is fitted through data processing, so that the four-parameter curve can reflect upper and lower asymptotes and half effective concentration value (EC)50Value) and slope.
In a third aspect, the invention provides the use of the method of the first aspect of the invention in the manufacture of a means for assessing the pharmaceutical biological activity of an anti-CTLA-4 monoclonal antibody.
Preferably, the means comprises a product, a device, a kit and a platform.
In a fourth aspect, the invention provides the use of the system of the second aspect of the invention in the manufacture of a means for assessing the pharmaceutical biological activity of an anti-CTLA-4 monoclonal antibody.
Preferably, the means comprises a product, a device, a kit and a platform.
The invention has the advantages and beneficial effects that:
(1) the invention uses the Raji cell strain which is constructed and stably expresses CD3scFv as a target cell for the first time, and the constructed Jurkat effect cell which stably expresses CTLA-4 gene and luciferase reporter gene is simultaneously applied to the detection of the pharmaceutical biological activity of the anti-CTLA-4 monoclonal antibody.
(2) Compared with the prior art, the RGA method for detecting the biological activity of the anti-CTLA-4 monoclonal antibody has the advantages of simple operation and short experimental period, and can obtain results on the same day of the experiment and avoid the problems of possibility of cell pollution caused by long-time incubation and the like.
(3) The RGA method for detecting the biological activity of the anti-CTLA-4 monoclonal antibody provided by the invention has the advantages of strong specificity, high accuracy, high precision and good durability, and can be used for determining the biological activity of the anti-CTLA-4 monoclonal antibody and quickly evaluating and screening the anti-CTLA-4 monoclonal antibody.
(4) The RGA method for detecting the biological activity of the anti-CTLA-4 monoclonal antibody provided by the invention is sensitive to the structural change of the anti-CTLA-4 monoclonal antibody, and can be used for stability test of the anti-CTLA-4 monoclonal antibody, the drug activity evaluation of the monoclonal antibody and batch quality release.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a graph showing the results of flow analysis of Raji cell Raji-CD3scFv transfected with CD3 scFv;
FIG. 2 is a graph showing the results of flow analysis of CTLA-4-transfected Jurkat cells Jurkat-CTLA-4-NFAT-luc;
FIG. 3 is a dose-effect graph of the biological activity assay of anti-CTLA-4 monoclonal antibody;
FIG. 4 is a graph showing the comparison of the dilution ratios of different monoclonal antibodies in the assay of the biological activity of anti-CTLA-4 monoclonal antibodies;
FIG. 5 is a graph showing the comparison of the effect-target ratio and the induction time in the assay of the biological activity of anti-CTLA-4 monoclonal antibody, wherein, A is a graph: effective target ratio, panel B: the induction time;
FIG. 6 is a graph showing the results of different effective target ratios and signal to noise ratios corresponding to the induction time in the detection of the biological activity of the anti-CTLA-4 monoclonal antibody;
FIG. 7 is a graph showing the results of the specificity verification of the method for detecting the biological activity of anti-CTLA-4 monoclonal antibody;
FIG. 8 is a graph of the results of a regression line fit of theoretical and actual values of relative titer of a sample;
fig. 9 is a graph showing the results of the secondary stability verification of target cells and effector cells, in which a-graph: target cells, panel B: an effector cell;
FIG. 10 is a graph showing the results of dose-effect curves obtained by testing the biological activities of different anti-CTLA-4 monoclonal antibodies;
FIG. 11 is a graph showing the results of dose-response curves obtained by assaying the biological activities of anti-CTLA-4 monoclonal antibodies with different degrees of denaturation.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Example 1 construction of Raji-CD3scFv cell line and Jurkat-CTLA-4-NFAT-luc cell line
1. Construction of Raji cell line stably expressing CD3scFv
Using NEONTMThe electroporation transfection system (Invitrogen) performed CD3scFv plasmid transfection on Raji cells. 48h after transfection, Hygromycin B was added for pressure selection. After cell density and viability were restored, 5 96-well plates were plated per 0.5/well. When the confluency of the cell clones in the wells is more than 30%, a cell line stably expressing the CD3scFv is obtained by flow screening and is named as a Raji-CD3scFv cell.
2. Construction of Jurkat cell line stably expressing CTLA-4 Gene and luciferase reporter Gene expressed driven by NFAT response element
(1) Jurkat cells stably transfected with NFAT responsive element driven expressed luciferase reporter
Using NEONTMElectroporation transfection system (Invitrogen) Jurkat cells were transfected with the luciferase reporter plasmid expressed by the NFAT response element. 48h after transfection, Hygromycin B was added for pressure selection. After cell density and viability were restored, 5 96-well plates were plated per 0.5/well. When the cell clones in the wells grow to confluencyAnd when the expression rate is more than 30 percent, screening cell clones by using PMA (polymethyl methacrylate) with the concentration of 20ng/mL, and screening to obtain a monoclonal cell line which stably expresses the luciferase reporter gene driven by the NFAT response element and is named as Jurkat-NFAT-luc cells.
(2) Jurkat-NFAT-luc cells stably transfected CTLA-4-Y165F gene
Since wild-type CTLA-4 does not readily reside on the cell surface, but mutated CTLA-4(CTLA-4-Y165F) can stably reside on the cell surface, CTLA-4-Y165F, which is transfected in this example, is a mutant CTLA-4, CTLA-4-Y165F obtained by mutating CTLA-4 according to the methods in the literature (Leung H T, Bradshaw J, Cleaveland J S, et al.
Using NEONTMElectroporation transfection system (Invitrogen) Jurkat cells were transfected with the luciferase reporter plasmid expressed by the NFAT response element. 48h after transfection, G418 was added for pressure selection. After cell density and viability were restored, 5 96-well plates were plated per 0.5/well. When the confluency of cell clones in the wells is more than 30%, the cell clones are screened by Raji-CD3 scFv. Resuspension of Raji-CD3scFv cell centrifugation basal medium to a density of 5X 105Cloning cells in a 96-well plate, taking 150 mu L of cell suspension, centrifuging, using the Raji-CD3scFv cell suspension to resuspend to 40 mu L, adding 20 mu L/well into the left and right multiple wells of a 384-well plate; the left replicate wells were supplemented with 20. mu.L of basal medium, and the right replicate wells were supplemented with 20. mu.L of CTLA-4 antibody (diluted with basal medium) at 10. mu.g/mL; 37 ℃ and 5% CO2Culturing for 6-16 h. Adding ONE-Glo, 40 mu L/hole, detecting chemiluminescence value, and screening to obtain Jurkat monoclonal cell line stably expressing CTLA-4 gene and luciferase reporter gene expressed by NFAT response element, named Jurkat-CTLA-4-NFAT-luc cell.
The expression conditions of Raji cell Raji-CD3scFv stably expressing CD3scFv, Jurkat cell Jurkat-CTLA-4-NFAT-luc stably expressing CTLA-4 gene and luciferase reporter gene driven to be expressed by NFAT response element are analyzed by flow cytometry.
3. Results of the experiment
The results of flow analysis showed the successful construction of Raji cells stably expressing CD3scFv, Jurkat cells stably expressing CTLA-4 gene and luciferase reporter gene driven by NFAT response element (see fig. 1 and 2).
EXAMPLE 2 establishment of RGA method for assaying biological Activity of anti-CTLA-4 monoclonal antibody
1. Experimental methods
(1) Sample preparation: diluting the anti-CTLA-4 monoclonal antibody to 200 mu g/mL by using a diluent, and then carrying out reaction according to the weight ratio of 1:4, setting 10 concentration gradients, each concentration 2-3 multiple wells, and adding 50 μ L/well into a 96-well white plate.
(2) Preparation of Jurkat-CTLA-4-NFAT-luc effector cell suspension: collecting effector cells with good growth state, centrifuging to remove supernatant, resuspending cells with diluent, counting, adjusting cell density to 6 × 106one/mL for use.
(3) Preparing a Raji-CD3scFv target cell suspension: collecting target cells with good growth state, centrifuging to remove supernatant, resuspending the cells with diluent, counting, adjusting cell density to 6 × 105one/mL for use.
(4) Cell plating: the diluted effector cells and target cells were mixed at a ratio of 1:1, and 50. mu.L/well was added to a 96-well white plate.
(5) Cell incubation: 37 ℃ and 5% CO2Incubate in incubator for 6 h.
(6) Reading a plate: taking out the cell plate, balancing at room temperature, adding 100 μ L/hole of fluorogenic substrate, and measuring the fluorescence intensity of each hole with a multifunctional microplate reader after 5min at room temperature. And fitting a four-parameter S-shaped curve by taking the concentration of the antibody as an abscissa and the fluorescence signal value as an ordinate.
2. Statistical analysis
The Fold Induction (FI) was calculated according to the following formula: FI ═ RLU (reaction well mean-background well mean)/RLU (negative control well mean-background well mean). Analysis of the data by GraphPad Prism software against CTLA-4 monoclonal antibodyAnd fitting a dose-effect curve of the anti-CTLA-4 monoclonal antibody by using a four-parameter equation regression model, wherein the concentration logarithm is an x axis, and the corresponding FI value is a y axis. Relative titer (%) was expressed as EC of reference product50EC with sample50In which EC50Is half the effective concentration. The Signal to noise ratio (S/N) was calculated as the ratio of the D value to the A value (D/A) in the four parameter curve.
3. Results of the experiment
And fitting a dose-effect curve of the anti-CTLA-4 monoclonal antibody by using a four-parameter equation regression model with the concentration logarithm of the anti-CTLA-4 monoclonal antibody as an x axis and the corresponding FI value as a y axis, wherein the obtained data are analyzed to accord with a four-parameter equation: y ═ a-D/[ 1+ (x/C)B]+ D, typical S-shaped curve on the semi-logarithmic axis (see FIG. 3), R2>0.99。
Example 3 optimization of RGA detection method
1. Optimization of antibody dose-effect range
And (3) performing serial dilution by taking 200 mu g/mL as a dilution starting point of the anti-CTLA-4 monoclonal antibody, setting the dilution ratios of serial dilutions to be 1:3, 1:4 and 1:5 respectively, setting 10 concentration points, and determining the action range of the anti-CTLA-4 monoclonal antibody according to a four-parameter curve fitted by the determined chemiluminescence values.
The experimental method comprises the following steps: experiments were performed according to the method described in example 2, and finally the dose-effect curves of the different dilution schemes were compared, and the optimal dilution ratio was selected based on whether the four-parameter curve contained an upper and a lower plateau and whether the points distributed on the upper and lower plateaus and the linear part were uniform.
The experimental results are as follows: the experimental results are shown in FIG. 4, and the results show that the points distributed on the upper and lower platforms and the linear portion are more uniform when the dilution ratio is 1:4, and thus, the optimal dilution ratio is determined to be 1:4, and the working concentration range is 0.01-200. mu.g/mL.
2. Optimization of Effect-target ratio (E: T) and Induction time
6 x 4 factorial design experiments are carried out on the set 6 effective target ratio groups and 4 induction time groups, 24 groups of experiments are respectively carried out, different effective target ratios and induction times are analyzed, the signal-to-noise ratio (S/N) is calculated by the ratio of the D value and the A value in the four-parameter curve, and the highest S/N group is selected as the optimal experiment condition.
The experimental method comprises the following steps: the number of the fixed Jurkat-CTLA-4-NFAT-luc effector cells is 15 multiplied by 104The number of target cells of Raji-CD3scFv is set to be 7.5 multiplied by 10 respectively per hole4One/hole, 3X 1041.5X 10 units/hole41 x 10 per hole47.5X 10 pieces/hole3Number/hole, 6X 103The number/hole mode is that the effective target ratio is set to be 2:1, 5:1, 10:1, 15:1, 20:1 and 25:1 respectively, the induction time is set to be 4h, 5h, 6h and 7h respectively, namely, all the plates arranged at different effective target ratios are respectively at 37 ℃ and 5% CO2And (3) incubating in an incubator for 4, 5, 6 and 7 hours, and then carrying out biological activity detection on the anti-CTLA-4 monoclonal antibody, wherein the specific detection method is carried out according to the method described in the example 2.
The experimental results are as follows: the results are shown in fig. 5A and B, and the results of the signal to noise ratio are shown in fig. 6, which shows that the effective target ratio E: T is 10:1, the signal to noise ratio is highest when the induction time is 6h, and the presented dose-effect curve is also better, therefore, 10:1 is selected as the optimal E: T, and 6h is selected as the optimal induction time.
Example 4 validation of RGA detection method
1. Attribute validation
In this example, specificity of target cells, specificity of effector cells and specificity of antibodies were verified separately (see table 1).
TABLE 1 Attribute validation
Figure BDA0002988358210000101
The experimental method comprises the following steps:
(1) verifying the specificity of target cells, and replacing Raji-CD3scFv cells with Raji cells which are not transfected with CD3scFv and diluent as a negative control group to perform a biological activity detection experiment on the anti-CTLA-4 monoclonal antibody;
(2) validation of specificity of effector cells: taking Jurkat cells which are not transfected with CTLA-4-NFAT-luc and diluent as a negative control group to replace the Jurkat-4-NFAT-luc cells to carry out a biological activity detection experiment of the anti-CTLA-4 monoclonal antibody;
(3) the antibody specificity is verified, and an irrelevant monoclonal antibody and a diluent are used as a negative control to replace an Ipilimumab (Iplilimumab, the target point is CTLA-4) to carry out a biological activity detection experiment, wherein the irrelevant monoclonal antibody comprises monoclonal antibody medicaments with different target points: nivolumab (target PD-1), Rituximab (Rituximab, target CD 20).
The specific detection method was performed as described in example 2.
The experimental results are as follows: the results are shown in fig. 7, the RGA method established in the present invention is based on specific target cells and effector cells, and specific anti-CTLA-4 mab, and once the target cells or effector cells are replaced with natural cells or diluent that is not transfected with any plasmid, there is no dose-response curve, and when ipilimumab is replaced with diluent or a mab drug against other target, there is no dose-response curve, and only when target cells Raji-CD3scFv, effector cells Jurkat-CTLA-4-NFAT-luc, and ipilimumab whose target is CTLA-4 are present at the same time, a good dose-response curve can be obtained, demonstrating that the specificity of the RGA method established in the present invention is better.
2. Accuracy verification
The recovery rate is expressed as the ratio of the measured value of the relative titer of the sample to the theoretical value of the relative titer thereof, i.e., the recovery rate is 100% of the measured value of the relative titer/the theoretical value of the relative titer, and is a parameter that can be used to express the accuracy of the detection method.
The experimental method comprises the following steps: the relative titer was calculated as described in example 2 using 100% samples as reference and 5 samples of different titer levels were prepared by diluting the stock solution to different initial concentrations in dilution medium, 50%, 75%, 100%, 125% and 150% for 5 recovery groups, each sample being tested in 3 replicates. And (3) counting the average value and the Relative Standard Deviation (RSD) of the relative titer of each group of samples, and further calculating the recovery rate according to the formula of the recovery rate according to the 3-time repeated detection results of the 5 groups of recovery rate samples so as to verify the accuracy of the method.
The experimental results are as follows: the statistical experimental results are shown in Table 2, and the results show that the recovery rate is 92.12-103.44%, and the RSD is less than 7%, which indicates that the RGA method established by the invention has better accuracy.
TABLE 2 accuracy verification
Figure BDA0002988358210000121
3. Linearity verification
The experimental method comprises the following steps: the theoretical values of relative titer of the five groups of samples in the accuracy verification according to this example were fitted to the measured values to form a regression line.
The experimental results are as follows: the experimental results are shown in FIG. 8, R of regression line between theoretical and measured values20.9985, indicating a better linear fit with a better linear relationship.
4. Precision verification
Repeatability and intermediate precision were verified separately.
The experimental method comprises the following steps: two experimenters performed 100% titer level samples on 3 different days, each time in parallel for 3 plates, according to the method described in example 2.
The experimental results are as follows: the experimental results are shown in Table 3, and show that the RSD of 18 repeated experiments is less than 10 percent, and the RSD between plates/days/experimenters is less than 10 percent, which proves that the RGA method established by the invention has better precision.
TABLE 3 precision verification
Figure BDA0002988358210000131
5. Durability verification
The incubation time is +/-1 h, the number of target cells is +/-15%, the number of effector cells is +/-15%, and different generations of the target cells and the effector cells (the target cells are respectively 12 generations, 27 generations and 42 generations, and the effector cells are respectively 21 generations, 33 generations and 53 generations) are respectively adopted to evaluate the influence on the stability of the method established by the invention. Only one parameter was changed at a time, and other experimental conditions were not changed.
The experimental method comprises the following steps: the assay was performed as described in example 2.
The experimental results are as follows: the experimental result shows that when the incubation time is +/-1 h, the absolute deviation is less than 20%, when the number of target cells is +/-15%, the absolute deviation is less than 10%, when the number of effector cells is +/-15%, the absolute deviation is less than 10% (see table 4), the result of fig. 9A shows that the target cells Raji-CD3scFv have better secondary stability, the result of fig. 9B shows that the effector cells Jurkat-CTLA-4-NFAT-luc have better secondary stability, and the results show that the RGA method established by the invention has better durability.
TABLE 4 durability verification
Figure BDA0002988358210000141
Example 5 practical application of RGA method for detecting biological Activity of anti-CTLA-4 monoclonal antibodies
1. Detecting the biological activity of different anti-CTLA-4 monoclonal antibodies
Taking the original anti-CTLA-4 monoclonal antibody ipilimumab as a reference substance, selecting different types of anti-CTLA-4 monoclonal antibodies 1 and 2, and determining the biological activity of different types of anti-CTLA-4 monoclonal antibody samples by adopting an optimized and verified RGA method established by the invention.
The experimental method comprises the following steps: the assay was performed as described in example 2, with 3 replicates per sample.
The experimental results are as follows: the experimental results are shown in figure 10, and the results show that different types of anti-CTLA-4 monoclonal antibodies present better dose-effect curves, which indicates that the RGA method established by the invention can be applied to the detection and comparison of the biological activities of different types of anti-CTLA-4 monoclonal antibodies.
2. Detecting the biological activity of the anti-CTLA-4 monoclonal antibody with different denaturation degrees
The experimental method comprises the following steps: the ipilimumab samples were heated in a water bath at 70 ℃ for 1.5h and 2.5h, respectively, to prepare different denatured ipilimumab samples, which were then assayed separately from native ipilimumab as described in example 2.
The experimental results are as follows: the experimental result is shown in figure 11, and the result shows that the titer of the denatured ipilimumab is correspondingly reduced along with the prolonging of the heating time, which indicates that the RGA method established by the invention is sensitive to the structural change of the anti-CTLA-4 monoclonal antibody, and can be used for the stability test of the anti-CTLA-4 monoclonal antibody, the pharmaceutical activity evaluation of the monoclonal antibody and the batch quality release.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (16)

1. An RGA method for the detection of biological activity of an anti-CTLA-4 monoclonal antibody, comprising the steps of:
(1) constructing target cells and effector cells;
(2) mixing the target cell suspension and the effector cell suspension according to a certain proportion to obtain a mixed solution;
(3) diluting the anti-CTLA-4 monoclonal antibody drug sample and a reference substance in equal proportion, respectively transferring the diluted antibodies to the mixed solution in the step (2), and incubating and culturing;
(4) adding a luciferase substrate into the step (3), and fitting a four-parameter curve according to the signal value to determine the biological activity of the antibody;
the constructed target cell is a Raji cell stably expressing CD3 scFv;
the constructed effector cells are Jurkat cells which stably express CTLA-4 genes and reporter genes.
2. The method of claim 1, wherein the cell density of the prepared target cell suspension is 6 x 105one/mL.
3. The method of claim 1, wherein the reporter gene is a luciferase reporter gene.
4. The method of claim 3, wherein the luciferase reporter gene is a luciferase reporter gene whose expression is driven by an NFAT responsive element.
5. The method of claim 1, wherein the effector cell suspension is prepared at a cell density of 6 x 106one/mL.
6. The method of claim 1, wherein the anti-CTLA-4 monoclonal antibody is diluted at an equal ratio of 1:3 to 1: 5.
7. The method of claim 6, wherein the anti-CTLA-4 monoclonal antibody is diluted at an equal ratio of 1: 4.
8. The method of claim 1, wherein the incubation time is 4-7 hours.
9. The method of claim 8, wherein the incubation time is 6 hours.
10. The method of claim 1, wherein the ratio of effector cells to target cells is 2:1 to 25: 1.
11. The method of claim 10, wherein the ratio of effector cells to target cells is 10: 1.
12. A composition for evaluating the pharmaceutical biological activity of an anti-CTLA-4 monoclonal antibody, which comprises the following components:
(1) raji target cells stably expressing CD3scFv, Jurkat effector cells stably expressing CTLA-4 gene and reporter gene;
(2) anti-CTLA-4 monoclonal antibody drug samples and reference samples;
(3) a luciferase substrate.
13. The composition of claim 12, wherein the reporter gene is a luciferase reporter gene.
14. The composition of claim 13, wherein the luciferase reporter gene is a luciferase reporter gene whose expression is driven by an NFAT responsive element.
15. Use of the method of any one of claims 1 to 11 for the manufacture of a means for assessing the pharmaceutical biological activity of an anti-CTLA-4 monoclonal antibody;
the tool comprises a product and a device.
16. Use of the composition of any one of claims 12 to 14 for the manufacture of a means for assessing the pharmaceutical biological activity of an anti-CTLA-4 monoclonal antibody;
the tool comprises a product and a device.
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