WO2021135780A1

WO2021135780A1 - Method for screening antibody binding to membrane protein based on cellular level

Info

Publication number: WO2021135780A1
Application number: PCT/CN2020/132574
Authority: WO
Inventors: 杜靓; 张红艳; 袁纪军
Original assignee: 上海吉倍生物技术有限公司
Priority date: 2019-12-31
Filing date: 2020-11-30
Publication date: 2021-07-08
Also published as: TW202129275A; CN112067819A; CN113358877A; CN112067819B

Abstract

The present invention relates to the field of immunological detection. In particular, the present invention provides a method for determining the binding activity of an antibody to a target membrane protein on the cell surface. By means of the method, high-throughput direct detection of a hybridoma supernatant sample at the cellular level can be achieved, and this detection is not interfered with by a ''hook effect'', thereby having wide clinical and research uses.

Description

A screening method based on cell-level binding membrane protein antibodies

Technical field

The present invention relates to immunological detection. Specifically, the present invention provides a method for measuring the binding activity of an antibody to a target membrane protein on the cell surface.

Background technique

Antibody drugs are currently important products for disease treatment. Among the Top 10 global drug sales in 2018, there are 8 antibody drugs, including 6 monoclonal antibody drugs and 2 fusion proteins. Antibody drug research and development methods currently include fusion tumors, phage display, and single B cell sequencing. Fusion tumors are the mainstream. More than 95% of monoclonal antibody drugs currently on the market are discovered through fusion tumor methods. The supernatant antibody library generated by the fusion tumor method is as small as thousands to tens of thousands, and appropriate antibody screening methods are required to find antibodies that specifically bind to the target antigen. The classic antibody screening method is to find antibodies that bind to the antigen at the protein level by enzyme-linked immunosorbent assay (ELISA), and then use flow cytometry (FCM) to further verify the binding of candidate antibodies at the cellular level. ELISA uses the protein form of antigen. For membrane proteins, the structure of the recombinantly expressed protein and its natural structure on the cell membrane are inevitably different, which results in the false positive of ELISA screening for subsequent FCM screening (ELISA Horizontal binding but FCM level not binding)/false negative (FCM level binding but not being screened due to ELISA level not binding). In order to avoid false positives/false negatives in ELISA screening, cell-level combined screening based on fluorescence microanalysis, represented by Mirrorball, came into being. However, both traditional ELISA and fluorescence microanalysis are affected by the hook effect (Hook effect), which brings great problems to the screening of fusion tumor supernatant.

The hook effect, or Hook effect, refers to the phenomenon of false negatives due to the inappropriate ratio of antigen to antibody. When the antigen and antibody react specifically, the amount of conjugate produced is related to the concentration of the reactant. Whether adding different amounts of antigen to a certain amount of antibody or adding different amounts of antibody to a certain amount of antigen, it can be found that the strongest antigen-antibody reaction occurs only when the ratio of the two molecules is appropriate. Taking the precipitation reaction as an example, if a certain amount of antibody is added to a row of test tubes, and then an increasing amount of corresponding soluble antigen is added to each tube in turn, the reaction curve can be drawn according to the ratio between the formed precipitate and the antigen-antibody ( figure 1). The peak part of the curve is the range where the ratio of antigen-antibody molecules is appropriate, which is called the equivalent band of antigen-antibody reaction. Within this range, the antigen and antibody are fully bound, and the most precipitates are formed. The excess of antibodies and antigens before and after the equivalent band affects the formation of precipitates. This phenomenon is called band phenomenon. Banding phenomenon often occurs in clinical tests, especially in ELISA reactions. When the antibody is excessive, it is called the front zone, and when the antigen is excessive, it is called the posterior zone. In clinical testing, especially the previous band effect is obvious, the usual solution is to further dilute the sample.

The Hook effect also appeared in the cell-level binding detection based on fluorescence microanalysis, represented by Mirrorball. When cells and antibodies are homogeneously mixed, the amount of antibody and cell binding is related to the concentration of both. Take Mirrorball and the similar ABI 8200 FMAT detection platform as an example. The concentration-dependent fluorescence intensity curve for detecting the binding of anti-EGFT antibody to A431 cells is shown in Figure 2. Different amounts of antibodies are added to a certain amount of cell system. If the antibody concentration exceeds 0.1μg/mL, the signal value will have a strong fluorescence decrease. When using fluorescent microanalysis to screen antibodies in the supernatant of fusion tumors, positive clones with high expression and strong affinity will be missed due to the signal reduction of the "hook effect". The solution is to further dilute the supernatant, but doing so will greatly increase the workload.

Therefore, there is a need in the art to develop improved methods for screening antibodies, especially antibodies that bind to membrane proteins.

Summary of the invention

In order to solve the above problems, the present invention provides an improved method for screening antibodies against membrane proteins. This method can achieve high-throughput direct detection of fusion tumor supernatant samples at the cellular level without interference from the "hook effect" and has a broad Clinical and research applications.

Screening method

In one aspect, the present invention provides a method for determining the binding activity of an antibody against a membrane protein to a target membrane protein on the cell surface, which comprises the following steps:

(1) Provide a solid phase carrier with cells expressing the target membrane protein immobilized on the surface;

(2) Contacting the sample to be tested containing the antibody to be tested with the surface of the solid phase carrier;

(3) Contacting a secondary antibody with a detectable label on the surface of the solid-phase carrier, and the secondary antibody can specifically bind to the antibody to be tested;

(4) Detect the detectable label on the surface of the solid phase carrier.

In certain embodiments, the detectable label is a fluorescent label.

In some embodiments, step (4) includes the following steps: determining the number of cells with a detectable label (such as a fluorescent label) on the surface of the solid-phase carrier, and determining the total number of cells on the surface of the solid-phase carrier, In this way, the proportion of cells with a detectable label (such as a fluorescent label) in all cells is obtained. In some embodiments, step (4) includes the following steps: measuring the number of cells with a detectable label (such as a fluorescent label) on the surface of the solid support in one field of view, and measuring the number of cells in the same field of view. The total number of cells on the surface of the solid support, thereby obtaining the proportion of cells with a detectable label (such as a fluorescent label) in all cells.

In some embodiments, step (4) includes the following steps: obtaining a fluorescent image of the solid-phase carrier surface to obtain a cell count of the fluorescence image, and obtaining a bright-field image of the solid-phase carrier surface to obtain all State the cell count of the brightfield image to obtain the proportion of fluorescently labeled cells in all cells.

In some embodiments, the method further includes: (5) comparing the obtained ratio with a reference value, or generating a dose-response curve based on the ratio, and thereby obtaining a test antibody EC ₅₀ , and compare the EC ₅₀ with a reference value to determine the binding activity of the antibody to be tested to the target membrane protein.

In some embodiments, the reference value is the ratio obtained after performing the above steps (1)-(4) on the positive control sample or the EC ₅₀ obtained based on the ratio. In some embodiments, the positive control sample is an antibody known to specifically bind to the target membrane protein. In some embodiments, if the ratio is not lower than the reference value, it is determined that the antibody to be tested has the activity of specifically binding the target membrane protein.

In some embodiments, the EC _{50 of} the test antibody is obtained by the following method: Repeat steps (1)-(4) with a series of test samples containing different amounts of the test antibody, thereby generating a dose-response curve From this, the EC ₅₀ of the antibody was determined.

In some embodiments, the solid phase carrier is a microplate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. In certain embodiments, the solid phase carrier is a 96-well plate.

In some embodiments, step (4) is performed using a cell imaging microplate detector. In some embodiments, the cell imaging microplate detector is selected from CQ1 laser confocal imaging cell quantitative analysis system, BioTek Cytation 5 cell imaging microplate detection system, SpectraMax MiniMax300 cell imaging system, Celigo Image Cytometer full field of view Cell scanning analyzer.

In some embodiments, the cell density (cell confluence) on the surface of the solid carrier is 20%-80%, for example, 30%-80%, 40%-80%, 50%-80%, 60%- 80%, or 70%-80%.

In some embodiments, step (1) includes the following steps: (1a) providing cells expressing the target membrane protein; (1b) fixing the cells on the surface of a solid phase carrier.

In the present invention, the cells expressing the target membrane protein can be prepared by various methods known in the art, for example, obtained by genetic engineering recombination technology. For example, a DNA molecule encoding the target membrane protein is inserted into an expression vector, and then a host cell is transfected to obtain a cell expressing the target membrane protein.

In certain embodiments, the cell expressing the target membrane protein is a eukaryotic cell, such as a mammalian cell, such as a mouse cell, a human cell, and the like. In certain embodiments, the cell is a mammalian cell, such as MDA-MB-231, HEK293, Hela, CHO (e.g. CHO-K1, CHO-S, CHO DG44) and the like.

In certain embodiments, the cells expressing the target membrane protein are adherent cells or suspension cells.

In some embodiments, the cells expressing the target membrane protein immobilized on the surface of the solid phase carrier express substantially the same level of the target membrane protein.

In some embodiments, the step (1b) includes: seeding the cells on the solid-phase carrier, and allowing the cells to settle to the surface of the solid-phase carrier; using a cell fixative to fix the cells that have settled on the surface of the solid-phase carrier.

In some embodiments, the seeding density of the cells is 1.5×10 ⁴ /cm ² to 5×10 ⁴ cm ² .

In some embodiments, the cells are sedimented to the surface of the solid support by centrifugation.

In this context, "cell fixation agent" means a solution that is usually used for fixation of cells. Examples of cell fixatives include fixatives based on ethanol, methanol, formaldehyde, and glacial acetic acid, such as 4% paraformaldehyde solution, 10% formaldehyde solution, formaldehyde/NaCl solution, formaldehyde/acetic acid solution, alcohol/formaldehyde/acetic acid fixative, etc. Wait.

In some embodiments, the fixative is selected from 95% alcohol, 4% paraformaldehyde, glacial acetic acid, methanol, ether alcohol, and any combination thereof. In certain embodiments, the term "glacial acetic acid/glacial acetic acid" refers to acetic acid having a purity of at least 99.5% by weight (eg, 99.8% by weight).

In some embodiments, after step (1b), a step of discarding the liquid in the solid phase carrier is further included.

In other embodiments, the step (1b) includes inoculating the cells to a solid carrier, and incubating the cells in a cell culture medium under conditions that promote adhesion of the cells to the surface of the solid carrier.

In certain embodiments, the surface of the solid support comprises an adhesion matrix, such as gelatin or polyguanylic acid.

In certain embodiments, the cell culture medium contains an adhesion matrix, such as gelatin or polyguanylic acid.

In certain embodiments, the cell is a suspension cell. In some embodiments, the step (1b) includes: seeding the cells on a solid support containing an adhesion matrix on the surface, and incubating in a cell culture medium. In some embodiments, the step (1b) includes seeding the cells on a solid carrier and incubating in a cell culture medium containing an adhesion matrix.

In some embodiments, the sample to be tested is a cell culture supernatant. In some embodiments, the sample to be tested is a fusion tumor cell culture supernatant or a fusion tumor subclonal culture supernatant. In some embodiments, the sample to be tested is the culture supernatant of host cells (such as CHO cells) that produce recombinant antibodies. In certain embodiments, the cell culture supernatant is undiluted.

As used herein, the expression "the cell culture supernatant is undiluted" means that the cell culture supernatant has not undergone any dilution treatment after being separated from the cell culture system.

In some embodiments, the sample to be tested contains not less than 0.001 μg/mL (for example, not less than 0.1 μg/mL) of the antibody to be tested.

In some embodiments, the sample to be tested contains no more than 30 μg/mL of the antibody to be tested. In some embodiments, the sample to be tested contains no more than 10 μg/mL of the antibody to be tested.

In some embodiments, the sample to be tested contains 0.001 μg/mL to 30 μg/mL of the antibody to be tested. In some embodiments, the sample to be tested contains 0.001 μg/mL to 10 μg/mL of the antibody to be tested.

In some embodiments, the sample to be tested contains 0.1 μg/mL to 30 μg/mL of the antibody to be tested. In some embodiments, the sample to be tested contains 0.1 μg/mL to 10 μg/mL of the antibody to be tested.

In certain embodiments, the secondary antibody is specific for the antibody of the species (e.g., mouse) from which the antibody to be tested is derived.

In certain embodiments, the secondary antibody is selected from an anti-immunoglobulin antibody, such as an anti-IgG antibody, an anti-IgM antibody, or an anti-IgA antibody.

In certain embodiments, the immunoglobulin is from an immunized animal, such as a mouse.

In some embodiments, before step (3), it further includes determining the antibody subtype of the antibody to be tested, and selecting the corresponding secondary antibody according to the determined antibody subtype. In some embodiments, the determination can be accomplished by a commercial kit.

In some embodiments, after step (2) and/or (3), the method further includes a step of discarding the liquid in the solid phase carrier. In certain embodiments, the step of washing the surface of the solid support is included or not after the step of discarding the liquid.

In some embodiments, after the step of discarding the liquid after step (3), the method further includes adding a buffer to the solid phase carrier. In some embodiments, the buffer is selected from PBS, Hanks BSS, Earles salt, DPBS, HBSS, EBSS, and any combination thereof.

Definition of Terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory procedures of virology, biochemistry, and immunology used in this article are routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (ie, a binding molecule and a target molecule), such as the reaction between an antibody and the antigen to which it is directed. The binding affinity between two molecules can be described by the _{K D value.} K _D value refers to the ratio of kd (specific binding molecule-target molecule interaction dissociation rate; also called koff) to ka (specific binding molecule-target molecule interaction rate of association; also called kon) The obtained dissociation constant, or kd/ka expressed as molar concentration (M). The _{smaller the K D} value, the tighter the binding of the two molecules and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody has a concentration of less than about 10 ^-5 M, for example, less than about 10 ^-6 M, 10 ^-7 M, The affinity (K _D ) of 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less binds the antigen. The K _D value can be determined by a method well known in the art, for example, measured in a BIACORE instrument using surface plasmon resonance (SPR).

As used herein, the term "detectable label" can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. It is particularly preferable that such a label can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Red protein (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds), magnetic beads (E.g,

), calorimetric markers such as colloidal gold or colored glass or plastic (for example, polystyrene, polypropylene, latex, etc.) beads, and avidin (for example, streptavidin) modified by combining the above-mentioned markers ) Of biotin.

As used herein, the term "antibody" refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a domain CL. Constant domains are not directly involved in the binding of antibodies and antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system. The VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR). Each V _H and V _L, the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. In certain embodiments, the host cell is preferably a mammalian cell.

Beneficial effect

The present invention provides an improved method for screening antibodies against membrane proteins. The method can realize high-throughput direct detection of fusion tumor supernatant samples at the cell level while ensuring the detection accuracy, greatly simplifying the operation steps, and does not Interfered by the "hook effect", it has broad clinical and research applications.

The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, but not to limit the scope of the present invention. According to the accompanying drawings and the following detailed description of the preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art.

Description of the drawings

Figure 1 shows a schematic diagram of the hook effect in the antigen-antibody immunoprecipitation detection method.

Figure 2 shows the antibody concentration-dependent curve of anti-EGFT antibody binding to A431 cells detected by the Mirrorball and ABI 8200 FMAT detection platform. The results show that when the antibody concentration exceeds 0.1 μg/mL, a strong fluorescence reduction occurs in the signal value, resulting in a hook effect.

Figure 3 shows the imaging results of the positive hole C3 and the negative hole B1 in Example 1.

Figure 4 shows the proportion of fluorescent cells in the supernatant of each fusion tumor on the test plate in Example 1, where H11 is the negative control well and H12 is the positive control well.

Figure 5 shows the fluorescence field imaging on the detection plate in Example 2.

Figure 6 shows the proportion of fluorescent cells in the supernatant of each fusion tumor on the test plate in Example 2, where G11 is a positive control well and H11 is a negative control well.

Figure 7 shows the antibody concentration-dependent curve of the method of the present invention for detecting anti-PD-L1 antibodies in Example 3-1. The results show that when the antibody concentration reaches 10 μg/mL, the detection value of the method of the present invention still does not decrease, and no hook effect is produced.

Figure 8 shows the antibody concentration-dependent curve of the method of the present invention for detecting anti-CD20 antibodies in Example 3-2. The results show that when the antibody concentration reaches 30 μg/mL, the detection value of the method of the present invention still does not decrease, and no hook effect is produced.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If no specific conditions are indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

Example 1: Screening of anti-PD-L1 monoclonal antibodies based on adherent cells

1. Cells expressing human PD-L1 HEK293-PD-L1 (in-house construction; using GK gene to construct human PD-L1 (Q9NZQ7) lentivirus to infect ATCC-derived HEK293 cells, the virus is transfected After 48 hours of staining, add resistance reagent to kill the cells that do not express the protein, and then after about 2 weeks of resistance screening to obtain the desired antigen-high-expressing cells), after digestion, wash twice with DPBS and count to 8000 cells per well. The cells were fixed on the bottom of a flat-bottomed 96-well plate (96-Well Corning ^TM 3596 Plate) and cultured overnight to make them adherent.

2. Take 50μL of anti-PD-L1 mouse fusion tumor supernatant and add it to the plate wells, add blank fusion tumor medium to the corresponding negative control wells, and add 50μL of the known specific binding to the antigen at a concentration of 0.5μg/mL in the positive control wells The positive control antibody (Abcam, ab238697), incubate at room temperature for 45-60 minutes.

3. After the incubation, the supernatant is removed from the clap board, and the strength of the clap board is subject to no visible liquid residue. Add 50μL to each well

488-labeled anti-mouse IgG (Abcam ab97003, Goat Anti-Human IgG Fc) was used as the secondary antibody. The secondary antibody was diluted in a serum-free medium with an incubation concentration of 2 μg/mL and incubated at room temperature for 30 minutes.

4. After the secondary antibody incubation is completed, the supernatant is removed from the plate and the plate strength is subject to no visible liquid residue. Add 100 μL DPBS to each well, and then perform imaging readings on the Celigo Image Cytometer cell imaging microplate detector. During imaging, two channels of bright field and fluorescence are set at the same time, and bright field imaging and fluorescence imaging are performed on the cells in each well. Condition setting: 96% reading hole area; 1μm pixels; fluorescence field, 200 enhancement, 150,000μms exposure; bright field, 0 enhancement, 3200μms exposure.

Figure 3 shows the imaging results of positive well C3 and negative well B1. It can be seen from the figure that there is a clear fluorescence signal on the cell position in the bright field in the C3 positive well, while there is no fluorescence signal on the cell position in the B1 negative hole corresponding to the bright field.

5. Use the cell imaging microplate detector to analyze the readings of the experiment plate. The imaging obtained by the fluorescence channel counts the antibody-bound cells according to the fluorescence-labeled cell morphology and the fluorescence intensity setting parameters, and the imaging obtained by the bright field channel counts the adherent cells according to the cell morphology setting parameters, and then two sets of data Divide it to obtain the percentage of cells that bind to the antibody and show green fluorescence to the total number of adherent cells (% fluorescent cells), and determine whether the fusion tumor supernatant antibody binds to HEK293-PD-L1 cells according to this ratio. Bright field cell counting conditions are: 15 intensity baseline, 2 accuracy, 10 size filtering, automatic background correction, automatic cell separation correction; fluorescence field cell counting conditions: 3 intensity baseline, 2 accuracy, 10 size filter automatic background correction, Automatic cell separation correction.

Figure 4 shows the proportion of fluorescent cells in the supernatant of each fusion tumor on the test plate, where H11 is the negative control well and H12 is the positive control well. For this screening, the defined value>20% is a strong positive, 2%<value<20% is a weak positive, and a value<2% is negative. The results showed that A10, B3, C3, D5, D6, and F7 were strong positive wells, and the corresponding fusion tumor cells were selected for subsequent subcloning and subcloning screening.

Example 2: Screening of anti-CD20 monoclonal antibodies based on suspension cells

1. The constructed CHO-CD20 cells expressing the antigen CD20 (in-house construction; the lentivirus constructed with human-derived CD20 (P11836) prepared by Jikai gene infects Thermo Fisher-derived CHO cells, 48 hours after virus transfection Add resistance reagents to kill the cells that do not express the protein, and then after about 2 weeks of resistance screening to obtain the desired antigen-high expression cells), wash twice with DPBS and count, and inoculate the cells to a flat bottom with a cell amount of 15000 cells per well A 96-well plate (96-Well Corning ^TM 3596 Plate) was centrifuged at 1000 rpm for 5 minutes, and then incubated with 4% paraformaldehyde solution for 15 minutes at room temperature for fixation.

2. Take 50μL of anti-CD20 mouse fusion tumor supernatant and add it to the plate wells, add blank fusion tumor medium to the corresponding negative control wells, and add 50μL positive control antibody (Abcam, ab78237) at a concentration of 0.5μg/mL to the positive control wells. Incubate at room temperature for 45-60 minutes.

488-labeled anti-mouse IgG (Abcam ab97003, Goat Anti-Human IgG Fc) was used as the secondary antibody. The secondary antibody was diluted in a serum-free medium with an incubation concentration of 3 μg/mL and incubated at room temperature for 30 minutes.

4. After the secondary antibody incubation is completed, the supernatant is removed from the plate and the plate strength is subject to no visible liquid residue. Add 100 μL DPBS to each well, and then perform imaging readings on the Celigo Image Cytometer cell imaging microplate detector. When imaging, two channels of bright field and fluorescence are set at the same time, and bright field imaging and fluorescence imaging are performed on the cells in each well. Condition setting: 96% reading hole area; 1μm pixels; fluorescence field, 200 enhancement, 150,000μms exposure; bright field, 0 enhancement, 3200μms exposure.

Figure 5 shows the fluorescence field imaging on a test panel. As can be seen from the figure, positive wells such as A1, C5, D7, G11, etc. have obvious fluorescence signals visible to the naked eye, while negative wells such as A7, B2, C4, and F1 have no observable fluorescence signals.

5. Use the cell imaging microplate detector to analyze the readings of the experiment plate. The imaging obtained by the fluorescence channel counts the antibody-bound cells according to the fluorescence-labeled cell morphology and the fluorescence intensity setting parameters, and the imaging obtained by the bright field channel counts the adherent cells according to the cell morphology setting parameters, and then two sets of data Divide it to obtain the percentage of cells that bind to the antibody and show green fluorescence to the total number of adherent cells (% fluorescent cells), and determine whether the fusion tumor supernatant antibody binds to CHO-CD20 cells or not. Bright field cell counting conditions are: 15 intensity baseline, 2 accuracy, 10 size filtering, automatic background correction, automatic cell separation correction; fluorescent field cell counting conditions: 7 intensity baseline, 2 accuracy, 10 size filter automatic background correction, Automatic cell separation correction.

Figure 6 shows the proportion of fluorescent cells in the supernatant of each fusion tumor on the test plate, where G11 is the positive control well and H11 is the negative control well. For this screening, we define a value> 30% as a strong positive, 2% <a value <30% as a weak positive, and a value <2% as a negative. The fusion tumor cells corresponding to the strong positive wells with a reading value of >30% are selected for subsequent subcloning and subcloning screening.

Example 3: Hook effect evaluation

3-1. According to the method described in Example 1, an anti-PD-L1 antibody with an initial concentration of 10 μg/mL, diluted 3 times, and a total of 11 concentration points (this antibody was screened in Example 1. For Example 1 The fusion tumor cells screened in the fusion tumor cell were sequenced to obtain the heavy and light chain variable region sequence, and then the antibody was recombinantly expressed. The mouse IgG1 antibody used in this example was used for detection, and the mouse IgG1 isotype control antibody was used as a control. The percentage of fluorescent cells obtained by the cell imaging microplate detector is the ordinate, and the antibody concentration is the abscissa, and a dose-response curve is drawn. The results are shown in Figure 7, when the antibody concentration reaches 10 μg/mL, the detection value of the method of the present invention still does not decrease. This result shows that the method of the present invention still does not produce the Hook effect when the detection concentration is as high as 10 μg/mL.

3-2. According to the method described in Example 2, the initial concentration of 30μg/mL, 4-fold dilution, a total of 8 concentration points of the anti-CD20 positive antibody-01/02/03 (the three strains of antibodies are described in Example 2 Screening results. The fusion tumor cells screened in Example 2 were sequenced to obtain the heavy and light chain variable region sequences, and the chimeric antibody was recombinantly expressed. The human IgG1 chimeric antibody used in this example was tested. The human IgG1 isotype control antibody was used as a control, and the percentage of fluorescent cells obtained by the cell imaging microplate detector was used as the ordinate and the antibody concentration was used as the abscissa to draw a dose-response curve. The results are shown in Figure 8. When the antibody concentration reaches 30 μg/mL, the detection value of the method of the present invention still does not decrease. This result shows that the method of the present invention still does not produce the Hook effect when the detection concentration is as high as 30 μg/mL.

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and these changes are within the protection scope of the present invention. . All of the present invention is given by the appended claims and any equivalents thereof.

Claims

The method for determining the binding activity of an antibody against a membrane protein to a target membrane protein on the cell surface includes the following steps:

(1) Provide a solid phase carrier with cells expressing the target membrane protein immobilized on the surface;

(2) Contacting the sample to be tested containing the antibody to be tested with the surface of the solid phase carrier;

(3) Contacting a secondary antibody with a detectable label on the surface of the solid-phase carrier, and the secondary antibody can specifically bind to the antibody to be tested;

(4) Detect the detectable label on the surface of the solid carrier;

Preferably, the detectable label is a fluorescent label.
The method of claim 1, wherein step (4) comprises the following steps: measuring the number of cells with detectable labels on the surface of the solid-phase carrier, and measuring the total number of cells on the surface of the solid-phase carrier, so as to obtain The proportion of cells with detectable markers in all cells;

Preferably, the step (4) includes the following steps: obtaining a fluorescent image of the solid-phase carrier surface to obtain a cell count of the fluorescent image, and obtaining a bright-field image of the solid-phase carrier surface to obtain the bright field Count the cells in the field image to obtain the proportion of fluorescently labeled cells in all cells.
The method of claim 2, wherein the method further comprises: (5) comparing the obtained ratio with a reference value, or generating a dose-response curve based on the ratio, and thereby obtaining a dose-response curve. Measure the EC 50 of the antibody and compare the EC 50 with the reference value to determine the binding activity of the antibody to be tested to the target membrane protein;

Preferably, the reference value is the ratio obtained after performing the above steps (1)-(4) on the positive control sample or the EC 50 obtained based on the ratio; preferably, the positive control sample is known to be specific An antibody that binds to the target membrane protein; preferably, if the ratio is not lower than a reference value, it is determined that the detected antibody has the activity of specifically binding to the target membrane protein;

Preferably, the EC 50 of the antibody to be tested is obtained by the following method: Repeat steps (1)-(4) with a series of test samples containing different amounts of the antibody to be tested, thereby generating a dose-response curve and determining it accordingly The EC 50 of the antibody.
The method of claims 1-3, wherein the solid phase carrier is a micro-culture plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate;

Preferably, the solid phase carrier is a 96-well plate.
The method according to any one of claims 1 to 4, wherein step (4) is performed using a cell imaging microplate detector.
The method of any one of claims 1 to 5, wherein step (1) comprises the following steps: (1a) providing cells expressing the target membrane protein; (1b) fixing the cells on the surface of a solid phase carrier.
The method of claim 6, wherein the step (1b) comprises: seeding the cells on the solid-phase carrier, and allowing the cells to settle to the surface of the solid-phase carrier; Cells are fixed;

Preferably, the seeding density of the cells is 1.5×10 4 /cm 2 to 5×10 4 /cm 2 ;

Preferably, the cells are sedimented to the surface of the solid carrier by centrifugation;

Preferably, the fixative is selected from 95% alcohol, 4% paraformaldehyde, glacial acetic acid, methanol, ether alcohol, and any combination thereof;

Preferably, after step (1b), a step of discarding the liquid in the solid phase carrier is further included.
The method of claim 6, wherein the step (1b) comprises: seeding the cells on a solid carrier, and incubating the cells in a cell culture medium under conditions that promote adhesion of the cells to the surface of the solid carrier;

Preferably, the seeding density of the cells is 1.5×10 4 /cm 2 to 5×10 4 cm 2 ;

Preferably, the surface of the solid phase carrier comprises an adhesion matrix, such as gelatin or polyguanylic acid;

Preferably, the cell culture medium contains an adhesion matrix, such as gelatin or polyguanylic acid;

Preferably, after step (1b), a step of discarding the liquid in the solid phase carrier is further included.
The method of any one of claims 1-8, wherein the cell expressing the target membrane protein is an adherent cell or a suspension cell;

Preferably, the cell is selected from MDA-MB-231, HEK293, Hela, CHO.
The method of any one of claims 1-9, wherein the sample to be tested is a cell culture supernatant;

Preferably, the sample to be tested is a fusion tumor cell culture supernatant or a fusion tumor subclonal culture supernatant;

Preferably, the sample to be tested is the culture supernatant of host cells (such as CHO cells) that produce recombinant antibodies.
The method of any one of claims 1-10, wherein the sample to be tested contains not less than 0.001 μg/mL (for example, not less than 0.1 μg/mL) of the antibody to be tested;

Preferably, the sample to be tested contains no more than 30 μg/mL of the antibody to be tested;

Preferably, the sample to be tested contains 0.001 μg/mL to 30 μg/mL of the antibody to be tested;

Preferably, the sample to be tested contains 0.1 μg/mL to 30 μg/mL of the antibody to be tested.
The method of any one of claims 1-11, wherein the secondary antibody is specific for the antibody of the species from which the antibody to be tested comes from;

Preferably, the secondary antibody is selected from an anti-immunoglobulin antibody, such as an anti-IgG antibody, an anti-IgM antibody or an anti-IgA antibody;

Preferably, the immunoglobulin is from an immunized animal, such as a mouse.
The method according to any one of claims 1-12, wherein, after step (2) and/or (3), the method further comprises a step of discarding the liquid in the solid phase carrier;

Preferably, the step of washing the surface of the solid phase carrier is included or not included after the step of discarding the liquid.
The method of claim 13, wherein, after the step of discarding the liquid after step (3), the method further comprises adding a buffer to the solid phase carrier;

Preferably, the buffer is selected from PBS, Hanks BSS, Earles salt, DPBS, HBSS, EBSS and any combination thereof.