Method for screening blocking antibody

Abstract

The invention provides a one-step screening method for rapidly screening the affinity and the blocking function of the candidate antibody at low cost and high throughput. The method comprises the following specific steps: constructing an antibody gene to be screened in an expression vector, transfecting the vector into cells for expression, optionally using a high-concentration LiCl cell lysis solution to lyse the cells expressing the antibody in the cells, fully curing the antibody in a cell lysis supernatant or a cell culture supernatant by using a BLI sensor, immersing the sensor into an antigen so that the sensor is fully combined with the antibody, and detecting the antibody in the cell lysis supernatant by using the BLI sensor. After balancing, immersing into receptor protein for competitive experiment, and calculating the affinity constant of the antibody and the antigen and the efficiency of blocking the combination of the receptor and the antigen according to the detection signal of the sensor. According to the method, the tedious step of antibody purification in traditional screening is omitted, and the accuracy and the sensitivity which are the same as those of the purified antibody determination affinity and the blocking activity can be achieved; and step-by-step detection of traditional antibody affinity determination and function determination is combined, and the affinity and blocking efficiency data of the antibody can be obtained through one experiment. Meanwhile, the invention discloses a cell lysis solution formula, sample types of the method are expanded to comprise intracellular expression antibodies, BLI is used for determining the affinity of the antibodies and antigens in lysis cell supernatant, and the sensitivity of the cell lysis solution formula is equivalent to that of purified antibodies.

Description

[0001] Reference to relevant applications

[0002] This application claims priority to the invention patent application filed on September 30, 2024, with application number 202411388933.2 and entitled "A Method for Screening Blocking Antibodies", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the functional screening of candidate antibodies during the antibody discovery phase, such as binding affinity and / or blocking efficiency. Specifically, this invention relates to a rapid, high-throughput method for screening blocking antibodies. Background Technology

[0004] Biopharmaceuticals account for an increasingly large proportion of innovative drugs, with antibody drugs, such as monoclonal antibodies, polyclonal antibodies, and antibody-drug conjugates (ADCs), becoming increasingly popular research topics. However, antibody discovery remains the first hurdle hindering the rapid development of antibody drugs. Current antibody discovery pathways include hybridoma, single-cell B-cell screening, phage display library screening, and AI-based antibody generation. High-throughput screening methods, such as phage display libraries, yield hundreds of antibody candidate sequences. To screen these sequences for antibodies with high affinity and targeting specific antigen sites, four steps are required: first, synthesizing the antibody gene into an expression vector; second, expressing and purifying the antibody; third, screening the purified antibody based on antibody affinity; and fourth, performing functional experiments on the purified antibodies with binding capacity, such as blocking activity testing. Completing these four steps is time-consuming and costly, with the expression and purification of a large number of antibody sequences requiring significant production costs and representing the longest process. Therefore, high-throughput, rapid, sensitive, and low-cost antibody screening methods are urgently needed. This invention designs a screening method that reduces the above four steps to a single step, eliminating the need for antibody expression and purification. It utilizes only antibodies from cell culture supernatant or intracellularly expressed antibodies to simultaneously test the affinity and blocking function of candidate antibodies via biolayer interference (BLI). Some antibodies, when expressed in cell lines, fail to be properly secreted extracellularly due to folding or secretion peptides, leading to missed screenings when using cell culture supernatant. However, classic cell lysis buffers, after lysing cells, contain a large amount of irrelevant proteins, nucleic acids, lipids, and various complexes, making them incompatible with BLI for determining antibody affinity and blocking activity. Therefore, a superior cell lysis buffer is urgently needed to disrupt protein-nucleic acid and lipid complexes or precipitate nucleic acids. This invention attempts to add lithium chloride to the cell lysis buffer, effectively ensuring the BLI sensor's ability to specifically immobilize antibodies and the antibody's affinity for antigens.

[0005] BLI is a label-free technique that converts optical interference signals occurring on the surface of a biosensor into real-time response signals. When the optical layer binds to or dissociates from specifically binding molecules, the interference spectrum shifts to the right and left respectively, allowing for real-time recording of the binding and dissociation processes. The magnitude of the interference spectrum shift is positively correlated with the concentration of specifically binding molecules. BLI is commonly used for antibody affinity assays, but the antibodies are usually expressed and purified. This invention achieves the same accuracy and sensitivity as purified antibodies using antibodies from secretion supernatant and cell lysate supernatant. Furthermore, it combines antibody affinity and blocking activity testing into one step, whereas typically these two steps are performed separately: first, antibody affinity is tested using SPR or BLI, and then antibody functional activity is detected using ELISA or cell-level competitive assays. This invention uses a BLI sensor to obtain affinity data and blocking activity determination in a single step, providing a convenient, rapid, and low-cost high-throughput functional screening method for antibodies. The BLI Octet RED384 instrument has a throughput of 16 channels per cycle, higher than the Biacore instrument's maximum throughput of 8 channels per cycle. Therefore, the BLI instrument can test a significantly larger number of samples after multiple cycles compared to the Biacore. The price of the BLI instrument's sensor, which incorporates the sample, is only 1 / 10 of that of the Biacore sensor, and its elimination of the supernatant filtration step also greatly saves experimental time. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the problems of cumbersome, inefficient and costly antibody screening processes, and to propose a one-step method for screening antibodies with high binding affinity and high blocking efficiency.

[0007] On one hand, the present invention provides a method for screening blocking antibodies, which includes the following steps:

[0008] (1) Antibody solidification

[0009] The BLI sensor is immersed in a liquid containing candidate antibodies, optionally for a time of 60–1800 s, such as 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, 960, 1020, 1080, 1140, 1200, 1260, 1320, 1380, 1440, 1500, 1560, 1620, 1680, 1720, or 1800 s, or the BLI signal is monitored to rise by more than 0.2 nm, such as 1–10 nm, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nm, preferably 3–7 nm, such as 3, 4, 5, 6, or 7 nm;

[0010] (2) Antigen binding

[0011] Immerse the BLI sensor in a liquid containing the antigen, optionally for a time ranging from 60 to 1800 seconds, for example at intervals of 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, 960, 1020, 1080, 1140, 1200, 1260, 1320, 1380, 1440, 1 500, 1560, 1620, 1680, 1720 or 1800s, or, monitoring the BLI signal rise by more than 0.2nm, preferably 0.2 to 3nm, such as 0.2, 0.5, 1, 1.5, 2.0, 2.5 or 3.0nm, or 0.2 to 1nm, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0nm;

[0012] (3) Antigen dissociation

[0013] Immerse the BLI sensor in PBST buffer for an optional time of 60–1800 s, for example, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, 960, 1020, 1080, 1140, 1200, 1260, 1320, 1380, 1440, 1500, 1560, 1620, 1680, 1720, or 1800 s. The BLI signal (e.g., the BLI signal of an irrelevant antibody control) is monitored to decrease, for example, to the BLI signal level after antibody binding (e.g., for an irrelevant antibody control) or the BLI signal level after antigen binding decreases by at least 5% relative to the increase in the BLI signal level after antibody binding, for example, 10% to 100%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (e.g., for a candidate antibody); and

[0014] (4) Antigen-pair competitive binding

[0015] Immerse the BLI sensor in a liquid containing the antigen-pair, optionally for 60–1800 s, for example, at intervals of 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, 960, 1020, 1080, 1140, 1200, 1260, 1320, 1380, 1440, 1500, 1560, 162. 0, 1680, 1720, or 1800 s, or monitoring an increase in the BLI signal (e.g., the BLI signal of an antigen-antigen pair control), for example, an increase of more than 0.2 nm, for example, 0.2–3 nm, for example, 0.2, 0.5, 1, 1.5, 2.0, 2.5, or 3.0 nm, preferably 0.2–1 nm, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 nm; and

[0016] (5) Optional, affinity calculation

[0017] Optionally, use the following formula to calculate the binding affinity of the antibody to the antigen.

[0018] K D =k off / k on (For example, k) on It is obtained in the antigen-binding step, k off (obtained during the antigen dissociation step); and

[0019] (6) Optional, blocking efficiency calculation

[0020] Optionally, the efficiency of antibody blocking antigen-antigen pair binding can be calculated using the following formula. (For example, Ag_response[test] is the response value of the candidate antibody binding the antigen on the test group sensor, Rec_response[test] is the response value of the antigen (ligand) binding the antigen couple (receptor) on the test group sensor, Ag_response[ctrl] is the response value of the antigen binding on the control group sensor, and Rec_response[ctrl] is the response value of the antigen (ligand) binding the antigen couple (receptor) on the control group sensor, where the control group has no antibody or has irrelevant antibody).

[0021] In one implementation, each step is accompanied by a wash, for example using a buffer such as PBS, PBST (PBS, 0.02% Tween-20, pH 7.4), TAE, TBE, or MOPS.

[0022] In one embodiment, the antigen dissociation step uses a wash buffer prepared in PBS, PBST, TAE, TBE, or MOPS buffer. In one embodiment, the wash buffer further contains cations. In one embodiment, the cation is a monovalent cation, such as sodium, potassium, or ammonium ions. In one embodiment, the cation is a divalent cation, such as calcium or magnesium ions. In one embodiment, the wash buffer further contains anions. In one embodiment, the anion is a monovalent anion, such as chloride, iodide, bromide, nitrate, nitrite, bicarbonate, bisulfate, bisulfite, or dihydrogen phosphate. In one embodiment, the anion is a divalent anion, such as carbonate, sulfate, sulfite, or hydrogen phosphate. In one embodiment, the anion is a trivalent anion, such as phosphate. In one embodiment, the wash buffer contains at least 10 mM, such as 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000 mM or even higher concentrations of anions. In one embodiment, the wash buffer is prepared in PBST buffer and additionally contains 10-1000 mM, such as 150-800 mM, preferably 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 mM NaCl.

[0023] In one embodiment, the candidate antibody carries an Fc region, and the BLI sensor is a protein A-sensor. In another embodiment, the candidate antibody carries a His tag, and the BLI sensor is an NTA-sensor.

[0024] In one embodiment, the liquid containing the candidate antibody is a culture supernatant. In one embodiment, the culture supernatant is obtained by culturing cells that secrete the candidate antibody in a culture medium. In one embodiment, the culture medium does not contain serum.

[0025] In one embodiment, the liquid containing the candidate antibody is a cell lysate supernatant. In one embodiment, the cell lysate supernatant is the supernatant of lysates obtained by lysing cells expressing the candidate antibody intracellularly using a lysis buffer. In one embodiment, the lysis buffer contains 10–1000 mM, preferably 200–800 mM, more preferably 500 mM LiCl in 25 mM Tris pH 7.4, 150 mM NaCl, 0.5% w / v sodium deoxycholate, and 1% v / v Triton X-100.

[0026] In one embodiment, the antigen is a ligand and the antigen-pair is a receptor.

[0027] In one embodiment, the candidate antibody is a single-domain antibody. In another embodiment, the candidate antibody is a single-domain antibody with an Fc region (e.g., the IgG4 Fc region).

[0028] In one implementation scheme, 2 to 500, for example 5 to 250, or even 10 to 100, candidate antibodies are screened simultaneously.

[0029] On the other hand, the present invention provides a lysis buffer containing 10 to 1000 mM, preferably 200 to 800 mM, and more preferably 500 mM LiCl in 25 mM Tris pH 7.4, 150 mM NaCl, 0.5% w / v sodium deoxycholate, and 1% v / v Triton X-100.

[0030] In one embodiment, the lysis buffer is used to lyse cells expressing antibodies intracellularly. In another embodiment, the lysis buffer is used to lyse cells secreting antibodies extracellularly. Attached Figure Description

[0031] Figure 1 : Detection results (sensor signals) of 41 candidate antibodies binding to antigens and blocking receptor binding ligands (i.e., antigens).

[0032] Figure 2 The results of comparing the affinity constant and blocking efficiency of purified antibodies with those of cell supernatants (sensor signals) were used to determine these parameters.

[0033] Figure 3 : Detection results (sensor signal) of affinity constant determination when cells are cultured in serum-free medium.

[0034] Figure 4 Effect of cell lysis buffers containing different concentrations of lithium ions on the affinity constant of cell lysis supernatant (sensor signal). Detailed Implementation

[0035] The technical problem to be solved by this invention is to overcome the problems of cumbersome, inefficient and costly antibody screening processes, and to propose a one-step method for screening antibodies with high binding affinity and high blocking efficiency.

[0036] To address the aforementioned technical problems, this invention discloses a method and application for high-throughput screening of antibody function and activity, the method comprising the following steps:

[0037] (1) Synthesize the antibody gene to be screened and clone it into a eukaryotic expression vector;

[0038] (2) The recombinant vector is transferred into the expression host to enable antibody expression;

[0039] (3) Sensor immobilization protein 1 (e.g., antibody);

[0040] (4) Protein 1 on the sensor binds to protein 2 (e.g., antigen, i.e., ligand);

[0041] (5) The binary complex on the sensor competes with protein 3 (e.g., receptor).

[0042] The specific operation of step (1) is as follows:

[0043] 1.1. Synthesize the gene and clone it into an expression vector with a secretory peptide and / or a tag. The protein tag is preferably Fc, histidine tag, myc, or Flag; the secretory peptide is preferably the IL-10 signal peptide. The expression vector is preferably pcDNA or PTT series vectors, and more preferably pcDNA3.4 or PPT5 vectors.

[0044] 1.2. The recombinant plasmid was transformed into E. coli for amplification.

[0045] 1.3. Sanger sequencing was performed on the recombinant plasmid to confirm that the synthesized gene was free of mutations.

[0046] 1.4. Extraction and purification of plasmids.

[0047] The specific operation of step (2) is as follows:

[0048] 2.1. Resuscitate cells, expressing the cells as CHO, HEK293 cell lines, preferably CHO-K1, CHO-S or HEK293T cells.

[0049] 2.2. When the cell confluence reaches 90% or more, follow the regimen of 3×10 5 Cells were seeded into 96-well plates at a density of 1 cell / mL and cultured at 37°C and 5% CO2 for 18–24 hours.

[0050] 2.3. Prepare the DNA transfection complex according to the instructions for the transfection reagent.

[0051] 2.4. Remove the cell culture medium from the wells and add the transfection complex. Incubate the cells at 37°C and 5% CO2 for 4–6 hours.

[0052] 2.5. In one embodiment, the lysis buffer is used to lyse cells expressing antibodies intracellularly.

[0053] 2.6. Remove the transfection complex and add 100 μL of culture medium, preferably with or without serum, and incubate for 24–72 hours, preferably 72 hours, until the antibody is secreted into the cell culture medium.

[0054] Steps (3), (4), and (5) are performed using a molecular interaction instrument.

[0055] Step (3) involves immobilizing protein 1 using the sensor, preferably an antibody secreted into the culture medium, while simultaneously adding a washing step to remove non-specifically bound or weakly bound molecules from the sensor. The specific steps are as follows:

[0056] 3.1. Fourteen ProA sensors were selected as the experimental group to determine antibody affinity and blocking activity;

[0057] Two NTA sensors were defined as the receptor reference group and used as a reference for calculating antibody blocking activity.

[0058] 3.2. Immerse the sensor in PBST buffer (PBS, 0.02% Tween-20, pH 7.4) for pre-wetting for more than 10 minutes, preferably 10–30 minutes, to remove sucrose from the sensor surface. Next, immerse the sensor in PBST buffer for equilibration for 30–120 seconds, preferably 60–90 seconds.

[0059] 3.3. The experimental group sensor was immersed in cell culture supernatant or cell lysis supernatant to solidify the antibody. The receptor control group was immersed in PBST buffer for 100-1000 seconds, preferably 400-600 seconds. The experimental group sensor biological layer captured the antibody, and the BLI signal increased.

[0060] 3.4. The sensor is immersed in PBST buffer for equilibration, preferably for 60 to 1000 seconds, more preferably 200 to 600 seconds.

[0061] Step (4) involves binding protein 1 to protein 2 on the sensor, where protein 2 is preferably an antigen, to saturate the antibody with the antigen. The specific steps are as follows:

[0062] 4.1. Immerse the experimental group sensor in an antigen at a concentration of 10–1000 nM, preferably 20–200 nM, to allow the antibody on the sensor to bind to the antigen. Immerse the receptor reference group sensor in a receptor protein at a concentration of 10–1000 nM, preferably 20–200 nM, for 30–600 seconds, preferably 60–200 seconds. The antibody on the experimental group sensor binds to the antigen, and the receptor reference group sensor solidifies the receptor protein. The thickness of the biological layer increases, and the BLI signal rises.

[0063] 4.2. Immerse the sensor in PBST buffer and equilibrate for 60–400 seconds, preferably 60–200 seconds.

[0064] Step (5) involves performing an antigen-antigen binary complex on the sensor and protein 3 to perform an antigen epitope competition analysis. Protein 3 is preferably a receptor. The specific operation is as follows:

[0065] 5.1. The experimental group sensor is immersed in 10-1000 nM of receptor, preferably 20-200 nM, and the antigen on the sensor may or may not bind to the receptor; the receptor reference group sensor is immersed in 10-1000 nM of antigen, preferably 20-200 nM, for 30-600 seconds, preferably 60-200 seconds.

[0066] 5.2. The sensor is immersed in 10mM, pH 2.0 glycine for 5 seconds, followed by immersion in PBST buffer for 5 seconds. These two steps are repeated to ensure that the antibodies, antigens, and receptors on the sensor are fully eluted in the glycine solution, and the PBST signal drops to 0, completing sensor regeneration. The regenerated sensor is then ready for a new round of experiments.

[0067] In some embodiments of the present invention, the proportion of antigen-binding receptors in the absence of antibodies can be calculated using a receptor reference group, as follows:

[0068] Re c_response[ctrl] / Ag_response[ctrl]

[0069] Rec_response[ctrl] represents the receptor reference group sensor's response value when binding to the receptor, and Ag_response[ctrl] represents the receptor reference group sensor's response value when binding to the antigen. The above formula can be used to calculate the proportion of antigen-receptor binding in the experimental group, i.e., in the presence of antibodies.

[0070] Furthermore, the efficiency of antibody blocking receptor binding to antigen was obtained by comparing the ratio of the experimental group to the receptor control group, and the calculation formula is as follows:

[0071]

[0072] Rec_response[test] represents the response value of the sensor binding the receptor in the experimental group, and Ag_response[test] represents the response value of the sensor binding the antigen in the experimental group.

[0073] In some embodiments of the present invention, the KD value and blocking activity of the purified antibody and the antibody-antigen binding in the cell expression supernatant were compared. The specific embodiments were carried out according to (3)(4)(5). The antibody samples in step (3) were the purified antibody and the antibody secreted into the culture medium, respectively. The concentration of the purified antibody was preferably 1 to 50 μg / ml.

[0074] In some embodiments of the present invention, antibodies were expressed using serum-free culture medium and their KD values ​​were determined, and specific embodiments were carried out according to (3) and (4).

[0075] In some embodiments of the present invention, a modified cell lysis buffer is used to release intracellular proteins and to determine the KD values ​​of antibodies and antigens in the cell lysis supernatant using BLI. The specific steps are as follows: X.1. Obtain CHO-K1 or 293T cells expressing antibodies intracellularly, referring to (1) and (2).

[0076] X.2. Preparation of cell lysis buffer. Cell lysis buffer A consists of: 25 mM Tris (pH 7.4), 150 mM NaCl, 0.5% (w / v) sodium deoxycholate, 1% (v / v) Triton X-100 and 1000 mM LiCl;

[0077] The LiCl concentration in cell lysis buffer B was 500 mM; the LiCl concentration in cell lysis buffer C was 100 mM.

[0078] The LiCl concentration in cell lysate D is 0.

[0079] X.3. Resuspend and lyse the cells using cell lysis buffer to release the antibodies in the cytoplasm, centrifuge, and collect the supernatant. X.4. Measure the KD value according to (3) and (4).

[0080] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Specific details not specified in the examples are performed under conventional conditions in the art or conditions recommended by the manufacturer.

[0081] Example 1: One-step method for determining the affinity and blocking activity of 41 candidate antibodies

[0082] (I) Experimental Methods

[0083] 1. Construction of recombinant expression vectors

[0084] 1.1. Forty-one candidate antibody genes were synthesized and cloned into the eukaryotic secretory expression vector pcDNA3.4 (carrying human IL-10 signal peptide and human IgG4 Fc gene).

[0085] 1.2. The recombinant plasmid was transformed into Escherichia coli (ThermoFisher, EC0112) for amplification.

[0086] 1.3. Sanger sequencing was performed on the recombinant plasmid to confirm that the synthesized gene was free of mutations.

[0087] 1.4. Extraction and purification of plasmids.

[0088] 2. Cell transfection

[0089] 2.1. Resuscitate CHO-KI (ATCC, CCL-61) cells.

[0090] 2.2. When the cell confluence reaches 90% or higher, dilute the cells to 3 × 10⁶ cells using IMDM medium (Gibco) containing 10% FBS (Gibco, A5669701). 5 Cells / ml: Seed 100 μL of cells into 96-well plates (Corning, 3599) and incubate at 37°C with 5% CO2 for 18–24 hours.

[0091] 2.3. Prepare the DNA transfection complex according to the instructions of the transfection reagent (SinoBiological, STF02).

[0092] 2.4. Remove the cell culture medium from the well and add 50 μL of the transfection complex. Incubate the cells at 37°C and 5% CO2 for 4–6 hours.

[0093] 2.5. Remove the transfection complex and add 100 μL of IMDM medium (Gibco) containing 10% FBS (Gibco, A5669701), and incubate for 48–72 h.

[0094] 2.6. Collect the cell culture supernatant and wait for the next experiment.

[0095] 3. Affinity and blocking activity assay

[0096] 3.1. Fourteen ProA sensors (Sartorius, 18-5010) were selected as the experimental group to determine the affinity and blocking activity of the antibody (including Fc); two NTA sensors (Sartorius, 18-5101) were selected as the control group to be used as a reference for calculating the antibody blocking activity.

[0097] 3.2. Immerse the sensor in PBST buffer (PBS + 0.02% Tween 20) for 10 minutes to remove sucrose from the sensor surface.

[0098] 3.3. Experiments were conducted using a molecular interaction apparatus (Sartorius, Octet Red384):

[0099] 3.3.1. Immerse the sensor in PBST buffer for equilibration for 60 seconds.

[0100] 3.3.2. The experimental group's sensor was immersed in the cell culture supernatant, while the control group was immersed in PBST buffer for 600s. This allowed the sensor to bind to the antibody in the culture medium, solidify the antibody in the sensor's biological layer, increase its thickness, and raise the BLI signal.

[0101] 3.3.3. The sensor was immersed in PBST buffer for 400 seconds for cleaning.

[0102] 3.3.4. The experimental group sensor was immersed in 100 nM human TSLP (R&D SYSTEMS, 1398-TS / CF), and the antibody on the sensor bound the antigen; the control group sensor was immersed in 100 nM receptor human IL7RA & TSLPR (SinoBiological, CT115-H08H1, with His tag) to immobilize the receptor for 90 s.

[0103] 3.3.5. All sensors were immersed in PBST buffer for 90 seconds for dissociation.

[0104] 3.3.6. The experimental group sensor was immersed in 100 nM receptor-human IL7RA & TSLPR for competitive binding experiments; the control group sensor was immersed in 100 nM human TSLP, and the receptor on the sensor bound to the antigen for 90 s.

[0105] 3.3.7. Immerse the sensor in a 10mM glycine solution at pH 2.0 for 5 seconds to regenerate it, then rinse it with PBST buffer for 5 seconds. Repeat this step 3 times.

[0106] 3.3.8. Repeat steps 3.3.1 to 3.3.7 6 times to complete the screening of 41 antibody strains.

[0107] 4. Data Processing

[0108] 4.1. The affinity data of antibody binding to antigen were obtained by analyzing the 1:1 fitting model.

[0109] 4.2. Calculation using antigen response value (Ag_response) and receptor response value (Rec_response)

[0110] The efficiency of antibody blocking antigen-receptor binding is calculated using the following formula:

[0111]

[0112] (II) Experimental Results

[0113] Table 1 shows the affinity data for antigens bound to the cell expression supernatants of 41 antibody strains and the data on blocking antigen-receptor binding. Eleven of these antibodies bound to the antigens; three had a KD value in the 10.0 nM range, and eight had a KD value in the 1.0 nM range (Table 1). Analysis of the blocking activity of these antibodies revealed that three antibodies achieved 100% efficiency in blocking antigen-receptor binding, while the remaining eight antibodies showed blocking activities exceeding 98%.

[0114] Table 1: Detection results of antibody binding to antigen and blocking receptor binding for 41 strains (ND = Not detected)

[0115]

[0116]

[0117] This invention enables the acquisition of affinity and blocking activity data for 41 antibodies from cell expression supernatant within a short time (2.5 hours, three rounds of experiments, 16 probes per round), and three high-affinity, completely blocking antibodies were screened out, demonstrating the high efficiency and high throughput of this method.

[0118] Example 2: Comparison of the sensitivity of methods for determining affinity and blocking activity using cell expression supernatant and purified antibody.

[0119] (I) Experimental Methods

[0120] The 20 μg / ml purified product solutions, 1 μg / ml purified product solutions, and cell culture supernatants corresponding to antibodies 42, 43, and 44 were tested according to "1. Antibody gene synthesis", "2. Cell transfection", and "3. Affinity and blocking activity detection" in Example 1. Data processing was performed according to "4. Data processing" in Example 1 to obtain the affinity and blocking activity data of each group of antibodies.

[0121] (II) Experimental Results

[0122] Affinity data and data on blocking antigen-receptor binding of antibodies 42, 43, and 44 at 20 μg / ml and 1 μg / ml concentrations, as well as cell culture supernatant, are shown in Table 2 and [Table data would be inserted here]. Figure 2 .

[0123] Table 2: Affinity constants and blocking efficiencies of purified antibodies 42, 43, and 44 and cell culture supernatants.

[0124] Antibody number <![CDATA[K D (M)]]> Blocking efficiency, % 42-H 1.23E-08 100 42-L 1.10E-08 100 42-SN 1.17E-08 100 43-H 1.17E-08 100 43-L 8.20E-09 100 43-SN 1.12E-08 100 44-H 8.44E-09 100 44-L 8.34E-09 100 44-SN 1.06E-08 100

[0125] SN = Culture supernatant

[0126] H = corresponding to a high concentration (20 μg / ml) purified antibody solution

[0127] L = the corresponding low concentration (1 μg / ml) purified antibody solution.

[0128] Experimental results showed that the affinity constants of the purified antibody and the cell culture supernatant differed by less than three times, and there was no difference in blocking activity. This indicates that the affinity and blocking activity measured in the cell culture supernatant can reach the level of the purified antibody, demonstrating that this is a highly sensitive screening method. Antibodies 42, 43, and 44 in Example 2 correspond to antibodies 12, 5, and 1 in Example 1, respectively. The affinity constants tested in the two experiments differed by less than three times, and there was no difference in blocking activity, indicating that the method of this invention is highly substitutable for existing purified antibody testing techniques.

[0129] Example 3: Sensitivity of antibody affinity assay in serum-free culture medium

[0130] (I) Experimental Methods

[0131] Following step "1" in Example 1, the antibody gene was synthesized; following step "2", transfection was performed, and after 4–6 hours of transfection, the culture medium (Opium Med, P83059) was replaced and cultured for 72 hours. Affinity testing was performed following step "3" in Example 1, adjusting the washing time in "3.3.3" to 200 seconds. Data processing was performed following step "4" in Example 1 to obtain affinity data.

[0132] (II) Experimental Results

[0133] Affinity data for the antibodies expressed in serum-free culture medium from 8 strains are shown in Table 3 and 4. Figure 3 .

[0134] Table 3: Affinity constants of 8 antibodies expressed in serum-free culture medium (ND = Not detected)

[0135] Antibody number <![CDATA[K D (M)]]> Antibody number <![CDATA[K D (M)]]> 45 3.29E-12 49 2.03E-08 46 ND 50 2.73E-08 47 1.35E-08 51 1.72E-08 48 2.56E-08 52 ND

[0136] Experimental results show that the Protein A sensor immobilizes antibodies secreted in serum-free medium. During the sensor washing step, the antibodies immobilized on the sensor do not dissociate, which helps to shorten washing and screening times. Since the protein immobilized by the sensor has higher purity, the theoretically measured affinity constant is more accurate. Furthermore, due to the high cost of serum, using serum-free medium significantly reduces screening costs, demonstrating the low cost and high efficiency of the method of this invention. In the "sensor captures antibody" step, antibody number 52 did not show an increase in BLI signal, indicating that the antibody was not expressed and this sequence can be discarded.

[0137] Example 4: Effect of different concentrations of lithium-ion cell lysis buffer on antibody affinity assay in cell lysis supernatant

[0138] (I) Experimental Methods

[0139] 1. Following the procedure in Example 1, construct a recombinant vector containing only a histidine tag antibody, and obtain a recombinant plasmid through extraction and purification.

[0140] 2. Following step “2” in Example 1, recombinant CHO-K1 cells transfected for 72 hours were obtained. The cell pellet was collected and flash-frozen at -80°C.

[0141] 3. Prepare the cell lysis buffer, namely, 25 mM Tris (pH 7.4) in double-distilled water, 150 mM NaCl, and 0.5%...

[0142] Sodium deoxycholate (w / v), 1% (v / v) Triton X-100, and different concentrations of LiCl were used as cell lysis buffers. Cell lysis buffer A had a LiCl concentration of 1000 mM; cell lysis buffer B had a LiCl concentration of 500 mM; cell lysis buffer C had a LiCl concentration of 100 mM; and cell lysis buffer D had a LiCl concentration of 0 mM.

[0143] 4. Add 1 mM PMSF (Shanghai Sangon Biotech, A610425) to the cell lysis buffer, mix well, then add lysis buffer to the cell pellet, mix by pipetting, and lyse on ice for 30 minutes. After complete lysis, centrifuge at 10000-14000g for 3-5 minutes at 4℃, and use the supernatant to determine antibody affinity.

[0144] 5. Refer to “3” in Example 1 and use Octet Red384 to perform affinity determination.

[0145] 6. Take 5 NTA sensors and immerse them in PBST buffer for 10 minutes to remove sucrose from the sensor surface.

[0146] 7. Immerse the sensor in PBST buffer for equilibration for 60 seconds.

[0147] 8.4 NTA sensors (experimental group) were immersed in the supernatant of 4 different cell lysates, and 1 NTA sensor (positive control) was immersed in purified antibody (prepared with PBST, concentration: 5ug / ml) for 300s. Nickel ions on the sensor bind to the histidine tag on the antibody, increasing the thickness of the sensor's biological layer, which is reflected in an increase in BLI signal.

[0148] 9. Immerse the sensor in PBST buffer for 120 seconds.

[0149] 10. Immerse the sensor in PBST buffer and perform baseline operation for 60 seconds.

[0150] 11. When the sensor is immersed in a 100 nM antigen solution for 120 s, the antibody on the sensor binds to the antigen, the thickness of the sensor's biological layer increases, which is reflected in an increase in the BLI signal.

[0151] 12. The sensor was immersed in PBST buffer for 60 seconds. The antigen on the sensor dissociated, the thickness of the biological layer decreased, and the BLI signal decreased.

[0152] (II) Experimental Results

[0153] The results were analyzed using a 1:1 fitting model to obtain the affinity results for different cell lysis buffer groups. The experimental results are shown in the table below.

[0154] Table 4: Affinity constants of antibodies in the lysis supernatant of four cell lysis buffers

[0155]

[0156]

[0157] Experimental results showed that the KD value of cell lysis buffer group B was within three times that of the purified antibody group, and there was no significant difference in affinity between the two. Cell lysis buffer containing 500mM LiCl effectively ensured the affinity of the antibody for the antigen in the cell lysis supernatant. The antibody KD values ​​of cell lysis buffer groups containing 1000mM, 100mM LiCl, and no LiCl were more than three times that of the purified antibody group; these conditions are not suitable for intracellular antibody expression.

[0158] This invention provides a rapid, high-throughput method for screening antibody functional activity and its applications. The four examples above illustrate the advantages of this method, including high efficiency, high throughput, high sensitivity, and low cost. Example 4 discloses a new lysis buffer formulation, extending the applicability of this method to cell lysis buffer samples. This method can be applied to high-throughput screening of antibodies, proteins, etc., facilitating the early discovery of antibody drugs.

[0159] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various modifications to the method in form and detail are all within the scope of protection of the present invention.

Claims

1. A method for screening blocking antibodies, comprising the following steps: (1) Antibody solidification Immerse the BLI sensor in a liquid containing the candidate antibody, optionally for 60 to 1800 s, or monitor the BLI signal rise by more than 0.2 nm, preferably 1 to 10 nm. (2) Antigen binding Immerse the BLI sensor in a liquid containing the antigen, optionally for 60 to 1800 s, or monitor the BLI signal rise by more than 0.2 nm, preferably 0.2 to 3 nm; (3) Antigen dissociation Immerse the BLI sensor in PBST buffer, optionally for 60–1800 s, or monitor for a decrease in the BLI signal of an unrelated antibody control; and (4) Antigen-pair competitive binding Immerse the BLI sensor in a liquid containing the antigen-pair, optionally for 60–1800 s, or monitor for an increase in the antigen-pair control BLI signal of 0.2 nm or more, preferably 0.2–3 nm; and (5) Optional, affinity calculation Optionally, use the following formula to calculate the binding affinity of the antibody to the antigen. K D =k off / k on ;and (6) Optional, blocking efficiency calculation Optionally, the efficiency of antibody blocking antigen-antigen pair binding can be calculated using the following formula.

2. The method according to claim 1, wherein (1) The candidate antibody has an Fc region, and the BLI sensor is a protein A-sensor; or (2) The candidate antibody is tagged with His, and the BLI sensor is an NTA-sensor.

3. The method according to claim 1, wherein the liquid containing the candidate antibody is culture supernatant or cell lysate supernatant.

4. The method of claim 3, wherein the culture supernatant is obtained by culturing cells that secrete candidate antibodies in a culture medium.

5. The method of claim 4, wherein the culture medium is serum-free.

6. The method of claim 3, wherein the cell lysate supernatant is the supernatant of lysate obtained by lysing cells expressing candidate antibodies intracellularly using a lysis buffer.

7. The method according to claim 6, wherein the lysis buffer contains 10 to 1000 mM, preferably 200 to 800 mM, more preferably 500 mM LiCl in 25 mM Tris pH 7.4, 150 mM NaCl, 0.5% w / v sodium deoxycholate, and 1% v / v Triton X-100.

8. The method according to claim 1, wherein (1) The antigen is the ligand, and the antigen's partner is the receptor; or (2) The antigen is the receptor, and the antigen's partner is the ligand.

9. The method of claim 1, wherein the candidate antibody is a single-domain antibody.

10. The method according to claim 1, wherein 2 to 500, for example 5 to 250, or even 10 to 100, candidate antibodies are screened simultaneously.

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