An in vitro method for detecting antibody internalization efficiency

Abstract

The application discloses an in-vitro detection method for antibody internalization efficiency, comprising the following steps: after antigen-positive cells and antigen-negative cells are digested and AO / PI-stained and counted, the cells are resuspended with complete culture medium, inoculated and cultured; an incubation system is prepared by using a to-be-detected antibody which has no killing effect on the antigen-positive cells and a recombinant protein DT3C, and the antibody-DT3C conjugate is prepared by incubation; the cultured antigen-positive cells and antigen-negative cells are each divided into a no-antibody control group, an antibody treatment group and a complete killing group, and 0.1% Triton X-100, the antibody-DT3C conjugate and a protein-free incubation culture medium are respectively added into the three groups; the antigen-positive cells are co-cultured, the cell activity of the antigen-positive cells is detected, and the cell survival rate and the antibody internalization efficiency are calculated. By adding low-concentration Ficoll70 and PEG6000, the coupling efficiency is significantly improved, the detection sensitivity and repeatability are improved, the result determination is accurate, and the flux is high.

Description

Technical Field

[0001] This invention relates to the field of antibody-drug conjugate (ADC) detection technology. More specifically, it relates to an in vitro method for detecting antibody internalization efficiency. Background Technology

[0002] Antibody-drug conjugates (ADCs) are innovative cancer treatments that combine monoclonal antibodies with highly effective cytotoxic agents, enabling precise recognition and efficient killing of tumor cells. Their design philosophy integrates the superior targeting capabilities of antibodies with the powerful cytotoxic effects of small-molecule toxins, thereby enhancing drug selectivity for tumor tissues, reducing non-specific damage to normal cells, and more efficiently killing tumor cells.

[0003] The antitumor mechanism of ADC drugs can be mainly divided into three key steps: First, the drug is partially and specifically recognized and bound to the target antigen highly expressed on the surface of tumor cells by antibodies; then, through receptor-mediated endocytosis, the ADC-antigen complex is internalized into the tumor cell. In the lysosomal or endosome environment, the linker between the antibody and the toxin molecule is specifically broken, thereby releasing the active cytotoxin; finally, the free small molecule toxin interferes with the key physiological processes of tumor cells, such as mitosis, nucleic acid metabolism, or protein synthesis, thereby inducing cell cycle arrest and apoptosis, achieving the purpose of eliminating tumor cells.

[0004] The efficacy of antibody-drug conjugates (ADCs) largely depends on their internalization efficiency, i.e., whether the antibody can be effectively and efficiently internalized by target cells and release the drug. Antibody internalization refers to the process by which an antibody, after specifically binding to an antigen on the cell membrane surface, is transported into the cell interior (such as endosomes or lysosomes) through an antigen-antibody complex. Accurately determining the internalization efficiency of antibodies is an important prerequisite for evaluating their drug-likeness and efficacy. An ideal detection method should have high throughput, high sensitivity, and a wide range of applications. Existing detection technologies mainly fall into the following categories: The first is the traditional acid elution method, which uses a low-pH buffer to elute uninternalized antibodies bound to the cell surface and detects the residual fluorescence signal inside the cell by flow cytometry. However, this method is cumbersome, cells are easily damaged in an acidic environment, and incomplete elution can easily lead to false positive results. The second is the confocal microscopy imaging method, which directly observes whether the fluorescence signal has entered the cell interior after the cell is labeled with antibodies using a high-resolution microscope. However, this method relies on expensive and sophisticated instruments, has low throughput, is difficult to perform quantitative analysis, and is highly subjective, making it unsuitable for large-scale screening. The third is the radioactive isotope labeling method. The first method uses labeled antibodies such as 125I to separate intracellular and extracellular radioactivity. This method carries the risk of radioactive contamination, requires specialized qualifications, and isotope decay may affect antibody bioactivity. The second method is an indirect method using enzyme-linked immunosorbent assay (ELISA). This method detects the amount of antibody inside cells after lysis, but it cannot distinguish between membrane-bound antibodies and fully internalized antibodies. The third method is an indirect detection method using toxin conjugation. Currently, FabFc-ZAP is widely used. It is an immunotoxin composed of an antibody that recognizes the human IgG Fc fragment conjugated to the ribosome-inactivating protein saporin. When it binds to a human primary antibody, it can be internalized by cells expressing the target antigen, thereby specifically killing the cell. However, this protein has a large molecular weight, which can affect the tertiary structure of the antibody and create steric hindrance, affecting the antigen-antibody recognition and binding process.

[0005] To overcome the shortcomings of traditional detection techniques, recombinant protein DT3C is applied to the field of antibody internalization efficiency detection. Recombinant protein DT3C is a DT3C recombinant fusion protein composed of a diphtheriatoxin (DT) without a receptor-binding domain and the C1, C2, and C3 (3C) domains of streptococcus protein G. It can bind to antibodies, attaching to the cell surface through antigen-antibody specific recognition and entering the cell via endocytosis. Intracellularly, it undergoes hydrolysis, releasing a toxic toxin payload that inhibits normal protein translation, leading to cell death. DT3C can bind to any IgG antibody, thus it can be used to detect the internalization efficiency of antibodies from different species. Furthermore, DT3C has a small molecular weight and does not affect antigen-antibody recognition and binding. The mAb-DT3C conjugate formed after DT3C binds to an antibody mimics the mechanism of action of ADC drugs in vitro, which helps to evaluate antibody internalization capacity and drug release efficiency in the early stages of drug development. However, during the formation of mAb-DT3C conjugates, the binding efficiency of recombinant DT3C protein to antibodies is affected by various factors, including protein conformational stability, solution environment, and the degree of protein aggregation. How to further improve the conjugation efficiency of DT3C to antibodies, thereby enhancing the sensitivity and reproducibility of detection, remains a pressing technical problem to be solved in this field.

[0006] Therefore, developing an optimized in vitro detection method for antibody internalization efficiency, and performing a preliminary screening step for ADC drug antibodies in vitro, can efficiently and effectively screen out superior candidate antibodies with high specificity and high internalization efficiency. Summary of the Invention

[0007] One objective of this invention is to provide an efficient, rapid, accurate, and high-throughput in vitro detection method for antibody internalization efficiency, particularly by optimizing the formation conditions of antibody-DT3C conjugates to improve conjugation efficiency and further enhance detection sensitivity and reproducibility.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides an in vitro detection method for antibody internalization efficiency, comprising the following steps: Antigen-positive and antigen-negative cells were digested, AO / PI stained and counted, then resuspended in complete culture medium and seeded into culture plates for culture. Antibodies that do not kill antigen-positive cells alone were selected and incubated with recombinant protein DT3C in a protein-free incubation medium to prepare antibody-DT3C conjugates. After adhering to the culture vessel, antigen-positive cells and antigen-negative cells were divided into a control group without antibody, an antibody-treated group, and a complete killing group. The antibody-DT3C conjugate was added to the antibody-treated group, an equal volume of protein-free incubation medium was added to the control group without antibody, and an equal volume of 0.1% Triton X-100 was added to the complete killing group to construct a co-culture system. The co-culture systems of each group were co-cultured. The antibody-DT3C conjugate had no killing effect on antigen-negative cells but had a killing effect on antigen-positive cells. The cell viability of antigen-positive cells was detected, and the cell viability was calculated according to the measured signal value using the following formula: Cell viability = (Signal value of antibody-treated group - Signal value of completely killed group) / (Signal value of control group without antibody - Signal value of completely killed group) × 100%; The antibody internalization efficiency is calculated based on the cell viability using the following formula: Antibody internalization efficiency = 100% - Cell viability.

[0009] In a specific embodiment of the present invention, the recombinant protein DT3C is the DT3C recombinant protein expressed by the *E. coli* expression system, comprising 33-417 aa (P00588) & 291-497 aa (P19909), with a 6×His tag at the N-terminus. It is composed of a diphtheria toxin (DT) without a receptor-binding domain and the C1, C2, and C3 (3C) domains of streptococcus protein G. It can bind to the Fc fragment of an antibody, replacing the small molecule drug in an ADC (antibody-drug conjugate) drug, and is an immunotoxin. The resulting antibody-DT3C conjugate mimics the ADC drug, and the antibody internalization efficiency is evaluated. After the antibody-DT3C conjugate recognizes and binds to the cell surface antigen, it is internalized. Then, the recombinant protein DT3C is cleaved by furin protease, and the catalytic domain of the recombinant protein DT3C is released into the cytoplasm, leading to ADP-ribosylation of elongation factor (EF)-2, subsequently causing cytotoxicity through inhibition of protein translation.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the recombinant protein DT3C is shown in SEQ ID NO.1, and the N-terminus contains a 6×His tag.

[0011] In a specific embodiment of the present invention, the reagent kit used for detecting cell viability is selected from at least one of an MTT assay kit, a CCK-8 assay kit, and a chemiluminescence assay kit; the signal value is absorbance or chemiluminescence intensity, specifically as follows: When using the MTT assay kit or the CCK-8 assay kit, the signal value is absorbance. When using a chemiluminescence detection kit, the signal value is the chemiluminescence intensity.

[0012] In a specific embodiment of the present invention, the detection method for the antibody to be tested and the recombinant protein DT3C, which have no killing effect on antigen-positive cells, is as follows: the antibody to be tested and the recombinant protein DT3C are co-cultured with cultured antigen-positive cells, respectively, the cell activity of the antigen-positive cells is detected, the cell viability and the internalization efficiency of the antibody to be tested are calculated, and the antibody to be tested and the recombinant protein DT3C, which have no killing effect on antigen-positive cells, are obtained.

[0013] In a specific embodiment of the present invention, the protein-free incubation medium includes a basal medium and additives, wherein the additives include the following components in weight-volume percentages: 1-3% (w / v) Ficoll and 0.5-1.5% (w / v) PEG6000. In this invention, the Ficoll content in the incubation system is 1-3% by mass and volume. As a non-specific protein stabilizer, Ficoll effectively maintains the native conformation of the DT3C recombinant protein and antibody in the incubation system, reduces non-specific aggregation between protein molecules, and thus promotes full contact and efficient binding between the 3C domain of DT3C and the Fc segment of the antibody, significantly improving the formation efficiency of antibody-DT3C conjugates. The PEG6000 content in the incubation system is 0.5-1.5% by mass and volume. PEG6000 is a highly hydrophilic and inert macromolecule that mainly plays a exclusion volume effect in the incubation system. It can compress the effective space. PEG6000 itself occupies a large volume of solution, indirectly "squeezing" the protein into the remaining small volume, thereby rapidly increasing the local protein concentration, improving the effective concentration, and significantly increasing the frequency of intermolecular collisions. Protein interactions become easier to occur in this crowded environment, and the formed complexes are not easily dissociated, thus enhancing the formation of antibody-DT3C conjugates.

[0014] In a preferred embodiment of the present invention, the additive comprises the following components in weight-volume percentages: 3% (w / v) Ficoll and 1.5% (w / v) PEG6000.

[0015] In a specific embodiment of the present invention, the complete culture medium comprises the following components in volume fractions: 80%-90% basal culture medium and 20%-10% fetal bovine serum (FBS).

[0016] In a preferred embodiment of the present invention, the complete culture medium comprises the following components in volume fractions: 90% basal culture medium and 10% fetal bovine serum (FBS).

[0017] In a specific embodiment of the present invention, the basal culture medium is selected from at least one of McCoy's 5A medium, DMEM high glucose medium, and Leibovitz L-15 medium.

[0018] In a specific embodiment of the present invention, the concentration of the antibody to be detected in the incubation system is 0-30 μg / mL, and not 0 μg / mL (e.g., 0.001 μg / mL, 0.005 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL, 20 μg / mL, and 30 μg / mL can be selected); the molar ratio of the antibody to be detected to recombinant protein DT3C is 1:1-1:4; and the solvent for both the antibody to be detected and the recombinant protein DT3C is a protein-free incubation medium.

[0019] In a preferred embodiment of the present invention, the concentration of the antibody to be detected in the incubation system is 20 μg / mL, and the molar ratio of the antibody to be detected to recombinant protein DT3C is 1:2.

[0020] In a specific embodiment of the present invention, the incubation is performed at 37°C for 30-60 minutes.

[0021] In a specific embodiment of the present invention, the number of antigen-positive cells and antigen-negative cells inoculated during the culture and co-culture is 1,000-10,000 cells / well, such as 1,000, 3,000, 5,000, 7,000, and 10,000 cells / well.

[0022] In a preferred embodiment of the present invention, the number of antigen-positive cells and antigen-negative cells inoculated during the culture and co-culture is 2000-9000 per well, respectively.

[0023] In a more preferred embodiment of the present invention, the number of antigen-positive cells and antigen-negative cells inoculated during the culture and co-culture is 3000-8000 per well.

[0024] In the most preferred embodiment of the present invention, the number of antigen-positive cells and antigen-negative cells inoculated during the culture and co-culture is 5000 per well.

[0025] In a specific embodiment of the present invention, the antibody to be detected is an antibody against an antigen; the antigen includes tumor therapeutic target proteins or immune checkpoint proteins.

[0026] In a preferred embodiment of the present invention, the antigen includes, but is not limited to, HER2, HER3, EGFR, TROP2, CD22, CD30, PD-L1, B7H3, B7H4, CTLA4, and LAG3.

[0027] In a specific embodiment of the present invention, the antigen-positive cells and antigen-negative cells are cells with a fusion degree ≥ 80%.

[0028] In a preferred embodiment of the present invention, the antigen-positive cells are cancer cells that highly express the corresponding antigen on their cell surface or artificially modified cells that overexpress the corresponding antigen; the antigen-negative cells are cancer cells that express the corresponding antigen very low on their cell surface; wherein, "high expression" means that, when detected by flow cytometry, the positive expression rate of the cancer cell surface antigen is ≥70%, and the mean fluorescence intensity (MFI) is ≥5 times that of the isotype control; and "very low expression" means that, when detected by the same method, the positive expression rate of the cancer cell surface antigen is ≤5%, and the MFI is ≤1.5 times that of the isotype control.

[0029] In a preferred embodiment of the present invention, the antigen is HER2; the antibody to be detected is an anti-HER2 antibody, specifically trastuzumab; the antigen-positive cell is SK-BR-3 breast cancer cell, which is a cancer cell that highly expresses 2HER, and the antigen-negative cell is MDA-MB-468 breast cancer cell, which is a cancer cell that expresses very low HER2.

[0030] In a specific embodiment of the present invention, the culture plate is a 96-well culture plate with white well walls and white well bottom.

[0031] In a specific embodiment of the present invention, the culture is carried out for 18-24 hours to ensure that the cells can stably adhere to the wall and grow.

[0032] In a specific embodiment of the present invention, the co-culture is carried out at 37±1℃ and 5±0.5%CO2 for 72-96 hours, such as 72 hours, 78 hours, 84 hours, 90 hours or 96 hours.

[0033] In this invention, the killing effect refers to the cell viability after treatment with a certain substance being at least 20% lower than that of cells under the same culture conditions without treatment with the certain substance; the non-killing effect refers to the cell viability after treatment with a certain substance being within 20% lower than that of cells under the same culture conditions without treatment with the certain substance.

[0034] The beneficial effects of this invention are as follows: This invention's in vitro antibody internalization efficiency detection method effectively improves the conjugation efficiency of DT3C and antibodies by adding 1-3% Ficoll and 0.5-1.5% PEG6000 to the antibody-DT3C incubation system. This results in a more complete and stable formation of the antibody-DT3C conjugate, directly enhancing the detection sensitivity and reproducibility of subsequent cell killing experiments. Furthermore, this invention's in vitro antibody internalization efficiency detection method includes measuring the inhibitory effect on antigen-positive cells, making the in vitro detection of antibody internalization efficiency more intuitive, direct, and accurately quantifiable. It can realistically simulate the cell killing process after ADC drug endocytosis, effectively evaluating whether the antibody can be internalized by target cells and efficiently release the drug, thereby improving the consistency of antibody internalization efficiency detection results in vitro and in vivo. In addition, this invention's method is simple to operate, time-saving, has few interfering factors, a wide range of applications, accurate result interpretation, and high throughput. It can perform high-throughput parallel testing on multiple antibodies, significantly improving the antibody and target screening efficiency in the early stages of ADC drug development. Attached Figure Description

[0035] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0036] Figure 1 Figure 1 shows the preliminary experimental results of trastuzumab internalization efficiency in the HER2-overexpressing cell line SK-BR-3 in Examples 1, 2, and 3. In this figure, A represents the killing efficiency of different trastuzumab treatment concentrations in the HER2-overexpressing cell line SK-BR-3; B represents the killing efficiency of different concentrations of trastuzumab combined with a fixed concentration of recombinant protein DT3C and different concentrations of recombinant protein DT3C alone in the HER2-overexpressing cell line SK-BR-3; and C represents the internalization efficiency of different molar ratios of trastuzumab to recombinant protein DT3C in the HER2-overexpressing cell line SK-BR-3.

[0037] Figure 2 The graph shows the internalization efficiency test results of Example 4, Comparative Example 1, and Comparative Example 2.

[0038] Figure 3 The graph shows the results of detecting the effects of adding different concentrations of Ficoll and PEG6000 to the incubation system on the conjugation efficiency and antibody internalization efficiency of trastuzumab-DT3C conjugates in Examples 4-7 and Comparative Examples 3-5.

[0039] Figure 4 The graph shows the antibody internalization efficiency detection results of the DT3C method and FabFc-ZAP method in Example 4 and Comparative Example 6. Detailed Implementation

[0040] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the test methods used in the following examples are conventional methods. All test materials are commercially available; the main sources for purchasing materials are as follows: Trastuzumab (Anti-ERBB2 / HER2 / CD340 Reference Antibody) was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd.

[0042] Rituximab (Anti-CD20 Reference Antibody (rituximab)) was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd.

[0043] The recombinant protein DT3C was synthesized, expressed, and purified in-house. Its sequence information is 33-417aa (P00588)+linker(GGGGSGGGGSGGGGS)+ 291-497aa (P19909). The full length of the protein is shown in SEQ ID NO.1, and it contains a 6×His tag at the N-terminus.

[0044] The FabFc-ZAP was purchased from Advanced Targeting Systems (ATS), part number: IT-65.

[0045] Ficoll 70 was purchased from Sigma-Aldrich, catalog number: F8016, cell culture grade.

[0046] Polyethylene glycol 6000 (PEG6000) was purchased from Sigma-Aldrich, catalog number: 81260, BioUltra grade.

[0047] Basic culture media: McCoy's 5A medium, purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number: PM150710; DMEM high glucose medium, purchased from Beijing TransGen Biotech Co., Ltd., catalog number: FI101-01; Leibovitz L-15 medium, purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number: PM151010.

[0048] The 96-well culture plate has white well walls and a white well bottom. It was purchased from Wuxi Guosheng Biotechnology Co., Ltd., product number: CIH-C8W.

[0049] The complete culture medium was a mixture of 90% of the corresponding basal medium and 10% FBS. The FBS was purchased from Beijing TransGen Biotech Co., Ltd., catalog number: FS301-02.

[0050] The chemiluminescence intensity was detected using a chemiluminescence cell viability assay kit (catalog number FC401) from Beijing TransGen Biotech Co., Ltd.

[0051] Example 1: Test to determine whether the antibody to be tested is suitable for internalization efficiency detection using the DT3C method. This embodiment is a preliminary experiment for the in vitro detection of trastuzumab internalization efficiency, used to determine whether the antibody to be tested is suitable for the DT3C method for internalization efficiency detection. The specific steps are as follows: 1. Prepare the culture medium Complete culture medium: 90% McCoy's 5A medium + 10% FBS; 2. Cell treatment SK-BR-3 cells with approximately 80% confluence and good growth status in the logarithmic proliferation phase were taken, digested, and counted by AO / PI staining. The cells were resuspended in the complete culture medium prepared in step 1 and the cell density was adjusted. The cells were then seeded into 96-well plates with a total of 5000 cells per well and cultured. 3. Preparation of trastuzumab Nine working solutions of trastuzumab were prepared by diluting the solutions with McCoy's 5A medium and incubating at 37°C for 30 min to obtain nine corresponding concentrations. The concentration gradients of trastuzumab were set up as follows: 0.001 μg / mL, 0.005 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL and 20 μg / mL.

[0052] 4. Antibody treatment After 24 h of culture, SK-BR-3 cells adhered to the culture vessel and were divided into three groups: the control group without antibody was treated with McCoy's 5A medium, the antibody-treated group was treated with nine different concentrations of trastuzumab working solution, and the complete killing group was treated with 0.1% Triton X-100. The volume of each group was equal, and a co-culture system was constructed.

[0053] 5. Detection and Calculation After co-culturing each group of cell culture systems at 37℃ and 5% CO2 for 96 hours, cell viability was assessed using a chemiluminescence assay kit. Cell viability and antibody internalization efficiency were calculated based on the measured chemiluminescence intensity. Cell viability = (Chemiluminescence intensity of antibody-treated group - Chemiluminescence intensity of complete kill group) / (Chemiluminescence intensity of control group without antibody - Chemiluminescence intensity of complete kill group) × 100%; Antibody internalization efficiency = 100% - cell viability.

[0054] Test results as follows Figure 1 As shown in Figure A, the results indicate that trastuzumab alone has no killing effect on SK-BR-3 cells, and the internalization efficiency of the antibody is not apparent.

[0055] Example 2: Determination of the optimal concentration of trastuzumab This example is a preliminary experiment for in vitro detection of trastuzumab internalization efficiency, used to determine the optimal concentration of trastuzumab. The specific steps are as follows: 1. Prepare the culture medium Complete culture medium: 90% McCoy's 5A medium + 10% FBS.

[0056] 2. Cell treatment SK-BR-3 cells with approximately 80% confluence and good growth status in the logarithmic proliferation phase were taken, digested, and counted by AO / PI staining. The cells were resuspended in the complete culture medium prepared in step 1 and the cell density was adjusted. The cells were then seeded into 96-well plates with a total of 5000 cells per well and cultured.

[0057] 3. Preparation of coupling compounds DT3C concentration gradients of 0.001 μg / mL, 0.005 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL, 20 μg / mL, and 30 μg / mL were prepared and diluted with McCoy's 5A medium to obtain individual DT3C solutions of the corresponding concentrations. A fixed concentration of 5 μg / mL of recombinant DT3C was set, and trastuzumab concentration gradients of 0.001 μg / mL, 0.005 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL, 20 μg / mL, and 30 μg / mL were prepared using McCoy's 5A medium. The incubation system was prepared by diluting 5A medium and incubating at 37°C for 30 min to form a trastuzumab-DT3C conjugate (trastuzumab-DT3C conjugate), thus obtaining the working solution of trastuzumab-DT3C conjugate at the corresponding concentration.

[0058] 4. Antibody treatment After 24 h of culture, SK-BR-3 cells adhered to the culture vessel and were divided into four groups: a control group without antibody was treated with McCoy's 5A medium, a DT3C-only treatment group was treated with the corresponding concentration of recombinant protein DT3C solution, a trastuzumab-DT3C treatment group was treated with the corresponding concentration of trastuzumab-DT3C conjugate working solution, and a complete killing group was treated with 0.1% Triton X-100. The volume of each group was equal, and a co-culture system was constructed.

[0059] 5. Detection and Calculation After co-culturing each group of cell cultures at 37℃ in a 5% CO2 incubator for 96 hours, cell viability was assessed using a chemiluminescence assay kit. Cell viability was calculated based on the measured chemiluminescence intensity. Cell viability = (Chemiluminescence intensity of treatment group - chemiluminescence intensity of complete kill group) / (Chemiluminescence intensity of control group without antibody - chemiluminescence intensity of complete kill group) × 100%.

[0060] Test results as follows Figure 1 As shown in Figure B, the results indicate that the recombinant DT3C protein alone in the DT3C-only treatment group (represented as "DT3C" in the figure) did not kill SK-BR-3 cells; however, in the trastuzumab-DT3C treatment group (represented as "trastuzumab-DT3C" in the figure), the killing effect reached a maximum of 90% when the concentration of trastuzumab was higher than 20 μg / mL. Therefore, using a trastuzumab concentration of 20 μg / mL can achieve a high killing level without excessive waste of experimental materials.

[0061] Example 3: Determination of the optimal molar ratio of trastuzumab to recombinant protein DT3C This example is a preliminary experiment for in vitro detection of trastuzumab internalization efficiency, which can be used to determine the optimal molar ratio of trastuzumab to DT3C. The specific steps are as follows: 1. Prepare complete culture medium 90% McCoy's 5A medium + 10% FBS.

[0062] 2. Cell treatment SK-BR-3 cells with approximately 80% confluence and good growth status in the logarithmic proliferation phase were taken, digested, and counted by AO / PI staining. The cells were resuspended in the complete culture medium prepared in step 1 and the cell density was adjusted. The cells were then seeded into 96-well plates with a total of 5000 cells per well and cultured.

[0063] 3. Preparation of coupling compounds Incubation systems with trastuzumab to recombinant DT3C molar ratios of 1:1, 1:2, and 1:3 were prepared using McCoy's 5A medium. The determined trastuzumab concentration was 20 μg / mL. The calculated concentration of DT3C was 10 μg / mL at a 1:1 molar ratio, 20 μg / mL at a 1:2 molar ratio, and 40 μg / mL at a 1:4 molar ratio. These incubation systems were combined and incubated at 37°C for 30 min to form three different ratios of trastuzumab-DT3C conjugate stock solutions. The three stock solutions were then independently serially diluted using McCoy's 5A medium to achieve trastuzumab concentrations of 0.001 μg / mL, 0.005 μg / mL, and 0.01 μg / mL, respectively. Nine gradients of trastuzumab-DT3C conjugate working solutions were obtained, namely 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL and 20 μg / mL.

[0064] 4. Antibody treatment After 24 h of culture, SK-BR-3 cells adhered to the culture vessel and were divided into three groups: a control group without antibody was given McCoy's 5A medium, a treatment group was given the corresponding concentration of trastuzumab-DT3C, and a complete killing group was given 0.1% Triton X-100. The volume of each group was equal, and a co-culture system was constructed.

[0065] 5. Detection and Calculation After co-culturing each group of cell culture systems at 37℃ and 5% CO2 for 96 hours, cell viability was assessed using a chemiluminescence assay kit. Cell viability and antibody internalization efficiency were calculated based on the measured chemiluminescence intensity. Cell viability = (Chemiluminescence intensity of treatment group - Chemiluminescence intensity of complete kill group) / (Chemiluminescence intensity of control group without antibody - Chemiluminescence intensity of complete kill group) × 100%; Antibody internalization efficiency = 100% - cell viability.

[0066] Test results as follows Figure 1As shown in Figure C, the results indicate that the antibody internalization efficiency detected by recombinant DT3C is almost identical when the molar ratio of trastuzumab to recombinant DT3C is 1:2 (represented as "trastuzumab-2xDT3C" in the figure) and 1:4 (represented as "trastuzumab-4xDT3C" in the figure). A molar ratio of 1:2 is sufficient for the experiment. At a molar ratio of 1:4, recombinant DT3C is in excess, resulting in material waste. Therefore, a molar ratio of 1:2 for trastuzumab to recombinant DT3C was chosen for subsequent antibody internalization efficiency testing.

[0067] Therefore, when testing the internalization efficiency of antibodies in vitro, the antibody concentration, DT3C concentration, or the molar ratio of the two should all be tested in advance to find a suitable concentration range.

[0068] Example 4 The difference from Example 3 is that only the incubation system with a molar ratio of 1:2 for trastuzumab and recombinant protein DT3C was selected, while the rest were the same.

[0069] Comparative Example 1 The difference from Example 4 is that the cell type was changed from SK-BR-3 to MDA-MB-468, which is an antigen-negative cell with extremely low HER2 expression, and served as a negative control; The complete culture medium is: 45% Leibovitz L15 medium + 45% DMEM high glucose medium + 10% FBS; Everything else is the same.

[0070] Comparative Example 2 The difference from Example 4 is that the antibody type is changed from trastuzumab to rituximab. This antibody can recognize CD20 positive cells and is an antibody unrelated to HER2. It serves as a negative control for the antibody. Everything else is the same.

[0071] The antibody internalization efficiency detection results for Example 4 and Comparative Examples 1 and 2 are as follows: Figure 2 As shown, the results indicated that trastuzumab-DT3C had no killing effect on HER2 antigen-negative cells, meaning that no internalization of trastuzumab-DT3C occurred in HER2 antigen-negative cells MDA-MB-468 (Comparative Example 1); the HER2-unrelated antibody rituximab-DT3C had no killing effect on HER2 antigen-positive cells SK-BR-3, meaning that no internalization of rituximab-DT3C occurred in HER2 antigen-positive cells (Comparative Example 2); and trastuzumab-DT3C had a killing effect on HER2 antigen-positive cells SK-BR-3, meaning that significant internalization of trastuzumab-DT3C occurred in HER2 antigen-positive cells (Example 4).

[0072] Example 5 This embodiment aims to optimize the concentrations of Ficoll70 and PEG6000 in the incubation system and verify the effect of their combined addition on improving the conjugation efficiency of recombinant protein DT3C and antibody. The specific steps are as follows: 1. Prepare the culture medium Protein-free incubation medium: McCoy's 5A basal medium containing 3% Ficoll70 and 1.5% PEG6000 by weight and volume. Take the basal medium, Ficoll70 and PEG6000 and dissolve them thoroughly before use. Complete culture medium: 90% McCoy's 5A medium + 10% FBS.

[0073] 2. Cell treatment SK-BR-3 cells with approximately 80% confluence and good growth status in the logarithmic proliferation phase were collected, digested, and counted by AO / PI staining. The cells were resuspended in the complete culture medium prepared in step 1 and the cell density was adjusted. The cells were then seeded into 96-well plates with a total of 5000 cells per well and cultured.

[0074] 3. Preparation of coupling compounds Using the protein-free incubation medium from step 1, a trastuzumab concentration of 20 μg / mL and a recombinant protein DT3C concentration of approximately 20 μg / mL were prepared, resulting in an incubation system with a trastuzumab to DT3C molar ratio of 1:2. This system was incubated at 37°C for 30 min to form the trastuzumab-DT3C conjugate stock solution. The conjugate stock solution was then serially diluted using the protein-free incubation medium to achieve nine concentrations of trastuzumab: 0.001 μg / mL, 0.005 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL, and 20 μg / mL, thus obtaining the corresponding trastuzumab-DT3C conjugate working solutions.

[0075] 4. Antibody treatment After 24 h of culture, SK-BR-3 cells adhered to the culture vessel and were divided into three groups: a control group without antibody was given only protein-free basal medium, a treatment group was given trastuzumab-DT3C conjugate, and a complete killing group was given 0.1% TritonX-100. The volume of each group was equal, and a co-culture system was constructed.

[0076] 5. Detection and Calculation After co-culturing each group of cell culture systems at 37℃ and 5% CO2 for 96 hours, cell viability was assessed using a chemiluminescence assay kit. Cell viability and antibody internalization efficiency were calculated based on the measured chemiluminescence intensity. Cell viability = (Chemiluminescence intensity of treatment group - Chemiluminescence intensity of complete kill group) / (Chemiluminescence intensity of control group without antibody - Chemiluminescence intensity of complete kill group) × 100%; Antibody internalization efficiency = 100% - cell viability.

[0077] Example 6 The difference from Example 5 is that the protein-free incubation medium is a basal medium containing 2% Ficoll70 and 1% PEG6000, while the rest are the same.

[0078] Example 7 The difference from Example 5 is that the protein-free incubation medium is a basal medium containing 1% Ficoll 70 and 0.5% PEG 6000, while the rest are the same.

[0079] Comparative Example 3 The difference from Example 5 is that the protein-free incubation medium is a basal medium containing 0.5% Ficoll70 and 0.1% PEG6000, while the rest are the same.

[0080] Comparative Example 4 The difference from Example 5 is that the protein-free incubation medium is a basal medium containing only 4% Ficoll 70, while the rest are the same.

[0081] Comparative Example 5 The difference from Example 5 is that the protein-free incubation medium is a basic medium containing only 2% PEG6000, while the rest are the same.

[0082] The antibody internalization efficiency detection results of Examples 5-7 and Comparative Examples 3-5 are as follows: Figure 3As shown in Table 1, the concentrations of trastuzumab-DT3C conjugate with an internalization efficiency of 50% and their relative concentrations compared to Example 4 are as follows. The results show that when the Ficoll70 concentration is 1-3% and the PEG6000 concentration is 0.5-1.5% (Examples 5-7), the antibody internalization efficiency is significantly improved compared to Example 4. The mAb-DT3C conjugate concentration in Example 4 with an internalization efficiency of 50% is 0.08476 μg / mL. After adding the additives, the concentration of the trastuzumab-DT3C conjugate decreased to 0.05608 μg / mL and 0.05827 μg / mL, respectively. The concentrations were 66% and 69% of the concentrations in Example 4, respectively. The group with 3% Ficoll70 and 1.5% PEG6000 (Example 5) had the highest internalization efficiency. Specifically, when the internalization efficiency was 50%, the concentration of trastuzumab-DT3C conjugate was 0.03474 μg / mL, which was 41% of the concentration in Example 4. This indicates that the combined addition of Ficoll70 and PEG6000 to the incubation system can significantly affect the formation efficiency of trastuzumab-DT3C conjugate and thus affect the internalization efficiency of mAb-DT3C conjugate. When the concentrations of Ficoll70 and PEG6000 were both low (0.5% and 0.1%, respectively, Comparative Example 3), representing 88% of the concentration in Example 4, the improvement in conjugation efficiency was not significant. When the concentrations of the two additives were high (4% and 2%, Comparative Examples 4 and 5), the internalization efficiency was 50% at concentrations of 0.118 μg / mL and 0.1271 μg / mL, respectively, representing 139% and 150% of the concentration in Example 4, indicating a decrease in internalization efficiency. High concentrations of additives may affect protein-protein interactions. Therefore, adding 1-3% Ficoll70 and 0.5-1.5% PEG6000 to the antibody-DT3C incubation system can effectively stabilize protein conformation, reduce aggregation, increase effective concentration, and significantly improve the conjugation efficiency of DT3C and antibody, thereby enhancing detection sensitivity. The preferred Ficoll70 concentration is 3%, and the preferred PEG6000 concentration is 1.5%.

[0083] Table 1. Concentration of mAb-DT3C conjugate with an internalization efficiency of 50% and its relative concentration compared to Example 4.

[0084] Comparative Example 6: Antibody internalization efficiency was detected using the FabFc-ZAP method.

[0085] The difference from Example 4 is that trastuzumab is not incubated with recombinant protein DT3C, but DT3C is replaced with FabFc-ZAP, incubated at room temperature for 60 min, and then co-cultured with cells. Everything else is the same.

[0086] The antibody internalization efficiency detection results for Example 4 and Comparative Example 6 are as follows: Figure 4 As shown, under the same treatment conditions, the sensitivity of detecting antibody internalization efficiency is significantly lower for trastuzumab FabFc-ZAP conjugates (represented as "trastuzumab-FabFc-ZAP" in the figure) formed by incubation using the FabFc-ZAP method than for trastuzumab-DT3C conjugates (represented as "trastuzumab-DT3C" in the figure) formed by incubation using the DT3C method. This is related to the large molecular weight of the FabFc-ZAP protein itself, which has a significant impact on the structure and site occupancy of the antibody, hindering antigen-antibody recognition and binding, thus affecting the antibody internalization process and failing to accurately and completely display the antibody internalization efficiency.

[0087] In summary, the DT3C method of this invention is more suitable for more accurate detection of antibody internalization efficiency, while the DT3C method optimized with the addition of Ficoll70 and PEG6000 further improves the detection sensitivity.

[0088] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. An in vitro detection method for antibody internalization efficiency, characterized in that, The in vitro detection method includes the following steps: Antigen-positive and antigen-negative cells were digested, AO / PI stained and counted, then resuspended in complete culture medium and seeded into culture plates for culture. Antibodies that do not kill antigen-positive cells alone were selected and incubated with recombinant protein DT3C in a protein-free incubation medium to prepare antibody-DT3C conjugates. After adhering to the culture vessel, antigen-positive cells and antigen-negative cells were divided into a control group without antibody, an antibody-treated group, and a complete killing group. The antibody-DT3C conjugate was added to the antibody-treated group, an equal volume of protein-free incubation medium was added to the control group without antibody, and an equal volume of 0.1% Triton X-100 was added to the complete killing group to construct a co-culture system. The co-culture systems of each group were co-cultured. The antibody-DT3C conjugate had no killing effect on antigen-negative cells but had a killing effect on antigen-positive cells. The cell viability of antigen-positive cells was detected. Based on the measured signal values, the cell viability and the internalization efficiency of the antibody to be tested were calculated according to the following formulas: Cell viability = (Signal value of antibody-treated group - Signal value of completely killed group) / (Signal value of control group without antibody - Signal value of completely killed group) × 100%, Antibody internalization efficiency = 100% - Cell viability.

2. The in vitro detection method according to claim 1, characterized in that, The amino acid sequence of the recombinant protein DT3C is shown in SEQ ID NO.1, and it contains a 6×His tag at the N-terminus.

3. The in vitro detection method according to claim 1, characterized in that, The protein-free incubation medium comprises a basal medium and additives, wherein the additives comprise the following components in weight-volume percentages: 1-3% Ficoll and 0.5-1.5% PEG6000; Preferably, the additive comprises the following components in the following mass-volume percentages: 3% Ficoll and 1.5% PEG6000.

4. The in vitro detection method according to claim 1, characterized in that, The complete culture medium comprises the following components in volume fractions: 80%-90% basal culture medium and 20%-10% fetal bovine serum; Preferably, the complete culture medium comprises the following components in volume fractions: 90% basal culture medium and 10% fetal bovine serum; More preferably, the basal culture medium is selected from at least one of McCoy's 5A medium, DMEM high glucose medium, and Leibovitz L-15 medium.

5. The in vitro detection method according to claim 1, characterized in that, The detection method for the test antibody and recombinant protein DT3C that have no killing effect on antigen-positive cells is as follows: the test antibody and recombinant protein DT3C are co-cultured with cultured antigen-positive cells, respectively, the cell viability of antigen-positive cells is detected, and the cell viability and internalization efficiency of the test antibody are calculated to obtain the test antibody and recombinant protein DT3C that have no killing effect on antigen-positive cells.

6. The in vitro detection method according to claim 1, characterized in that, The reagent kit used to detect cell viability is selected from at least one of the following: MTT assay kit, CCK-8 assay kit, and chemiluminescence assay kit; the signal value is absorbance or chemiluminescence intensity. When using the MTT assay kit or the CCK-8 assay kit, the signal value is absorbance. When using a chemiluminescence detection kit, the signal value is the chemiluminescence intensity.

7. The in vitro detection method according to claim 1, characterized in that, The concentration of the antibody to be tested in the incubation system is 0-30 μg / mL; the molar ratio of the antibody to be tested to recombinant protein DT3C is 1:1-1:

4. Preferably, the concentration of the antibody to be tested in the incubation system is 20 μg / mL, and the molar ratio of the antibody to be tested to recombinant protein DT3C is 1:

2.

8. The in vitro detection method according to claim 1, characterized in that, The antibody to be detected is an antibody against an antigen, and the antigen includes tumor therapeutic target proteins or immune checkpoint proteins. Preferably, the antigen includes HER2, HER3, EGFR, TROP2, CD22, CD30, PD-L1, B7H3, B7H4, CTLA4 and / or LAG3; More preferably, the antigen-positive cells are cancer cells that highly express the corresponding antigen on their cell surface or cells that have been artificially modified to overexpress the corresponding antigen; the antigen-negative cells are cancer cells that express the corresponding antigen at very low levels on their cell surface.

9. The in vitro detection method according to claim 8, characterized in that, The antigen is HER2; Preferably, the antibody to be detected is trastuzumab; More preferably, the antigen-positive cells are SK-BR-3 breast cancer cells; and the antigen-negative cells are MDA-MB-468 breast cancer cells.

10. The in vitro detection method according to claim 1, characterized in that, The culture is carried out for 18-24 hours; Preferably, the co-culture is carried out at 37±1℃ and 5±0.5%CO2 for 72-96 hours.

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