Method for comprehensive evaluation of immune checkpoint antibody in vitro function and efficacy and application thereof

Abstract

The present application relates to the present application belongs to the field of biological medicine and immunological oncology technology, specifically relates to the comprehensive evaluation method and application of immune checkpoint antibody in vitro function and efficiency by integrating signal pathway blocking, T cell activation, immune regulation, tumor microenvironment simulation and tumor cell killing and other multiple functional dimensions. The present application improves the integrity of the in vitro function evaluation of the candidate antibody; Establishes a comprehensive potency index (CPI), realizes the unified evaluation and horizontal comparison of the overall in vitro function of different candidate antibodies, improves the objectivity, repeatability and consistency of the evaluation results; Establishes a standardized evaluation process of "qualification access-comprehensive score-research and development decision", provides a standardized, quantifiable and repeatable comprehensive evaluation method for the selection of immune checkpoint antibody candidate molecules, improves the screening efficiency of candidate antibodies and the scientificity of research and development decision.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and immuno-oncology technology, specifically relating to a comprehensive evaluation method for systematically evaluating the in vitro function and efficacy of immune checkpoint antibodies. In particular, it relates to a comprehensive evaluation method for the in vitro efficacy of immune checkpoint antibodies by integrating multiple functional dimensions such as signal pathway blocking, T cell activation, immune regulation, tumor microenvironment simulation, and tumor cell killing, and its application. Background Technology

[0002] Programmed death receptor 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), constitute an important immune checkpoint pathway in the body, playing a crucial role in maintaining immune tolerance and regulating T cell function. PD-1 is mainly expressed on the surface of activated T cells and B cells, while PD-L1 is widely expressed on the surface of various cells, including tumor cells and antigen-presenting cells. When PD-L1 binds to PD-1, it can activate inhibitory signaling pathways within T cells, reducing T cell proliferation, cytokine secretion, and cytotoxic activity, thereby suppressing the body's anti-tumor immune response. Tumor cells can evade immune system surveillance and clearance by overexpressing PD-L1.

[0003] In recent years, PD-1 / PD-L1 immune checkpoint antibodies developed based on this mechanism of action have been widely used in the treatment of various solid tumors. Therefore, in the process of biosimilar drug development and candidate drug molecular screening, there is an urgent need for a method to systematically evaluate their in vitro function and efficacy.

[0004] Currently, the following methods are mainly used to evaluate the in vitro activity of immune checkpoint antibodies:

[0005] (1) Detection of PD-1 / PD-L1 signaling pathway blockade. Engineered cell models expressing PD-1 and PD-L1 are usually used to evaluate the ability of antibodies to block the PD-1 / PD-L1 signaling pathway by detecting changes in luciferase reporter genes or other downstream signals;

[0006] (2) Mixed Lymphocyte Reaction (MLR): By co-culturing immune cells from different donor sources, the release of cytokines, expression of T cell activation markers, or T cell proliferation are detected to evaluate the ability of antibodies to promote T cell activation.

[0007] (3) Superantigen stimulation system: Peripheral blood mononuclear cells (PBMCs) or whole blood were stimulated with superantigens such as Staphylococcus enterotoxin B (SEB), and changes in cytokine release or activation markers were detected to evaluate the T cell immune regulation capacity.

[0008] (4) PBMC-mediated tumor cell killing system: evaluate the ability of antibodies to enhance the anti-tumor killing ability of immune cells.

[0009] The methods described above evaluate antibody function at different immune stages, but they are usually independent of each other and lack a unified systematic evaluation framework. Specifically: blocking assays only reflect target binding function and cannot reflect the recovery of immune effect; the MLR and SEB systems reflect T cell activation but cannot simulate the real tumor environment; killing assays, while close to the endpoint effect, cannot reflect the early immune regulation process. Therefore, existing technologies are insufficient to achieve a systematic evaluation of immune checkpoint antibodies from "target blocking—immune activation—effect exertion," and also lack unified quantitative comparison standards. Summary of the Invention

[0010] To address the above problems, this invention provides a comprehensive evaluation method for assessing the in vitro function and potency of immune checkpoint antibodies. The technical solution of this invention is as follows:

[0011] Firstly, this invention provides a method for comprehensively evaluating the in vitro function and potency of immune checkpoint antibodies. The comprehensive evaluation method involves calculating a Comprehensive Potency Index (CPI) based on evaluation indicators obtained from a signal blocking evaluation module, an immune activation evaluation module, an immune checkpoint antibody immunomodulation module, and a terminal immune effect evaluation module. This comprehensive evaluation assesses the overall in vitro function of candidate immune checkpoint antibodies. The CPI is calculated using the following formula: CPI = W1*BS + W2*AS + W3*IMS + W4*TES; where W1, W2, W3, and W4 represent the weights of each functional module, satisfying W1 + W2 + W3 + W4 = 1; the weights are set according to the type of immune checkpoint antibody, the research and development objective, or the evaluation requirements. Specifically, BS represents the signal blocking score, AS represents the immune activation score, IMS represents the immune modulation score, and TES represents the terminal immune effect score.

[0012] In some embodiments, the method includes the following steps: (1) establishing a signal blocking evaluation module to detect the ability of immune checkpoint antibodies to block immune checkpoint signaling pathways and obtain a signal blocking score (BS); (2) establishing an immune activation evaluation module to detect the ability of immune checkpoint antibodies to promote T cell activation and cytokine release and obtain an immune activation score (AS); (3) establishing an immune checkpoint antibody immune regulation module to obtain an immune regulation score (IMS); (4) establishing a terminal immune effect evaluation module to detect the ability of immune checkpoint antibodies to enhance the killing of tumor cells mediated by immune cells and obtain a terminal immune effect score (TES); (5) establishing a comprehensive potency index (CPI) based on the evaluation indicators obtained in steps (1) to (4) to comprehensively evaluate the overall in vitro function of candidate immune checkpoint antibodies. In some embodiments, the signal blocking evaluation module uses an immune checkpoint signaling pathway reporter gene detection system to evaluate the ability of immune checkpoint antibodies to block signaling pathways and calculates the signal blocking score based on EC50. In some embodiments, the immune activation evaluation module uses a mixed lymphocyte reaction system to detect the release levels of one or both of the cytokines IL-2 and IFNγ, and calculates an immune activation score based on the IL-2 and IFNγ scores. In some embodiments, the immune regulation module is used to evaluate the regulatory capacity of immune checkpoint antibodies on an initiated immune response process, wherein the exogenous stimulation system is preferably a superantigen stimulation system. In some embodiments, the superantigen is staphylococcal enterotoxin B (SEB). In some embodiments, the immune regulation module measures the whole blood IL-2 and IFNγ release levels, and uses the maximum response value of each cytokine as the maximum release content to calculate the exogenous stimulation score. In some embodiments, the terminal immune effect evaluation module uses a PBMC-tumor cell co-culture system to detect the tumor cell killing rate, and calculates the terminal immune effect score based on the killing rate. In some embodiments, the tumor cells are tumor cells expressing immune checkpoint ligands. In some embodiments, the standardization process uses a reference antibody for normalization calculation. In some embodiments, the reference antibody is a known immune checkpoint antibody with the same target as the immune checkpoint antibody to be evaluated.In some embodiments, the immune checkpoint antibody is selected from one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, and anti-TIM-3 antibody, or is a bispecific or multispecific antibody targeting any two or more of the above-mentioned immune checkpoints. In some embodiments, the immune checkpoint antibody is an immune checkpoint antibody acting on the PD-1 / PD-L1 signaling pathway. In some embodiments, the immune checkpoint antibody is selected from one of anti-PD-1 antibody, anti-PD-L1 antibody, bispecific antibody that simultaneously binds PD-1 and PD-L1, and multispecific antibody containing a PD-1 or PD-L1 binding domain; the reference antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody comprises one of Pembrolizumab, Nivolumab, Cemiplimab, Sintilimab, Camrelizumab, Tislelizumab, Toripalimab, or Serplulimab, or a biosimilar thereof; the anti-PD-L1 antibody comprises one of Atezolizumab, Durvalumab, or Avelumab, or a biosimilar thereof. In some embodiments, the blocking score (BS) is calculated according to the following formula: ) ;

[0013] The Activation Score (AS) is calculated using the following formula: ;in,

[0014] , ;

[0015] The Immune Modulation Score (IMS) is calculated using the following formula:

[0016] ;

[0017] The Terminal Effector Score (TES) is calculated using the following formula: ;

[0018] Among them, the higher the BS value, the stronger the antibody's ability to block the PD-1 / PD-L1 signaling pathway; or the higher the AS value, the stronger the ability to restore T cell function; or the higher the IMS value, the stronger the ability of the immune checkpoint antibody to regulate the already initiated immune response process; or the higher the TES value, the stronger the ability of the antibody to restore the terminal killing function of PBMCs. In some embodiments, the candidate antibodies are first qualified according to the basic mechanism of action evaluation index, and then a comprehensive score is performed. In some embodiments, the development priority of candidate antibodies is determined according to the comprehensive efficacy index. In a second aspect, the present invention provides the application of the method described in the first aspect in the development of immune checkpoint drugs, wherein the comprehensive evaluation method can be applied to the following scenarios: (1) early screening and selection of candidate immune checkpoint antibodies; (2) candidate antibody research and development decision-making; (3) comparison of the overall function of different candidate antibodies; (4) antibody engineering optimization evaluation; (5) biosimilar consistency evaluation; (6) in vitro pharmacodynamic quality control and batch-to-batch consistency evaluation; (7) construction of a comprehensive evaluation platform for immune checkpoint antibodies; (8) other needs involving in vitro functional evaluation of immune checkpoint antibodies.

[0019] Based on the continuous biological process by which immune checkpoint antibodies exert their pharmacological effects, this method establishes a multidimensional functional evaluation system consisting of PD-1 / PD-L1 signal blocking ability, immune function recovery, immune regulation ability, and PBMC-mediated tumor cell killing ability. Furthermore, it establishes a standardized scoring method and a comprehensive efficacy index (CPI) to achieve unified evaluation, horizontal comparison, and standardized screening of candidate antibody functions in vitro.

[0020] Furthermore, this invention establishes standardized R&D decision-making rules, which comprehensively judge candidate antibodies based on the comprehensive efficacy index and the evaluation results of each functional module, providing a basis for candidate antibodies to enter subsequent in vivo efficacy evaluation, structural optimization, or termination of development.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention establishes a comprehensive evaluation system based on the continuous biological process of the pharmacological effects of immune checkpoint antibodies, realizing the continuous evaluation from basic mechanism of action, immune function recovery, immune regulation to the final anti-tumor effect, rather than a simple combination of multiple independent detection methods, thus improving the integrity of the in vitro functional evaluation of candidate antibodies.

[0023] (2) This invention establishes a unified and standardized scoring method, converts different experimental endpoints into functional evaluation indicators that can be compared horizontally, and further establishes a comprehensive efficacy index (CPI) to achieve unified evaluation and horizontal comparison of the overall in vitro function of different candidate antibodies, thereby improving the objectivity, repeatability and consistency of the evaluation results.

[0024] (3) This invention establishes a standardized evaluation process of “qualification access - comprehensive scoring - R&D decision-making”, and uses the comprehensive efficacy index as an important basis for candidate antibody screening and R&D decision-making. It provides a standardized, quantifiable and repeatable comprehensive evaluation method for the selection of immune checkpoint antibody candidate molecules, and improves the efficiency of candidate antibody screening and the scientific nature of R&D decision-making. Attached Figure Description

[0025] Figure 1 Figure 1 shows the results of antibody blocking activity against the PD-1 / PD-L1 signaling pathway.

[0026] Figure 2 Figure 1 shows the results of antibody-mediated T cell IL-2 release detection.

[0027] Figure 3 Figure 1 shows the results of antibody-mediated T cell IFNγ release detection.

[0028] Figure 4 Figure 1 shows the results of IL-2 detection mediated by antibody in the SEB system for whole blood release.

[0029] Figure 5 Figure 1 shows the detection results of IFNγ released from whole blood mediated by antibodies in the SEB system.

[0030] Figure 6 Figure 1 shows the results of the detection of the killing effect of PBMC on Calu-1 cells. Detailed Implementation

[0031] In some exemplary embodiments, the immune checkpoint antibody is an immune checkpoint antibody that acts on the PD-1 / PD-L1 signaling pathway. In some exemplary embodiments, the immune checkpoint antibody is an immune checkpoint antibody that acts on the PD-1 / PD-L1 signaling pathway. In some exemplary embodiments, the immune checkpoint antibody is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, bispecific antibodies that simultaneously bind PD-1 and PD-L1, and multispecific antibodies containing a PD-1 or PD-L1 binding domain; the reference antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. In conjunction with the content of this invention, in some embodiments described by those skilled in the art, the anti-PD-1 antibody includes one of Pembrolizumab, Nivolumab, Cemiplimab, Sintilimab, Camrelizumab, Tislelizumab, Toripalimab, or Serplulimab, or a biosimilar thereof; the anti-PD-L1 antibody includes one of Atezolizumab, Durvalumab, or Avelumab, or a biosimilar thereof. In conjunction with the content of this invention, in some embodiments described by those skilled in the art, the immune checkpoint antibody is selected from one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, and anti-TIM-3 antibody, or is a bispecific antibody or multispecific antibody targeting any two or more of the above-mentioned immune checkpoints.

[0032] Example

[0033] Example 1: Establishment of an evaluation module for immune checkpoint antibody signal blocking function (Signal Blocking Module)

[0034] This embodiment aims to establish an in vitro detection method for evaluating the ability of immune checkpoint antibodies to block the PD-1 / PD-L1 immune checkpoint signaling pathway.

[0035] 1.1 Experimental Objective

[0036] PD-1 / PD-L1 signal blocking ability is the initial step for immune checkpoint antibodies to exert their anti-tumor effects and is also an important functional indicator for evaluating the efficacy of immune checkpoint antibodies. Therefore, this embodiment uses signal blocking evaluation as the first functional module of the comprehensive evaluation platform to evaluate the ability of different immune checkpoint antibodies to relieve immunosuppressive signals and establish a blocking function score (BS).

[0037] 1.2 Experimental Principle

[0038] PD-1 is an immunosuppressive receptor expressed on the surface of activated T cells and B cells. When it binds to PD-L1 ligands expressed on the surface of tumor cells and antigen-presenting cells, TCR signaling and its mediated proliferation, transcriptional activation, and cytokine production are inhibited. Based on this principle, this experimental system used Jurkat cells stably expressing human PD-1 and NFAT-Luciferase, and CHO-K1 cells stably expressing human PD-L1 and a membrane-bound OKT3 single-chain antibody. When the two cell types were co-cultured, the PD-1 / PD-L1 interaction inhibited TCR signaling and NFAT-mediated luciferase activity. Adding antibodies that block PD-1 or PD-L1 release inhibition signals generated TCR signaling and NFAT-mediated luciferase activity. Therefore, by detecting the degree of recovery of luciferase luminescence intensity, the ability of immune checkpoint antibodies to block the PD-1 / PD-L1 signaling pathway can be quantitatively evaluated. Furthermore, a four-parameter logistic equation was used to fit the dose-response curve to calculate the half-maximal effective concentration (EC50). 50 And the blocking function score is obtained through standardization.

[0039] 1.3 Experimental Materials

[0040] (1) Cell

[0041] ① aAPC(OKT3)-PD-L1 CHO-K1 cells.

[0042] Stable expression of human PD-L1 and membrane-bound OKT3 single-chain antibodies were purchased from Jimon Biotechnology Co., Ltd., catalog number: GM-C05269.

[0043] ②H_PD-1 Reporter Jurkat cells.

[0044] Stable expression of human PD-1 receptor and NFAT-Luciferase reporter system, purchased from Jimon Biotechnology Co., Ltd., catalog number: GM-C07928.

[0045] (2) Test antibody

[0046] The following antibodies were used as representative immune checkpoint antibodies: Pembrolizumab, Atezolizumab, and the test antibody BT-YL-1 (a self-developed PD-1 / PD-L1 / VEGF trispecific antibody).

[0047] (3) Main reagents

[0048] Bright-Lite Luciferase Assay System (Vazyme, catalog number: DD1204-03), RPMI-1640 medium, fetal bovine serum (FBS), and PBS buffer.

[0049] 1.4 Experimental Methods

[0050] aAPC(OKT3)-PD-L1 CHO-K1 cells were seeded in 96-well flat-bottom plates at a density of 1.5 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C in a 5% CO2 incubator. The next day, after observing cell adhesion, the culture medium was discarded. 50 μL of H_PD-1 Reporter Jurkat cells were added to each well, at a cell density of 1.5 × 10⁻⁶ cells. 5 Each well contains one cell; simultaneously, add 50 μL of serially diluted Pembrolizumab, Atezolizumab, and BT-YL-1 (starting concentration 180 nM, 3-fold serial dilution, for a total of 9 concentrations), mix well, and incubate the cell plate at 37°C for 6 hours. After incubation, add Bright-Lite luciferase assay reagent to each well and detect the chemiluminescence signal using a multi-mode microplate reader.

[0051] 1.5 Data Processing

[0052] Using GraphPad Prism software, antibody concentration was plotted on the x-axis (Log10 transformation), and chemiluminescence signal on the y-axis. A four-parameter logistic regression (4PL) equation was used to fit dose-response curves, calculating the EC50 of each antibody in restoring reporter gene activity. 50 .

[0053] Using pembrolizumab as the standard reference antibody, the blocking score (BS) was calculated according to the following formula: .

[0054] Due to EC 50 The Blocking Score (BS) is negatively correlated with the antibody's blocking ability; therefore, a higher BS value indicates a stronger ability of the antibody to block the PD-1 / PD-L1 signaling pathway. The BS value is used as the output parameter of the first functional module of the comprehensive evaluation platform and is subsequently used in the calculation of the standardized evaluation framework and comprehensive efficacy index.

[0055] 1.6 Experimental Results

[0056] Specific results are as follows Figure 1As shown: All three antibodies can dose-dependently relieve PD-1 / PD-L1-mediated signal inhibition, and their EC50 values ​​are... 50 The values ​​were 6.79 nM, 1.34 nM, and 1.64 nM, respectively. Atezolizumab and BT-YL-1 exhibited lower EC values ​​in this system. 50 The value indicates that both have a high signal path blocking capability. Figure 1 The antibody demonstrated its blocking activity against the PD-1 / PD-L1 signaling pathway.

[0057] Table 1. Antibody blocking effect on the PD-1 / PD-L1 signaling pathway (EC5) 50 (nM)

[0058] <![CDATA[EC 50 (nM)]]> 6.79 1.34 1.64

[0059] Standardization was performed using Pembrolizumab as a reference antibody, according to the formula: Calculate the blocking function score.

[0060] Table 2. Calculation results of antibody BS at different immune checkpoints

[0061] BS 1.00 5.06 4.14

[0062] After standardization, the differences in blocking ability between different antibodies can be represented by a unified scoring format, providing standardized input parameters for subsequent comprehensive evaluation.

[0063] 1.7 Results Analysis

[0064] PD-1 / PD-L1 signaling blockade is the initiating step for immune checkpoint antibodies to exert their immunotherapeutic effects and a necessary prerequisite for restoring T cell function. Therefore, establishing a stable and reliable signaling blockade evaluation system is an important foundation for constructing a comprehensive evaluation platform for immune checkpoint antibodies.

[0065] In this embodiment, an in vitro signal blocking model was constructed using Jurkat cells stably expressing PD-1 and NFAT reporter genes and CHO-K1 cells stably expressing PD-L1. This model directly reflects the ability of immune checkpoint antibodies to relieve PD-1 / PD-L1 inhibitory signals, and thus evaluates the blocking effectiveness of antibodies from the perspective of the mechanism of action of immune checkpoint antibodies.

[0066] Furthermore, this embodiment uses EC 50 As the original evaluation parameters, and further converted into blocking function scores (BS) through standardization, the signal blocking capabilities of different antibodies can be compared using a unified dimension, providing a basis for subsequent data integration with other functional evaluation modules.

[0067] It should be noted that the blocking function score is not intended to replace EC. 50 Instead, it retains EC 50 Based on its pharmacological significance, this data is converted into standardized scores that can be directly used in comprehensive evaluation, thereby improving data consistency and comparability between different evaluation systems. In summary, this module establishes BS as a standardized output parameter for the comprehensive evaluation platform.

[0068] 1.8 Functional Access Determination

[0069] Since PD-1 / PD-L1 signaling blockade is the starting point for immune checkpoint antibodies to exert their effects and is also a necessary basis for subsequent T cell activation and terminal tumor killing, this invention first uses the blocking function score as the first screening indicator for candidate antibodies to enter the subsequent functional evaluation.

[0070] Considering the differences among candidate antibodies in terms of antigen-binding epitopes, molecular conformation, and affinity, a reasonable range of fluctuations in blocking ability is allowed, provided that the basic mechanism of action is maintained. This invention preferably uses Pembrolizumab as the standard reference antibody. When the BS of a candidate antibody is ≥0.80, it can be considered to have basic PD-1 / PD-L1 signal blocking ability and is qualified to proceed to subsequent comprehensive functional evaluation.

[0071] When the BS of the candidate antibody is less than 0.80, it indicates that its basic blocking ability is significantly lower than that of the standard reference antibody, and it is difficult to effectively relieve PD-1 / PD-L1-mediated immunosuppression. Therefore, it can be determined that it does not meet the qualification requirements of the comprehensive evaluation system of this invention, and it is not recommended to continue to carry out subsequent comprehensive evaluation.

[0072] This step serves as the sole qualification criterion in the comprehensive evaluation system of this invention. The candidate embodiments do not employ a multi-index, step-by-step elimination method because PD-1 / PD-L1 signal blocking capability is the fundamental mechanism by which immune checkpoint antibodies exert their pharmacological effects. For candidate antibodies lacking basic blocking capability, subsequent evaluation of their immune function lacks a biological basis; while for candidate antibodies possessing basic blocking capability, the performance of their different functional modules can be uniformly evaluated through a comprehensive efficacy index. Therefore, this invention adopts a "qualification criterion + comprehensive scoring" evaluation strategy to ensure that the evaluation system is both efficient in screening and avoids premature elimination of promising candidate antibodies due to differences in individual functions.

[0073] Example 2: Establishment of an Immune Activation Module for Evaluating Immune Checkpoint Antibody Activation Function

[0074] This embodiment aims to establish an in vitro detection method for evaluating the restoration of T-cell immune function by immune checkpoint antibodies.

[0075] 2.1 Experimental Objective

[0076] After immune checkpoint antibodies relieve PD-1 / PD-L1-mediated immunosuppression, their main pharmacological effects are promoting T cell reactivation, enhancing cytokine secretion, and restoring immune response. Therefore, evaluating signal blocking function alone is insufficient to fully reflect the efficacy of antibody drugs.

[0077] This embodiment uses a mixed lymphocyte reaction (MLR) to establish an immune activation function evaluation module, which is used to evaluate the ability of immune checkpoint antibodies to promote T cell activation and enhance cytokine secretion, and outputs an immune activation score (AS) as the second functional module of the comprehensive evaluation platform.

[0078] 2.2 Experimental Principle

[0079] Mixed lymphocyte reaction (MLR) is one of the most classic in vitro assays for evaluating cellular immune function. It involves co-culturing immune cells from two individuals to trigger "non-self" recognition regulated by immune checkpoints. When dendritic cells (APCs) bind to allogeneic T cells, they stimulate T cell activation and proliferation, initiating immune checkpoint activation, and PD-1 / PD-L1 signaling persistently suppresses T cell function. The addition of immune checkpoint antibodies can relieve this suppression, restoring T cell activation and further secreting effector cytokines such as IL-2 and IFNγ. Therefore, detecting cytokine release levels can evaluate the restorative effect of immune checkpoint antibodies on T cell immune function.

[0080] 2.3 Experimental Materials

[0081] (1) Cell

[0082] Mature dendritic cells (DCs) and CD3 from different healthy donors + T cells.

[0083] (2) Test antibody

[0084] The following antibodies were used as representative immune checkpoint antibodies: Pembrolizumab, Atezolizumab, the antibody to be tested BT-YL-1, and hIgG1 isotype control.

[0085] (3) Main reagents

[0086] Human IL-2 ELISA Kit, Human IFNγ ELISA Kit, RPMI-1640 medium, fetal bovine serum (FBS), PBS buffer.

[0087] 2.4 Experimental Methods

[0088] Mature dendritic cells (DCs) and allogeneic T cells were resuspended separately in RPMI 1640 complete medium (RPMI 1640 contains 10% inactivated FBS), and DCs were cultured at a rate of 1 × 10⁶ cells / year. 4 10 cells / well, 1×10 T cells 5 Cells were seeded per well in 96-well round-bottom cell culture plates. Then, different concentrations of hIgG1, Pembrolizumab, Atezolizumab, and BT-YL-1 (starting at 5 nM, serially diluted 10-fold, for a total of 3 concentrations) were added, gently mixed, and the cell culture plates were incubated at 37°C in a 5% CO2 incubator for 4 days. After incubation, the supernatant was collected from the wells, and the release levels of cytokines IL-2 and IFNγ were measured using a kit.

[0089] 2.5 Data Processing

[0090] The release levels of T cell cytokines mediated by different concentrations of the tested antibodies were analyzed using a Human IL-2 and IFNγ ELISA kit.

[0091] Using Pembrolizumab as the standard reference antibody, the overall immune activation capacity was calculated according to the following formula. , Further define the Immune Activation Score (AS): A higher AS value indicates a stronger ability of immune checkpoint antibodies to restore T cell function. The resulting Activation Score is used as an output parameter of the second functional module of the comprehensive evaluation platform and is further used for comprehensive efficacy evaluation.

[0092] 2.6 Experimental Results

[0093] The above methods were used to evaluate the ability of Pembrolizumab, Atezolizumab, and the test antibody BT-YL-1 to promote T cell activation. Results are as follows: Figure 2 and Figure 3 As shown, in the mixed lymphocyte reaction system, the hIgG1 isotype control only induced low levels of IL-2 and IFNγ release, while the addition of the tested antibodies promoted IL-2 and IFNγ secretion in a dose-dependent manner, indicating that all three antibodies could block PD-1 / PD-L1 signaling and restore T cell immune function. Among them, the BT-YL-1 antibody (0.05~5 nM) induced higher levels of IL-2 release than Pembrolizumab and Atezolizumab; at a concentration of 5 nM, Pembrolizumab induced the highest level of IFNγ release.

[0094] Figure 2 This demonstrates antibody-mediated IL-2 release from T cells. Figure 3 This demonstrates antibody-mediated IFNγ release from T cells.

[0095] Standardization was performed using Pembrolizumab as a reference antibody, according to the formula: , Calculate overall immune activation capacity, and further based on Calculate the immune activation score.

[0096] Table 3. Calculation results of AS of antibodies at different immune checkpoints

[0097] Pembrolizmab 1.00 1.00 1.00 Atezolizumab 0.51 0.45 0.48 BT-YL-1 1.64 0.65 1.14

[0098] After standardization, the differences in immune activation capacity among different antibodies can be represented by a unified scoring method, providing standardized input parameters for subsequent comprehensive evaluation.

[0099] 2.7 Results Analysis

[0100] This embodiment uses the MLR experimental system to reflect the ability of immune checkpoint antibodies to restore T cell activation by detecting the release levels of IL-2 and IFNγ by T cells.

[0101] It is noteworthy that the dose-response characteristics of T cell release levels of IL-2 and IFNγ induced by different immune checkpoint antibodies are not entirely consistent, suggesting that although these immune checkpoint antibodies act on the same signaling pathway, their functional characteristics in promoting T cell activation may differ. Therefore, evaluating antibody activity using only a single cytokine or a single concentration may not comprehensively reflect the overall immune activation capacity of the antibody. This module uses IL-2 and IFNγ as indicators of overall function evaluation. By integrating the response information of both, the influence of a single indicator on the evaluation results is reduced, making the comparison between different antibodies more stable and reliable. In summary, this module can stably evaluate the ability of immune checkpoint antibodies to restore T cell immune function, establish an immune activation score (AS), and standardize the characterization of the overall immune activation level of different antibodies.

[0102] 2.8 Functional Evaluation and Judgment

[0103] Mixed lymphocyte reaction (MLR) is used to evaluate the ability of candidate antibodies to restore T cell activation function after relieving PD-1 / PD-L1-mediated immunosuppression, and is an important component of the in vitro functional evaluation system for immune checkpoint antibodies.

[0104] Because different immune checkpoint antibodies differ in their target sites, binding epitopes, molecular conformations, and modes of action, their ability to promote T cell activation may vary. In addition, the results of MLR experiments are also affected by biological factors such as donor source, antigen-presenting cell status, and cell activity. Therefore, AS cannot fully represent the overall efficacy level of candidate antibodies.

[0105] For the reasons mentioned above, this invention does not use AS as the basis for qualification or elimination of candidate antibodies, but rather as a component of the Comprehensive Potency Index (CPI) in the overall scoring. The higher the AS value, the stronger the ability of the candidate antibody to restore T-cell immune function, and the greater its contribution to the comprehensive potency index; a lower AS value will correspondingly reduce the comprehensive potency index, but will not affect the candidate antibody's entry into the subsequent comprehensive evaluation.

[0106] It should be further explained that the MLR evaluation module primarily reflects the ability of immune checkpoint antibodies to restore T-cell immune function, serving as a crucial functional link connecting the basic mechanism of action with the terminal anti-tumor effect. This module can provide information on immune function recovery that cannot be obtained through signal blocking experiments and terminal tumor killing experiments. Together with other functional evaluation modules, it constructs a comprehensive evaluation system covering the entire action process of immune checkpoint antibodies, providing important basis for candidate antibody screening, mechanism of action analysis, and research and development decisions.

[0107] Therefore, this embodiment does not set separate functional admission criteria. After a candidate antibody completes the evaluation of this module, it continues to the next functional evaluation module. Finally, the comprehensive efficacy index (CPI) is combined to complete the unified evaluation and R&D decision.

[0108] Example 3: Establishment of an Immune Modulation Module for Evaluating Immune Checkpoint Antibody Immunomodulatory Characteristics

[0109] This embodiment aims to establish an in vitro detection method for evaluating the ability of immune checkpoint antibodies to regulate T cell function and enhance immune response under strong exogenous stimulation.

[0110] 3.1 Experimental Objective

[0111] This embodiment uses a human whole blood immune evaluation system stimulated by staphylococcal enterotoxin B (SEB) to simulate the state of the immune system when subjected to strong exogenous stimulation. It is used to evaluate the regulatory ability of immune checkpoint antibodies on the initiated immune response process, serving as the third functional module of the comprehensive evaluation platform.

[0112] 3.2 Experimental Principle

[0113] SEB, as a superantigen, nonspecifically bridges MHC-II molecules on the surface of APCs with the TCR Vβ chain, strongly activating a large number of T cells and synergistically activating monocytes, triggering the release of pro-inflammatory cytokine cascades, thereby establishing a model of an "initiated" excessive immune response.

[0114] In this embodiment, an immune regulation feature evaluation module is established using SEB stimulation of human whole blood system to evaluate the regulatory capacity of immune checkpoint antibodies on the initiated immune response process, and outputs the Immune Modulation Score (IMS) as the third functional module of the comprehensive evaluation platform.

[0115] 3.3 Experimental Materials

[0116] (1) Whole blood

[0117] Fresh peripheral venous blood was collected from healthy donors and treated with heparin anticoagulation to obtain human whole blood samples.

[0118] (2) Test antibody

[0119] The following antibodies were used as representative immune checkpoint antibodies: Pembrolizumab, Atezolizumab, and the test antibody BT-YL-1, while hIgG1 isotype control was set up.

[0120] (3) Main reagents

[0121] SEB, Human IL-2 ELISA Kit, Human IFNγ ELISA Kit.

[0122] 3.4 Experimental Methods

[0123] Fresh human whole blood was added at a rate of 100 μL / well to 96-well round-bottom plates, followed by 50 μL of SEB working solution (final concentration 0.5 μg / ml) to each well to induce T cell activation. Simultaneously, 50 μL of different concentrations of hIgG1, Pembrolizumab, Atezolizumab, and BT-YL-1 (starting concentration 10 nM, serially diluted 10-fold, for a total of 3 concentrations) were added. Cells were incubated at 37°C in a 5% CO2 incubator for 48 h. After culture, the culture supernatant was collected, and IL-2 and IFNγ levels were detected using ELISA.

[0124] 3.5 Data Processing

[0125] The levels of whole blood cytokine release mediated by different concentrations of the tested antibodies were analyzed using a Human IL-2 and IFNγ ELISA assay kit.

[0126] Because the release of different cytokines in the SEB stimulation system exhibits dose-dependent differences, IL-2, as a marker of early T cell activation, shows a peak release at lower stimulation concentrations, while IFNγ, as an effector cytokine, typically shows a peak release at higher stimulation concentrations. Therefore, this embodiment employs a multi-concentration combined assessment approach to capture the functional performance of the antibody at different stages of immune activation, thereby avoiding bias from single-concentration evaluation. First, the maximum response value of each cytokine was taken as the maximum release content at three concentrations: 0.1 nM, 1 nM, and 10 nM. Subsequently, using Pembrolizumab as the standard reference antibody, the Immune Modulation Score (IMS) was defined. IMS primarily reflects immune regulatory capacity. The obtained IMS data serves as the output parameter of the third functional module of the comprehensive evaluation platform and is further used for comprehensive efficacy evaluation.

[0127] 3.6 Experimental Results

[0128] The above methods were used to evaluate the regulatory capacity of Pembrolizumab, Atezolizumab, and the test antibody BT-YL-1 on the initiated immune response. Results are as follows: Figure 4 As shown, under SEB stimulation, the hIgG1 control group exhibited low IL-2 release levels. After the addition of immune checkpoint antibodies, each antibody promoted IL-2 release to varying degrees. Different antibodies showed different concentration-response patterns. Specifically, the IL-2 release level in the Pembrolizumab group gradually increased with increasing antibody concentration, reaching its peak at 10 nM; the Atezolizumab group showed a similar trend, reaching its maximum release level at 10 nM; the BT-YL-1 antibody induced a high level of IL-2 release even at low concentrations, peaking at 1 nM, and no further enhancement was observed after further increasing the antibody concentration. These results indicate that although different immune checkpoint antibodies can all enhance the release of early T cell activation-related cytokines, the concentrations required to achieve the optimal immune response differ.

[0129] IFNγ release level as Figure 5 As shown, in the SEB stimulation system, all immune checkpoint antibodies promoted IFNγ release. Unlike IL-2, IFNγ, as an effector cytokine, typically requires stronger or sustained immune activation to reach its release level. The results showed that Pembrolizumab, Atezolizumab, and BT-YL-1 antibodies all achieved high IFNγ release levels at 10 nM. Among them, the BT-YL-1 antibody induced a higher level of IFNγ release than the control antibody, suggesting a stronger tendency to enhance effector T cell function.

[0130] Figure 4 The antibody was shown to mediate the release of IL-2 from whole blood in the SEB system. Figure 5 This demonstrates that the antibody mediates the release of IFNγ from whole blood in the SEB system.

[0131] To further determine the maximum response value of each tested antibody, the maximum release amount of each cytokine was measured under three concentration conditions: 0.1 nM, 1 nM, and 10 nM.

[0132] Table 4. Average IL-2 release in each group

[0133] hIgG1 11039.32 11730.93 12093.36 Pembrolizumab 12027.23 12717.84 13440.50 Atezolizumab 11123.15 12393.52 12998.83 BT-YL-1 12575.99 13002.02 11134.10

[0134] Table 5. Average IFNγ release values ​​for each group

[0135] hIgG1 20242.60 20236.22 18994.10 Pembrolizumab 19898.42 208144.64 22100.96 Atezolizumab 18276.54 20966.83 21986.91 BT-YL-1 21007.50 22915.92 24217.57

[0136] As shown in Tables 4 and 5, the maximum release amounts of IL-2 from Pembrolizumab, Atezolizumab, and BT-YL-1 antibody were 13440.50, 12998.83, and 13002.02, respectively. The maximum release amounts of IFNγ were 22100.96, 21986.91, and 24217.57, respectively.

[0137] IMS is constructed based on the maximum response values ​​of IL-2 and IFNγ.

[0138] Table 6. IMS Calculation Results of Antibodies at Different Immune Checkpoints

[0139] IMS 1.00 0.98 1.05

[0140] After standardization, the regulatory differences between different antibodies on the initiated immune response process can be represented by a unified scoring method, providing standardized input parameters for subsequent comprehensive evaluation.

[0141] 3.7 Results Analysis

[0142] In this embodiment, SEB was used to stimulate a whole human blood system. By detecting the release levels of IL-2 and IFNγ, the ability of immune checkpoint antibodies to regulate immune responses under strong exogenous stimulation was evaluated.

[0143] Experimental results showed that Pembrolizumab, Atezolizumab, and BT-YL-1 antibodies could all promote the release of IL-2 and IFNγ under SEB stimulation, but there were certain differences among the different antibodies in terms of cytokine release levels and optimal response concentrations. BT-YL-1 antibody induced higher levels of IL-2 release at lower concentrations and also showed a higher maximum response value in IFNγ release, suggesting that different immune checkpoint antibodies have different immunomodulatory characteristics under exogenous stimulation.

[0144] It should be noted that IL-2 and IFNγ represent different stages of T cell activation and effector function, respectively. Evaluating the immunomodulatory capacity of candidate antibodies using only a single cytokine or a single concentration may lead to biased results. Therefore, this embodiment establishes an immunomodulatory score by combining the maximum response values ​​of IL-2 and IFNγ to standardize the evaluation of the overall immunomodulatory capacity of candidate antibodies under exogenous stimulation.

[0145] Furthermore, the biological processes focused on by the immunomodulation evaluation module differ from those of the MLR and PBMC-mediated tumor cell killing evaluation modules. MLR primarily evaluates the ability of candidate antibodies to restore T-cell immune function, PBMC-mediated tumor cell killing assays evaluate the ability of candidate antibodies to induce a terminal anti-tumor effect, while this embodiment focuses on evaluating the ability of candidate antibodies to regulate the immune response under an immune-activated state. Therefore, the IMS established in this embodiment can provide immunomodulatory information that other functional modules cannot obtain for the comprehensive evaluation system. Together with other functional evaluation modules, it enables continuous evaluation of candidate antibodies from their basic mechanism of action, immune function recovery, immunomodulation to their terminal anti-tumor effect, providing standardized functional parameters for subsequent comprehensive efficacy evaluation.

[0146] 3.8 Functional Evaluation and Judgment

[0147] The immune regulation evaluation module is mainly used to evaluate the ability of candidate antibodies to regulate immune responses in an immune-activated state, and is an important component of the comprehensive in vitro functional evaluation system for immune checkpoint antibodies.

[0148] Since the level of cytokine release is not only affected by the mechanism of action of candidate antibodies, but also by a variety of factors such as the intensity of exogenous stimulation, the activation state of immune cells, and the cytokine regulatory network, there is no simple linear correlation between the results and the final anti-tumor efficacy of candidate antibodies. It is difficult to comprehensively evaluate the overall efficacy of candidate antibodies based solely on IMS.

[0149] For the reasons stated above, this invention does not use IMS as a basis for qualification or elimination of candidate antibodies, but rather as a component of the Comprehensive Potency Index (CPI) in the overall scoring. A higher IMS value indicates a stronger ability of the candidate antibody to regulate the immune response under exogenous stimulation, and a greater contribution to the CPI; a lower IMS value will correspondingly reduce the CPI, but will not affect the candidate antibody's inclusion in subsequent comprehensive evaluation.

[0150] Therefore, this embodiment does not set separate functional admission criteria. After a candidate antibody completes the evaluation of this module, it continues to the next functional evaluation module. Finally, the comprehensive efficacy index (CPI) is combined to complete the unified evaluation and R&D decision.

[0151] Example 4: Establishing a Terminal Effector Module for Evaluating the Terminal Immune Effect of Immune Checkpoint Antibodies

[0152] This embodiment aims to establish an in vitro detection method for evaluating the ability of immune checkpoint antibodies to enhance PBMC-mediated tumor cell killing.

[0153] 4.1 Experimental Objective

[0154] The ultimate therapeutic goal of immune checkpoint antibodies is to restore the ability of immune cells to recognize and kill tumor cells. Therefore, tumor cell killing assays can directly reflect the ability of immune checkpoint antibodies to mediate terminal immune effects and are an important component in evaluating the overall in vitro efficacy of antibodies.

[0155] In this embodiment, a co-culture system of PBMCs and PD-L1 positive tumor cells was used to establish a terminal immune effect evaluation module, and the tumor cell killing rate was used to calculate the effect score (TES), which serves as the fourth functional module of the comprehensive evaluation platform of this invention.

[0156] 4.2 Experimental Principle

[0157] PBMCs contain various immune effector cells, including CD8⁺ T cells, CD4⁺ T cells, and natural killer cells. After pre-activation with anti-CD3 and anti-CD28 antibodies, PBMCs can restore their proliferative and cytotoxic functions. PD-L1-positive tumor cells can persistently inhibit the killing activity of PBMCs through PD-1 / PD-L1 signaling. The addition of immune checkpoint antibodies enhances the recognition and killing of tumor cells by PBMCs by relieving PD-1 / PD-L1-mediated immunosuppression, thereby reducing tumor cell survival. Therefore, by detecting tumor cell survival or killing rate, the ability of immune checkpoint antibodies to restore terminal immune effects can be directly evaluated.

[0158] 4.3 Experimental Materials

[0159] (1) Cell

[0160] Effector cells: healthy human PBMCs; target cells: Calu-1 cells (human lung cancer cells that highly express PD-L1).

[0161] (2) Test antibody

[0162] The following antibodies were used as representative immune checkpoint antibodies: Pembrolizumab, Atezolizumab, and the test antibody BT-YL-1, while hIgG1 isotype control was set up.

[0163] (3) Main reagents

[0164] OKT3 antibody, anti-CD28 antibody, CellTrace™ Violet (CTV) dye, Fixable Viability DyeeFluor™ 780 live / dead cell dye, RPMI-1640 medium, fetal bovine serum (FBS), and PBS buffer.

[0165] 4.4 Experimental Methods

[0166] The revived PBMCs were cultured overnight in RPMI-1640 complete medium. The next day, OKT3 antibody was coated onto 6-well plates at a final concentration of 1 μg / mL and incubated at 37 °C for 2 h. The coating solution was discarded, and the plates were washed twice with PBS. The PBMC density was adjusted to 2 × 10⁶ cells / well using RPMI-1640 complete medium. 6 Cells were added at a density of 2 ml / ml to each well, along with anti-CD28 antibody solution to a final concentration of 2 µg / ml. The cells were incubated at 37 ℃ in a 5% CO2 incubator for 3 days to activate PBMCs. After three days, PBMCs were collected, washed twice with PBS, and the cell density was adjusted to 0.2 × 10⁻⁶ cells / ml. 6 Cells / ml. Mix 50 µl of PBMC suspension with 50 µl of diluted antibody and add to a 96-well round-bottom plate. Incubate at 37 °C, 5% CO2 for 30 min. Adjust the density of logarithmic growth phase Calu-1 tumor cells to 1 × 10⁻⁶ cells / ml using PBS. 6 Cells were incubated at 0.5 nM CTV dye per ml, and incubated at 37 °C for 5 min. Staining was terminated by adding 5 times the volume of complete culture medium, followed by washing once with complete culture medium to remove excess dye. Subsequently, the cell density was adjusted to 1 × 10⁶ cells / ml using 1640 complete culture medium. 5Cells / ml, 100 µl per well, co-cultured with PBMCs for 3 days. After 3 days, remove the 96-well plate, mix by pipetting, centrifuge at 1500 rpm for 5 min, wash the cell pellet once with assay buffer, add 50 μl of Fixable Viability Dye eFluor™ 780 working solution to each well and resuspend, incubate at 4°C in the dark for 30 min. Then wash once with assay buffer, and resuspend in 100 μl of assay buffer to each well.

[0167] 4.5 Data Processing

[0168] Flow cytometry was used to analyze the death of CTV-positive tumor cells. 50 μl of sample was collected from each well, and scatter plots were used to delineate the principal cell population, single cell populations, and PB450. + APC-Cy7 + Double-positive cells accounted for PB450 + The percentage of cells killed by PBMCs represents the number of tumor cells killed by the tumor cells.

[0169] Using Pembrolizumab as the standard reference antibody, the Terminal Effector Score (TES) was calculated. The Killing Rate (test sample) represents the PBMC killing rate against tumor cells in the test antibody group, while the Killing Rate (standard) represents the PBMC killing rate against tumor cells in the standard reference antibody group. A higher TES value indicates a stronger ability of the antibody to restore the terminal killing function of PBMCs. The resulting immune response score serves as the fourth functional parameter in the comprehensive efficacy evaluation system.

[0170] 4.6 Experimental Results

[0171] The above methods were used to evaluate the effects of Pembrolizumab, Atezolizumab, and the test antibody BT-YL-1 on enhancing the PBMC-mediated killing ability of Calu-1 tumor cells. The results are as follows: Figure 6 As shown.

[0172] In the co-culture system of PBMC and Calu-1 cells, the addition of immune checkpoint antibodies enhanced the killing effect of PBMC on Calu-1 cells to varying degrees, indicating that blocking PD-1 / PD-L1 signaling can restore PBMC-mediated anti-tumor immune function. Figure 6 This demonstrates the cytotoxic effect of PBMCs on Calu-1 cells.

[0173] The tumor cell killing rate of each group was further statistically analyzed, and Pembrolizumab was used as a reference antibody for standardization. The Terminal Effector Score (TES) was calculated, and the results are shown in Table 7.

[0174] Table 7 Evaluation results of tumor cell killing mediated by PBMCs of different immune checkpoint antibodies

[0175] TES 1.00 0.99 1.04

[0176] The results showed that, in this experimental system, all three immune checkpoint antibodies effectively enhanced the killing effect of PBMCs on PD-L1 positive tumor cells. The overall differences between the different antibodies were small.

[0177] 4.7 Results Analysis

[0178] PBMC-mediated tumor cell killing assays are important in vitro models for evaluating the terminal efficacy of immune checkpoint antibodies, comprehensively reflecting multiple continuous biological processes such as immune cell recognition, activation, and cytotoxicity. Experimental results showed that all three immune checkpoint antibodies enhanced PBMC-mediated tumor cell killing.

[0179] Unlike Examples 1, 2, and 3, which evaluated signal blocking, immune activation, and immune response respectively, this example directly detects tumor cell death, which can more closely approximate the final effect of immune checkpoint antibodies in vivo in the fight against tumors.

[0180] While these assays can reflect the ability of immune checkpoint antibodies to restore the killing function of immune cells, the lack of a unified data standardization method makes it difficult to directly compare the results with signal blocking experiments and cytokine release experiments. Therefore, this embodiment does not simply establish a PBMC killing experiment, but uses PBMC-mediated tumor cell killing as the terminal functional module of a comprehensive evaluation system, and achieves standardized expression of the terminal immune effect by constructing a TES.

[0181] This module, as the fourth functional module of the comprehensive evaluation platform of this invention, together with the signal blocking function evaluation module, the immune activation function evaluation module, and the immune regulation evaluation module, constitutes a multi-dimensional functional evaluation system covering the complete action process of immune checkpoint antibodies, providing complete functional data for the subsequent establishment of a standardized evaluation framework and the calculation of the comprehensive efficacy index.

[0182] 4.8 Functional Evaluation and Judgment

[0183] The PBMC-mediated tumor cell killing evaluation module is used to evaluate the ability of candidate antibodies to restore the terminal anti-tumor effect of immune cells and is an important component of the in vitro comprehensive functional evaluation system for immune checkpoint antibodies.

[0184] Because PBMC-mediated tumor cell killing ability is influenced by multiple factors, including signaling pathway blockade, T cell activation, immune regulatory status, and effector cell function, the results reflect the comprehensive effect of the candidate antibody after the overall immune function is restored, rather than the effect of a single functional module. Although terminal tumor killing ability can reflect the overall in vitro efficacy of the candidate antibody well, it is still insufficient to evaluate all functional characteristics of the candidate antibody alone.

[0185] For the reasons mentioned above, this invention uses the Terminal Effector Score (TES) to quantitatively evaluate the terminal antitumor effect of candidate antibodies and incorporates it as a component of the Comprehensive Potency Index (CPI) for comprehensive scoring. A higher TES value indicates a stronger ability of the candidate antibody to restore PBMC-mediated tumor cell killing, and a greater contribution to the CPI; a lower TES value correspondingly reduces the CPI, but is not used as a sole basis for terminating the evaluation of candidate antibodies.

[0186] It should be further clarified that this module evaluates the final functional outcome of immune checkpoint antibodies after they have exerted their therapeutic effect. Together with the signal blocking evaluation module, MLR evaluation module, and immune regulation evaluation module, which correspond to the basic mechanism of action, immune function recovery, and immune regulation process, respectively, they constitute a continuous functional evaluation system covering the complete action process of immune checkpoint antibodies. TES not only reflects the final in vitro antitumor ability of candidate antibodies but also, in conjunction with other functional modules, analyzes key factors affecting the final efficacy, providing important evidence for the comprehensive evaluation of candidate antibodies and research and development decisions.

[0187] Therefore, this embodiment does not set separate functional admission criteria. After a candidate antibody completes the evaluation of this module, it continues to the next functional evaluation module. Finally, the comprehensive efficacy index (CPI) is combined to complete the unified evaluation and R&D decision.

[0188] Example 5: Establishment of a standardized evaluation framework for the in vitro function of immune checkpoint antibodies and a comprehensive efficacy index (CPI).

[0189] 5.1 Establishing the Purpose

[0190] Examples 1 to 4 established modules for evaluating signal blocking function, immune activation function, immune regulation, and terminal immune effect, respectively. Each module evaluates the in vitro functional characteristics of immune checkpoint antibodies from different perspectives. However, due to differences in experimental principles, detection indicators, data units, and result expression methods among the various detection systems, direct horizontal comparisons of experimental results are not possible, and a unified comprehensive evaluation standard is difficult to establish. Therefore, this example aims to establish a standardized evaluation framework. By uniformly normalizing the output results of each module, data generated by different experimental systems are converted into standardized scores, achieving a unified expression of multidimensional functional data. This leads to the construction of a comprehensive efficacy index, enabling a comprehensive quantitative evaluation of different immune checkpoint antibodies at the multidimensional functional level.

[0191] 5.2 Standardization Principles

[0192] Standardization was performed using a unified reference antibody. Pembrolizumab was used as the reference antibody, with each module's score defined as 1.00. Other antibodies to be evaluated were normalized using the corresponding indicators of the reference antibody to eliminate differences between different experimental platforms, batches, and testing units, thereby improving the comparability of evaluation results. The standardized output values ​​of each functional module were expressed as dimensionless scores, with higher values ​​indicating stronger corresponding functions.

[0193] 5.3 Standardized Calculation Methods for Each Functional Module

[0194] (1) Blocking Score (BS)

[0195] EC measured according to Example 1 50 Calculate the blocking capability score. Based on EC 50 It is negatively correlated with blocking ability, therefore the reciprocal normalization method is used: EC 50 The standard is pembrolizumab EC, which blocks the signaling pathway. 50 EC 50 The test sample is the EC signaling pathway blocking antibody to be evaluated. 50 EC 50 The lower the BS value, the higher the antibody's ability to block the PD-1 / PD-L1 signaling pathway.

[0196] (2) Immune Activation Score (AS)

[0197] Based on the T cell IL-2 and IFNγ release levels measured in Example 2, and using Pembrolizumab as a standard reference antibody, according to the formula: , Define the Immune Activation Score (AS): The higher the AS, the stronger the ability to restore T cell function.

[0198] (3) Immune Modulation Score (IMS)

[0199] Based on the whole blood IL-2 and IFNγ release levels measured in Example 3, the maximum response value of each cytokine was taken as the maximum release level. Pembrolizumab was used as the standard reference antibody to define the Immune Modulation Score (IMS): A higher IMS value indicates a stronger regulatory capacity of immune checkpoint antibodies on the initiated immune response process.

[0200] (4) Terminal Effector Score (TES)

[0201] Based on the PBMC-mediated tumor cell killing rate obtained in Example 4, and using Pembrolizumab as the standard reference antibody, the Terminal Effector Score (TES) was defined. The higher the TES value, the stronger the antibody's ability to restore the terminal killing function of PBMCs.

[0202] 5.4 Standardized Evaluation Results

[0203] Based on the experimental results obtained in Examples 1 to 4, the standardized scores corresponding to Pembrolizumab, Atezolizumab and the test antibody BT-YL-1 were calculated respectively, and the results are shown in Table 8.

[0204] Table 8 Antibody Standardized Scoring Results

[0205] Pembrolizmab 1.00 1.00 1.00 1.00 Atezolizumab 5.06 0.48 0.98 0.99 BT-YL-1 4.14 1.14 1.05 1.04

[0206] 5.5 Calculation of the Comprehensive Productivity Index (CPI)

[0207] In the processes of candidate antibody screening, biosimilar consistency evaluation, and drug development, it is necessary to use unified comprehensive evaluation indicators to quantitatively compare the overall in vitro functions of different antibodies. Therefore, by assigning a comprehensive efficacy index to the standardized evaluation results and weighting and integrating the four functional scores, the overall in vitro efficacy of immune checkpoint antibodies can be comprehensively evaluated.

[0208] The four scores in Examples 1 to 4 above correspond to different functional stages in the immune checkpoint antibody action process. Among them, (1) the PD-1 / PD-L1 signal blocking module (BS) is mainly used to evaluate whether the candidate antibody can effectively relieve the inhibitory signal mediated by the immune checkpoint, which is the basic mechanism for the candidate antibody to exert its efficacy. Since this module mainly reflects the mechanism of action itself and cannot directly reflect the subsequent recovery of immune function and the final anti-tumor effect, it is given a basic weight; (2) the immune activation module (AS) is used to evaluate the ability of the candidate antibody to restore T cell activation function, which is an important intermediate link connecting the basic mechanism of action and the final efficacy; the immune regulation module (IMS) is used to evaluate the ability of the candidate antibody to regulate the immune response in the immune activation state, which can supplement the reflection of the immune regulation characteristics of different candidate antibodies. The above two modules jointly reflect the immune function recovery process, so they are given the same medium weight; (3) the terminal immune effect module (TES) directly evaluates the ability of PBMC to kill tumor cells, and its results are closest to the final in vitro efficacy of the immune checkpoint antibody, so it is given the highest weight. The above weights satisfy the following conditions: the sum of the four weights is 1; the weight allocation conforms to the pharmacological process of immune checkpoint antibodies progressing from basic mechanisms of action to terminal antitumor effects; while ensuring the evaluation of basic mechanisms of action, the importance of terminal functional evaluation for overall efficacy prediction is emphasized; and the contributions of immune function recovery and immune regulation processes to the comprehensive efficacy evaluation of candidate antibodies are also considered. Based on these reasons, the weights of BS, AS, IMS, and TES are 0.20, 0.25, 0.25, and 0.30, respectively, to construct the comprehensive efficacy index CPI: CPI = 0.2*BS + 0.25*AS + 0.25*IMS + 0.3*TES, used to achieve a comprehensive quantitative evaluation of different immune checkpoint antibodies at multiple functional levels. A higher CPI value indicates stronger overall in vitro function of the immune checkpoint antibody. The antibody comprehensive efficacy index (CPI) was calculated based on the standardized scores, and the results are shown in Table 9.

[0209] Table 9. Antibody Overall Potential Index (CPI)

[0210] CPI 1.00 1.67 1.69

[0211] The calculation results show that different candidate antibodies have different advantages in each functional module. However, by integrating the results of different modules through a comprehensive efficacy index, each candidate antibody can obtain a unique comprehensive evaluation result, avoiding evaluation bias caused by a single experimental indicator. When two antibodies perform similarly in a certain functional module, their overall functional differences can still be reflected by the scores of other functional modules, thereby improving the distinguishing ability between different antibodies.

[0212] 5.6 Establish decision-making rules for candidate antibody development

[0213] This invention adopts an evaluation strategy of "qualification admission + comprehensive scoring". First, the PD-1 / PD-L1 signal blocking ability is used as the qualification admission standard for candidate antibodies to enter the comprehensive evaluation system. For candidate antibodies that meet the qualification admission requirements, a comprehensive efficacy index (CPI) is established by combining T cell activation ability, immune function recovery ability, immune regulation ability under exogenous stimulation and PBMC-mediated tumor cell killing ability. The priority ranking of candidate antibodies and research and development decisions are completed based on the CPI.

[0214] (1) Priority development

[0215] If a candidate antibody meets the eligibility criteria and its comprehensive efficacy index reaches the preset standard (e.g., CPI≥1), it indicates that the candidate antibody has a good basic mechanism of action, immune function recovery ability, immunomodulatory ability and terminal anti-tumor effect, and can be used as a priority candidate molecule for in vivo efficacy evaluation or subsequent development stage.

[0216] (2) Further optimization

[0217] The candidate antibody meets the eligibility criteria, but the overall potency index is within the preset range (e.g., 0.8 ≤ CPI < 0.9), indicating that the candidate antibody has certain development value, but there is still room for further optimization of the overall function. It can be further evaluated by combining structural optimization, affinity optimization or dosage optimization.

[0218] (3) Termination of development

[0219] If a candidate antibody fails to meet the eligibility criteria or its overall potency index is lower than the preset standard (e.g., CPI < 0.8), it indicates that the candidate antibody has insufficient overall in vitro function and is not recommended for further development.

[0220] Through the above-mentioned R&D decision-making rules, this invention realizes the transformation of candidate antibody from "functional testing" to "standardized screening - comprehensive scoring - R&D decision-making", enabling different candidate antibodies to be evaluated according to unified standards, and improving the consistency, objectivity and comparability of candidate antibody screening results.

[0221] 5.7 Application

[0222] The comprehensive evaluation system established by this invention can be applied to the following scenarios: (1) early screening and selection of candidate immune checkpoint antibodies; (2) decision-making for candidate antibody research and development; (3) comparison of the overall functions of different candidate antibodies; (4) evaluation of antibody engineering optimization; (5) consistency evaluation of biosimilars; (6) quality control of in vitro pharmacodynamics and batch-to-batch consistency evaluation; (7) construction of a comprehensive evaluation platform for immune checkpoint antibodies.

Claims

1. A method for comprehensively evaluating the in vitro function and potency of immune checkpoint antibodies, characterized in that, The comprehensive evaluation method calculates a Comprehensive Potency Index (CPI) based on evaluation indicators obtained from the signal blocking evaluation module, immune activation evaluation module, immune checkpoint antibody immunomodulation module, and terminal immune effect evaluation module. This CPI comprehensively evaluates the overall in vitro function of candidate immune checkpoint antibodies. The CPI is calculated using the following formula: CPI = W1*BS + W2*AS + W3*IMS + W4*TES; where W1, W2, W3, and W4 represent the weights of each functional module, satisfying W1 + W2 + W3 + W4 = 1. These weights are set according to the type of immune checkpoint antibody, the research objective, or the evaluation requirements. BS represents the signal blocking score, AS represents the immune activation score, IMS represents the immune modulation score, and TES represents the terminal immune effect score.

2. The method according to claim 1, characterized in that, The process includes the following steps: (1) Establishing a signal blocking evaluation module to detect the ability of immune checkpoint antibodies to block immune checkpoint signaling pathways and obtain a signal blocking score (BS); (2) Establishing an immune activation evaluation module to detect the ability of immune checkpoint antibodies to promote T cell activation and cytokine release and obtain an immune activation score (AS); (3) Establishing an immune checkpoint antibody immune regulation module and obtain an immune regulation score (IMS); (4) Establishing a terminal immune effect evaluation module to detect the ability of immune checkpoint antibodies to enhance the ability of immune cells to mediate tumor cell killing and obtain a terminal immune effect score (TES); (5) Establishing a comprehensive potency index (CPI) based on the evaluation indicators obtained in steps (1) to (4) to comprehensively evaluate the overall in vitro function of candidate immune checkpoint antibodies.

3. The method according to claim 2, characterized in that, The signal blocking evaluation module uses the immune checkpoint signaling pathway reporter gene detection system to evaluate the ability of immune checkpoint antibodies to block signaling pathways and calculates the signal blocking score based on EC50.

4. The method according to claim 2, characterized in that, The immune activation evaluation module uses a mixed lymphocyte reaction system to detect the release levels of one or two cytokines, IL-2 and IFNγ, and calculates the immune activation score based on the IL-2 and IFNγ scores.

5. The method according to claim 2, characterized in that, The immunomodulatory module is used to evaluate the ability of immune checkpoint antibodies to regulate an initiated immune response process, wherein the exogenous stimulation system is preferably a superantigen stimulation system.

6. The method according to claim 5, characterized in that, The superantigen is staphylococcal enterotoxin B (SEB).

7. The method according to claim 2, characterized in that, The immune regulation module measures the release levels of IL-2 and IFNγ in whole blood, and uses the maximum response value of each cytokine as the maximum release content to calculate the exogenous stimulus score.

8. The method according to claim 2, characterized in that, The terminal immune effect evaluation module uses a PBMC-tumor cell co-culture system to detect the tumor cell killing rate and calculates the terminal immune effect score based on the killing rate.

9. The method according to claim 8, characterized in that, The tumor cells are tumor cells that express immune checkpoint ligands.

10. The method according to claim 2, characterized in that, The standardization process uses a reference antibody for normalization calculation.

11. The method according to claim 10, characterized in that, The reference antibody is a known immune checkpoint antibody that has the same target as the immune checkpoint antibody to be evaluated.

12. The method according to claim 2, characterized in that, The immune checkpoint antibody is selected from one of the following: anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, and anti-TIM-3 antibody, or is a bispecific or multispecific antibody targeting any two or more of the above-mentioned immune checkpoints.

13. The method according to claim 12, characterized in that, The immune checkpoint antibody is an immune checkpoint antibody that acts on the PD-1 / PD-L1 signaling pathway.

14. The method according to claim 13, characterized in that, The immune checkpoint antibody is selected from one of the following: anti-PD-1 antibody, anti-PD-L1 antibody, bispecific antibody that binds both PD-1 and PD-L1, and multispecific antibody containing a PD-1 or PD-L1 binding domain; the reference antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

15. The method according to claim 14, characterized in that, The anti-PD-1 antibody includes one of Pembrolizumab, Nivolumab, Cemiplimab, Sintilimab, Camrelizumab, Tislelizumab, Toripalimab, or Serplulimab, or a biosimilar thereof; the anti-PD-L1 antibody includes one of Atezolizumab, Durvalumab, or Avelumab, or a biosimilar thereof.

16. The method according to claim 2, characterized in that, in, The Blocking Score (BS) is calculated using the following formula: ) ; The Activation Score (AS) is calculated using the following formula: ;in, ， ； The Immune Modulation Score (IMS) is calculated using the following formula: ； The Terminal Effector Score (TES) is calculated using the following formula: ; Among them, the higher the BS value, the stronger the antibody's ability to block the PD-1 / PD-L1 signaling pathway; or the higher the AS value, the stronger the ability to restore T cell function; or the higher the IMS value, the stronger the ability of the immune checkpoint antibody to regulate the already initiated immune response process; or the higher the TES value, the stronger the ability of the antibody to restore the terminal killing function of PBMCs.

17. The method according to claim 2, characterized in that, The candidate antibodies are first qualified based on the evaluation indicators of the basic mechanism of action, and then a comprehensive score is given.

18. The method according to claim 2, characterized in that, The priority of candidate antibody development is determined based on the comprehensive efficacy index.

19. The application of the method according to any one of claims 1-18 in the development of immune checkpoint drugs, characterized in that, The comprehensive evaluation method can be applied to the following scenarios: (1) early screening and selection of candidate immune checkpoint antibodies; (2) decision-making for candidate antibody development; (3) comparison of the overall function of different candidate antibodies; (4) evaluation of antibody engineering optimization; (5) consistency evaluation of biosimilars; (6) quality control of in vitro pharmacodynamics and batch-to-batch consistency evaluation; (7) construction of a comprehensive evaluation platform for immune checkpoint antibodies; (8) other needs involving in vitro functional evaluation of immune checkpoint antibodies.

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