Method for predicting immunogenicity of medicine in vitro based on peripheral blood mononuclear cells

Abstract

The invention discloses a method for predicting drug immunogenicity in vitro based on peripheral blood mononuclear cells. The method comprises the following steps: respectively mixing drugs with PBMCs from different donors, incubating, and determining the proportion of CD134 + CD25 + CD4 + cells in CD4 + cells; determining the proportion of CD134 < + > CD25 < + > CD4 < + > cells to CD4 < + > cells in PBMCs (peripheral blood mononuclear cells) from different donors, wherein the PBMCs are not incubated with drugs; determining a T cell activation index (TSI) according to the two proportions; the TSI is compared with a positive judgment threshold value, and whether the donor is a positive donor or not is determined; and determining the immunogenicity of the drug based on the proportion of the positive donor in all donors. When the method is used for measuring the immunogenicity of the medicine, the time cost is reduced, and the sensitivity is improved, so that the method has an important application value.

Description

Technical Field

[0001] This application relates to the field of biopharmaceutical technology, and particularly to a method for in vitro predicting the immunogenicity of drugs based on peripheral blood mononuclear cells. Background Art

[0002] Biological drugs such as antibody drugs play a key role in the treatment of major diseases such as cancer and autoimmune diseases. However, the safety and efficacy problems caused by the immunogenicity of the drugs themselves have hindered their development process to a certain extent. The most common form of this immunogenicity is the production of anti-drug antibodies (ADAs). ADAs can significantly reduce the therapeutic effect by neutralizing drug activity, altering pharmacokinetic properties or inducing allergic reactions (Wang, Y.-M. C. et al. Evaluating and Reporting the Immunogenicity Impacts for Biological Products--a Clinical Pharmacology Perspective. AAPS J 18, 395-403 (2016)). Therefore, it is of great significance to evaluate the immunogenicity of drugs in the early stage of drug development.

[0003] Immunogenicity assessment is a key link in the development of biological drugs. In vivo evaluation methods (such as animal models of mice, monkeys, etc.) can simulate the overall immune response of organisms and evaluate complex physiological processes such as antibody production, cellular immune response and potential toxicity. However, this method has a long experimental period (from several weeks to several months), high cost (especially for primate models) and is limited by the differences in the immune systems of animals and humans, and the results may be biased when extrapolated to humans. In addition, the high-throughput screening requirements (such as candidate drug libraries or engineered protein variants) are difficult to achieve through in vivo experiments. Therefore, the development of in vitro evaluation methods has become an important direction.

[0004] Currently, in vitro immunogenicity assessment is mainly based on the cascade reaction mechanism of innate immunity and adaptive immunity. The reported methods respectively conduct immunogenicity-related research on dendritic cells (DCs) and T cells (Gokemeijer, J. et al. Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development. AAPS J 25, 55 (2023)). As key antigen-presenting cells, the endocytosis efficiency of DCs is correlated with drug immunogenicity. By detecting the endocytosis intensity of drugs by DCs using the fluorescence labeling method and combining the expression levels of surface costimulatory molecules (such as CD40 / CD80 / CD86, etc.) and inflammatory factors (such as IL-6 / TNF-α, etc.), the risk of drugs triggering immune responses can be preliminarily evaluated (Melendez, R. et al. Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool. Bioanalysis 14, 703-713 (2022)). However, the occurrence of ADAs not only requires the activation of DCs but is also affected by antigen processing efficiency and T cell epitopes. Therefore, a direct assessment system based on the activation level of T cells has more definite biological significance.

[0005] Existing detection systems for T cell activation mainly include two modes: peripheral blood mononuclear cells (PBMCs) and DC-T co-culture (Cohen, S. & Chung, S. In vitro immunogenicity prediction: bridging between innate and adaptive immunity. Bioanalysis 13, 1071-1081 (2021)). The PBMCs system combines the test drug with PBMCs or CD8 +PBMCs co-cultured with exhausted T cells, and the drug immunogenicity was reflected by detecting T cell proliferation (isotope / BrdU labeling) or cytokine secretion (IL-2 / IFN-γ, etc.) (Goodell, V., dela Rosa, C., Slota, M., MacLeod, B. & Disis, M. L. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses. BMC Immunol 8, 21 (2007)). The DC-T co-culture system induced monocytes to differentiate into DCs by GM-CSF / IL-4, and after antigen loading, it was co-cultured with autologous T cells. The DC-T co-culture system could significantly improve the detection sensitivity of T cell activation, but this method was complex and time-consuming (Karle, A., Spindeldreher, S. & Kolbinger, F. Secukinumab, a novel anti-IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity. MAbs 8, 536-550 (2016)).

[0006] The PBMCs system represented by US 20230184747 A1 detected the proportion of CD134 and CD137 positive cells in viable CD4 + T cells by flow cytometry, and CD134 and CD137 were used as detection indicators for T cell activation. The positive rate predicted by this method had a low correlation with the actual incidence of ADA, and the prediction accuracy could be further improved; evaluating the cytokines induced by T cell activation, due to the variety of induced cytokines, multiple factor comprehensive analysis was required, which was difficult, and at the same time, the evaluation cost was also high, while the evaluation method for detecting T cell proliferation had a long experimental period.

[0007] Therefore, establishing an immunogenicity evaluation system that can not only shorten the culture time but also improve the sensitivity has important application value. Summary of the Invention

[0008] Based on this, it is necessary to provide at least one method for predicting the immunogenicity of drugs in vitro based on peripheral blood mononuclear cells.

[0009] In a first aspect of the present application, there is provided a method for predicting the immunogenicity of drugs in vitro based on PBMCs, which includes:

[0010] Mixing and incubating a drug with PBMCs from different donors respectively, and determining the proportion of CD134 + CD25 + CD4 + cells in CD4 + cells;

[0011] Determining the proportion of CD134 + CD25 + CD4 + cells in CD4 + cells of PBMCs from different donors not incubated with the drug;

[0012] Determining the T cell activation index (TSI) according to the two proportions;

[0013] Comparing the TSI with a positive judgment threshold to determine whether the donor is a positive donor; and,

[0014] Determining the immunogenicity of the drug based on the proportion of the number of positive donors in the total number of donors.

[0015] In some embodiments, the positive judgment threshold is obtained based on experimental data of low immunogenicity drugs.

[0016] In some embodiments, the 90 / 95 tolerance interval is calculated by the parametric method, and the TSI corresponding to the upper limit of the 90 / 95 tolerance interval is set as the positive judgment threshold.

[0017] In some embodiments, the positive judgment threshold is 1.0 - 2.0; for example, greater than or equal to 1.6.

[0018] In some embodiments, the positive judgment threshold is greater than or equal to 1.651786.

[0019] In some embodiments, the PBMCs are from at least 10 donors; exemplarily, at least 15 donors.

[0020] In some embodiments, the PBMCs are from at least 20 donors.

[0021] In some embodiments, the number of PBMCs mixed and incubated with the drug is 1×10 5 cells to 1×10 7 cells.

[0022] In some embodiments, the concentration of the drug is 10 μg / mL to 1000 μg / mL.

[0023] In some embodiments, the drug is a protein drug or a small molecule compound. Exemplarily, the protein drug may be, for example, a polypeptide, a monoclonal antibody or an antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate, etc.

[0024] In some embodiments, the incubation time of the drug with the PBMC is less than or equal to 72 hours.

[0025] In some embodiments, the incubation time is 36 hours to 48 hours.

[0026] In other embodiments, the incubation time is 48 hours to 72 hours.

[0027] In some embodiments, the CD134 in PBMC is determined by flow cytometry + CD25 + CD4 + The proportion of cells in CD4 + cells.

[0028] Using the method of the present application to measure the immunogenicity of the drug takes into account both reducing the time cost and improving the sensitivity. Therefore, this method has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or examples. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0030] Figure 1 It is the flow cytometry data analysis process in an embodiment of the present application.

[0031] Figure 2 It is the calculation of the single-label protein T cell activation index (TSI) in the PBMCs system in an embodiment of the present application.

[0032] Figure 3 It is the comparison of the clinical ADA positive rate of the drug and the CD134 in the PBMCs system in an embodiment of the present application + CD137 + Comparison of the positive rate of marker-characterized T cell activation.

[0033] Figure 4 Showing the CD134 in the PBMCs system in one embodiment of the present application + CD25 + The ratio of the marker characterizing the positive rate of T cell activation

[0034] Figure 5A Showing the correlation analysis between the positive rate of clinical ADA of the drug and the percentage of immunogenic positive individuals in the in vitro cell experiment in one embodiment of the present application

[0035] Figure 5B Showing the correlation analysis between the positive rate of clinical ADA of the drug and the percentage of immunogenic positive individuals in the in vitro cell experiment in one embodiment of the present application Detailed implementation manners

[0036] For ease of understanding the present application, the present application will be described more comprehensively below with reference to the relevant accompanying drawings. Preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that the understanding of the disclosure of the present application is more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs. The terms used in the description of the present application herein are for the purpose of describing specific embodiments only and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0038] In the present application, unless otherwise stated, "one or more" means any one of the listed items or any combination of the listed items. Similarly, other cases such as "one or more" which express "one or more" are also understood in the same way unless otherwise stated.

[0039] The "combinations thereof", "any combinations thereof", "any combination manners thereof", etc. used in the present application include all suitable combination manners of any two or more of the listed items.

[0040] In the present application, "suitable combination manners", "suitable manners", "any suitable manners", etc., the "suitable" is subject to being able to implement the technical solution of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application.

[0041] In this application, terms such as "specially", "for example", "such as", "example", and "exemplification" are used for descriptive purposes, indicating an association in terms of the covered content between the different technical solutions before and after, but should not be construed as a limitation on the previous technical solution, nor as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-restrictive example of A, and it can be understood that A is not limited to B.

[0042] The terms "comprising", "including", and "containing" used in this application are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-restrictive examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0043] In this application, in a technical feature or technical solution described in an open language, it includes a closed technical feature or technical solution composed of the listed content, and also includes an open technical feature or technical solution containing the listed content.

[0044] In this application, regarding exemplary descriptions such as "in some embodiments" or "in one embodiment", it can cover but is not limited to the following meanings: These solutions can be combined with other solutions in a suitable manner to form a new technical solution.

[0045] In this application, in "the first aspect", the term "first" is only used for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can it be construed as implicitly indicating the importance or quantity of the indicated technical feature. Moreover, "first" only serves the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0046] In this application, regarding a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the selectable numerical values within this numerical interval is considered continuous and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of this numerical interval, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as a percentage interval, a ratio interval, a ratio interval, etc.

[0047] In this application, for a method flow involving multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order limit, and they can be executed in an order other than the described one. Moreover, any step can include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same moment but can be executed at different moments, and their execution order is not necessarily sequential but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0048] T cell activation markers are important tools in immunological research. The expression of different markers can be used to evaluate the CD4 induced by biological drugs + T cell activation, providing an efficient detection scheme based on flow cytometry for optimizing the drug immunogenicity prediction model. In healthy and diseased states, the activation and functional regulation of T cells are crucial for the effectiveness and specificity of immune responses. Upregulation of the expression of the three receptors CD25, CD134, and CD137 all indicate that T cells are activated, and they play important roles in different immune responses. CD25 (IL-2Rα) is the alpha chain of the interleukin-2 receptor and is one of the early markers after T cell activation (Shatrova, A. N. et al. Time-Dependent Regulation of IL-2R α-Chain (CD25) Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling in Activated Human Blood T Lymphocytes. PLoS One 11, e0167215 (2016)). During T cell activation, the expression of CD25 rapidly upregulates and participates in IL-2 signal transduction, thus supporting the proliferation and differentiation of T cells. Since CD25 is in CD4 +It persists in regulatory T cells (Tregs) (accounting for 0.5% to 6% of total T cells), so CD25 must be used in combination with other markers to define antigen-specifically activated T cells, and there has been no previous study on using CD25 for predicting the in vitro immunogenicity of biologics. CD134 (OX40) is a member of the tumor necrosis factor receptor superfamily. OX40 is not expressed in unstimulated T cells but is expressed after T cell activation through the action of TCR signals, usually reaching a peak about 48 hours after activation (Gramaglia, I., Weinberg, A. D., Lemon, M. & Croft, M. Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 161, 6510-6517 (1998)). Its ligand OX40L plays an important role in the interaction between dendritic cells and T cells, helping to enhance T cell survival, proliferation, and effector functions. Therefore, CD134 is considered a key marker for evaluating sustained T cell activation and responses (Zaunders, J. J. et al. High levels of human antigen-specific CD4+ T cells in peripheral blood revealed by stimulated coexpression of CD25 and CD134 (OX40). J Immunol 183, 2827-2836 (2009)). CD137 (4-1BB) is a member of the TNF receptor superfamily and is upregulated through TCR signals during T cell activation (Cannons, J. L. et al. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol 167, 1313-1324 (2001)). By binding to the 4-1BBL (CD137L) ligand on the surface of antigen-presenting cells, it promotes T cell proliferation and cytokine secretion.4-1BB can be used to detect antigen-specific CD8+ T cells when combined with CD69 (Wölfl, M., Kuball, J., Eyrich, M., Schlegel, P. G. & Greenberg, P. D. Use of CD137 to study the full repertoire of CD8+ T cells without the need to know epitope specificities. Cytometry A 73, 1043-1049 (2008)), but also expressed in activated CD4. + T cells. All three markers can be used to characterize CD4 + T cell activation. Therefore, when using a single marker or a combination thereof, it is necessary to further determine which marker and combination can more accurately predict the immunogenicity of biopharmaceuticals. The higher the prediction accuracy (i.e., the correlation between the predicted immunogenicity positive rate and the clinical ADA positive rate), the more conducive it is to distinguish the possible immunogenicity differences between different biopharmaceutical candidate molecules, and it has a more definite guiding role in the selection of clinical candidate molecules.

[0049] This application proposes a method for detecting T cell activation based on flow cytometry. By studying the surface markers of T cell activation, the sensitivity and accuracy of different surface markers and their combinations for predicting drug immunogenicity are evaluated. Further optimize the PBMCs system to establish an efficient, rapid and sensitive in vitro biopharmaceutical immunogenicity evaluation system, providing stronger technical support for the research and development of biopharmaceuticals. The experimental technique can be used in the early stage of drug development to screen out molecules that may cause high immunogenicity in humans, guide the selection of lead molecules, reduce the risk and cost of drug development, and improve the development efficiency.

[0050] The inventor creatively changed the detection index indicating T cell activation and used the co-expression of CD134 and CD25 as the characteristic of T cell activation. Compared with the prior art using the co-expression of CD134 and CD137 as the characteristic of T cell activation, the correlation between the predicted positive rate and the actual incidence of ADA was significantly improved. The method for predicting the in vitro immunogenicity of biopharmaceuticals provided by this application has higher accuracy compared with existing similar methods.

[0051] One aspect of this application provides a method for predicting the immunogenicity of a drug in vitro based on peripheral blood mononuclear cells (PBMC), which includes:

[0052] S100: Mix and incubate the drug with PBMC from different donors respectively, and determine CD134 among them +CD25 + CD4 + CD4 + The proportion of cells;

[0053] S200: Determination of CD134 in PBMCs from different donors without incubation with drug + CD25 + CD4 + CD4 + The proportion of cells;

[0054] S300: T cell activation index (TSI) determined based on two ratios;

[0055] S400: comparing the TSI with a positive judgment threshold to determine whether the donor is a positive donor; and,

[0056] S500: Determine the immunogenicity of the drug based on the ratio of positive donors to all donors.

[0057] In some embodiments, in step S100, PBMCs are from at least 10 donors; illustratively: at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 donors.

[0058] In some embodiments, in step S100, the number of PBMCs mixed and incubated with the drug is 1×10 5 ~1×10 7 For example, 1×10 5 pcs, 2×10 5 pcs, 3×10 5 4×10 5 pcs, 5×10 5 pcs, 6×10 5 pcs, 7×10 5 8×10 5 pcs, 9×10 5 pcs, 1×10 6 pcs, 2×10 6 pcs, 3×10 6 4×10 6 pcs, 5×10 6 pcs, 6×10 6 pcs, 7×10 6 8×10 6 pcs, 9×10 6 pcs, 1×10 7 Or a value or range between any two values.

[0059] In some embodiments, in step S100, the concentration of the drug is 10 μg / mL to 1000 μg / mL. Exemplarily, for example, it is 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, 1000 μg / mL, or a range or value between any two values.

[0060] In some embodiments, in step S100, the drug is selected from the group consisting of polypeptides, small molecule compounds, and antibody-drug conjugates.

[0061] Exemplarily, the polypeptide is a biological macromolecule, such as an antibody or an antigen-binding fragment thereof.

[0062] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. A "native antibody" refers to an immunoglobulin molecule that occurs naturally. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N- to the C-terminus, each heavy chain has a variable region (VH, also known as the variable heavy domain, heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N- to the C-terminus, each light chain has a variable region (VL, also known as the variable light domain, or light chain variable domain), followed by a constant light domain (light chain constant region, CL). The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein.

[0063] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures, which can be classified into IgG-like bispecific antibodies and antibody fragment-based bispecific antibodies according to the integrity of the IgG molecule; according to the number of antigen-binding regions, they can be divided into bivalent, trivalent, tetravalent or more-valent bispecific antibodies, and according to whether the structure is symmetric or not, they can be divided into symmetric structure bispecific antibodies and asymmetric structure bispecific antibodies. Among them, the bispecific antibody based on antibody fragments, such as the Fab fragment lacking the Fc fragment, forms a bispecific antibody by binding two or more Fab fragments in one molecule. It has low immunogenicity, a small molecular weight, and high tumor tissue permeability. Typical antibody structures of this type include bispecific antibodies such as F(ab)2, scFv-Fab, (scFv)2-Fab, etc.; IgG-like bispecific antibodies (such as those with an Fc fragment), these antibodies have a relatively large molecular weight. The Fc fragment helps in the later purification of the antibody and improves its solubility and stability. The Fc part may also bind to the receptor FcRn, increasing the serum half-life of the antibody. Typical bispecific antibody structure models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

[0064] In some embodiments, in step S100, the time for the drug to be incubated with the PBMC is less than or equal to 72 hours.

[0065] Exemplarily, the incubation time of the drug with the PBMC is less than or equal to 72 hours, less than or equal to 70 hours, less than or equal to 68 hours, less than or equal to 66 hours, less than or equal to 64 hours, less than or equal to 62 hours, less than or equal to 60 hours, less than or equal to 58 hours, less than or equal to 56 hours, less than or equal to 54 hours, less than or equal to 52 hours, less than or equal to 50 hours, less than or equal to 48 hours, less than or equal to 46 hours, less than or equal to 44 hours, less than or equal to 42 hours, less than or equal to 40 hours, less than or equal to 38 hours, less than or equal to 36 hours, less than or equal to 34 hours, less than or equal to 32 hours, less than or equal to 30 hours, less than or equal to 28 hours, less than or equal to 26 hours, less than or equal to 24 hours, less than or equal to 22 hours, less than or equal to 20 hours, less than or equal to 18 hours, less than or equal to 16 hours, less than or equal to 14 hours, less than or equal to 12 hours, etc.

[0066] In some embodiments, the incubation time of the drug with PBMC is 36 hours to 48 hours.

[0067] In some embodiments, the incubation time of the drug with PBMC is 48 hours to 72 hours.

[0068] In some embodiments, in step S100 and / or step S200, the CD134 + CD25 + CD4 + cells in PBMC are determined by flow cytometry to account for the proportion of CD4 + cells.

[0069] In some embodiments, the TSI in step S300 is calculated with reference to the following calculation method:

[0070]

[0071] In some embodiments, in step S400, the positive judgment threshold is obtained based on the experimental data of low immunogenic drugs. Exemplarily, it is determined by the sum of outliers analysis or by linear regression.

[0072] In some embodiments, the parametric method is used to calculate the 90 / 95 tolerance interval, and the TSI corresponding to the upper limit of the 90 / 95 tolerance interval is set as the positive judgment threshold.

[0073] In some embodiments, low-immunogenicity drugs may be, for example, Bevacizumab, Secukinumab, Trastuzumab, Rituximab, Cetuximab, Atezolizumab, Pertuzumab, Adalimumab, Ustekinumab, Dupilumab, or Nivolumab, etc.

[0074] In some embodiments, the low-immunogenicity drugs include Bevacizumab and Secukinumab.

[0075] In some embodiments, the positive judgment threshold in step S400 is 1.0 - 2.0.

[0076] Exemplarily, for example, it is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or the range or value between any two numerical values.

[0077] In some embodiments, the positive judgment threshold is greater than or equal to 1.6.

[0078] In some embodiments, the positive judgment threshold is greater than or equal to 1.651786.

[0079] In some embodiments, in step S500, the immunogenicity of the drug is determined based on the proportion of positive donors among all donors.

[0080] Without wishing to be bound by any theory, it is considered that: according to the number of PBMC donors detected, the more the number of donors determined to be positive, the higher the possibility that the drug has immunogenicity, and vice versa. Exemplarily, when the proportion of positive donors among all donors is more than 95%, it is determined that the drug has immunogenicity; when the proportion of positive donors among all donors is more than 90%, 85%, 80%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or more, it is determined that the drug has immunogenicity, or the possibility of having immunogenicity is relatively high.

[0081] Some embodiments are provided below.

[0082] The embodiments of the present application will be described in detail below in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following embodiments, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or by referring to the experimental methods known in the art.

[0083] Example 1

[0084] 1. Co-incubation of antibody drug and PBMCs cells

[0085] Resuscitate PBMCs in AIM-V medium (containing 10% AB serum, 1% double antibody, 1% non-essential amino acids, 1% sodium pyruvate), and seed the cells at a cell density of 2.5×10 5 per well into a 96-well U-bottom cell culture plate (Guangzhou Jet Bio-Fil, Cat.TCP002096); place the cells in an incubator with 5% CO2 at 37°C for static culture. After 24 hours of PBMCs resuscitation, add the drug to be tested (see Table 1) at a concentration of 100 μg / mL to the PBMCs for co-incubation, gently pipette and mix well, and incubate for 36 - 48 hours.

[0086] Table 1 Incidence of anti-drug antibodies (ADA) of candidate biopharmaceuticals

[0087]

[0088] Note: Secukinumab can also be called Cosentyx, Bevacizumab can also be called Avastin, Pertuzumab can also be called Perjeta, Adalimumab can also be called Humira, Ixekizumab can also be called Taltz, Rituximab can also be called Rituxan, Bocozizumab can also be called Bococizumab, and Keyhole limpet hemocyanin can also be called KLH.

[0089] 2. Detection of T cell activation by flow cytometry

[0090] After incubation, the cells were collected by centrifugation at 400 g for 10 minutes. The supernatant was discarded, and the cells were rinsed once with flow cytometry buffer (PBS solution containing 2% FBS). The antibodies CD4 (Biolegend, 344604), CD134 (Biolegend, 350004), and CD25 (Biolegend, 356110) were diluted to the working concentration (1:20) according to the instructions in the flow cytometry buffer. 100 μL of the antibody solution was added to each well to resuspend the centrifuged cells, and the cells were gently pipetted and mixed well. The cells were incubated in the dark at 4 °C for 30 minutes. After incubation, the cells were collected by centrifugation at 400 g for 10 minutes, rinsed once with PBS, and the LIVE / DEAD Fixable Dead Cell Stain (Thermo fisher, L34994) was diluted in PBS solution according to the instructions. 100 μL of the dead cell dye dilution was added to each well to resuspend the cells, and the cells were incubated continuously in the dark at 4 °C for 30 minutes. After incubation, the cells were collected by centrifugation at 400 g for 10 minutes, rinsed once with PBS, and finally the cells were resuspended in PBS for flow cytometry analysis.

[0091] First, draw a gate to circle the lymphocyte population ( Figure 1 in A), remove cell adhesion ( Figure 1 in B), then circle the live cell population according to the staining of cell viability ( Figure 1 in C), then circle the CD4-FITC positive T cell population in the live cell population ( Figure 1 in D), and finally draw the boundary line of CD134-PE and CD25-APC in the CD4 + T cells, Figure 1 in E and Figure 1 in F, the cells in the Q6 quadrant represent the cells that are double positive for CD134 and CD25. The values shown in the figure are the proportions of the CD134 + CD25 + CD4 + cell population in CD4 + T cells, Figure 1 in E shows the cell staining of the medium negative control group, Figure 1 in F shows the cell staining of the KLH positive control group.

[0092] 3. Calculation of T cell activation index and determination of positive data

[0093] Using CD134 and CD25 as detection indicators for T cell activation, according to the flow cytometry results, the T cell stimulation index (TSI) was calculated. The calculation method of TSI is as follows: After treatment with the experimental group (test drug), the proportion of CD4+ The proportion of T cells among CD4 + T cells in the total population, divided by the proportion of positive cells for the labeled protein corresponding to the negative control group (culture medium blank control). The calculation formula is expressed as:

[0094]

[0095] Based on the TSI of low-immunogenic drugs Bevacizumab or Secukinumab, a determination threshold for positive donors was established. Based on the assumption that the data approximately follows a normal distribution, the parametric method was used to calculate the 90 / 95 tolerance interval (i.e., a statistical interval covering 90% of the data with a 95% confidence level) of the TSI values of low-immunogenic drugs. The TSI corresponding to the upper limit of this tolerance interval was set as the determination threshold for immunogenic positive samples. According to the reported incidence of ADA in clinical trials of drugs, the Pearson correlation coefficient r between the proportion of positive donors and the incidence of ADA was calculated, and it can be seen that the positive donor rate predicted by this system has a significant correlation with the ADA positive rate of drugs in clinical practice ( Figure 2 ).

[0096] The immunogenicity of 20 donors and 10 drugs was evaluated using different evaluation indicators in the PBMCs system. The experimental results showed that the evaluation indicator of T cell activation with a single labeled protein did not show good discrimination among different immunogenic drugs; compared with CD25 and CD137, CD134 had a slightly higher response to different drug stimulations ( Figure 2 ), so CD134 was further evaluated + CD137 + and CD134 + CD25 + The TSI of double-labeled proteins as an indicator of T cell activation and the positive rate of donors.

[0097] To obtain the optimal signal-to-noise ratio and detection sensitivity, the TSI value corresponding to the upper limit of the 90 / 95 tolerance interval was calculated and set as the determination threshold for low-immunogenic samples. The positive determination threshold of TSI under the condition of CD134 + CD137 + is 1.662789, that is, samples with TSI≥1.662789 are regarded as positive. CD134 + CD25 +Under the condition, the positive determination threshold of TSI is 1.651786, that is, the samples with TSI≥1.651786 are regarded as positive. The analysis results show that, compared with the single biomarker evaluation index, under the dual biomarker evaluation system, the positive donor ratio statistically analyzed by TSI has a high correlation with the immunogenicity of the drug. Molecules with high immunogenicity such as Bocozizumab and ATR107 also detected relatively high donor positive rates; molecules with low immunogenicity such as Bevacizumab, Secukinumab and Pertuzumab all had positive rates less than 10%; the positive rates of Ixekizumab and Rituximab were also close to the reported positive rates of ADA patients in clinical trials. Adalimumab is the only drug that the dual biomarker detection method classifies as having a lower immunogenicity risk than clinical ADA. This experimental result shows the feasibility and reliability of using T cell activation surface markers for predicting the immunogenicity of biological drugs, and compared with CD134 + CD137 + the detection index r = 0.8124 ( Figure 3 )(p = 0.0064), for CD134 + CD25 + under the detection index, the positive rate of the in vitro experimental detection results has a higher correlation with the clinical incidence of ADA, r = 0.9159 ( Figure 4 、 Figure 5A and Figure 5B )(p = 0.0002).

[0098] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0099] The above-described embodiments only represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for in vitro predicting the immunogenicity of a drug based on peripheral blood mononuclear cells (PBMCs), characterized in that, It includes: The drug was separately mixed and incubated with PBMC from different donors, and the proportions of CD134 + CD25 + CD4 + cells among CD4 + cells were detected; Determine CD134 in PBMCs from different donors that were not incubated with the drug + CD25 + CD4 + The proportion of cells among CD4 + cells; Determining the T cell activation index (TSI) according to two ratios; Comparing the TSI with a positive judgment threshold to determine whether the donor is a positive donor; and, Determining the immunogenicity of the drug based on the proportion of positive donors among all donors.

2. The method according to claim 1, wherein The positive judgment threshold is obtained based on the experimental data of low-immunogenicity drugs.

3. The method according to claim 2, characterized in that Calculating the 90 / 95 tolerance interval by the parametric method, and setting the TSI corresponding to the upper limit of the 90 / 95 tolerance interval as the positive judgment threshold.

4. The method according to claim 2, wherein The low-immunogenicity drugs include Bevacizumab and Secukinumab.

5. The method according to claim 2, wherein The positive judgment threshold is 1.0 - 2.

0.

6. The method according to claim 5, wherein The positive judgment threshold is greater than or equal to 1.

6.

7. The method according to claim 6, wherein The positive judgment threshold is greater than or equal to 1.651786.

8. The method according to any one of claims 1 to 7, characterized in that The PBMC is from a single donor.

9. The method according to any one of claims 1 to 7, characterized in that, The PBMC is from at least 10 donors.

10. The method according to claim 9, wherein The PBMC is from at least 15 donors.

11. The method according to claim 10, wherein The PBMC is from at least 20 donors.

12. The method according to any one of claims 1 to 7, characterized in that, The number of PBMCs mixed and incubated with the drug is 1×10 5 to 1×10 7 cells.

13. The method according to any one of claims 1 to 7, characterized in that, The concentration of the drug is 10 μg / mL - 1000 μg / mL.

14. The method according to any one of claims 1 to 7, characterized in that, The drug is a protein drug or a small molecule compound.

15. The method according to claim 14, wherein The protein drug is selected from the group consisting of polypeptides, monoclonal antibodies or their antigen-binding fragments, bispecific antibodies or their antigen-binding fragments, and antibody-drug conjugates.

16. The method according to any one of claims 1 to 7, characterized in that, The incubation time of the drug with the PBMC is less than or equal to 72 hours.

17. The method according to claim 16, wherein The incubation time of the drug with the PBMC is 36 hours - 48 hours.

18. The method according to claim 16, wherein The incubation time is 48 hours - 72 hours.

19. The method according to any one of claims 1 to 7, characterized in that, Determination of CD134 in PBMC by flow cytometry + CD25 + CD4 + The proportion of cells among CD4 + cells.

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