T cell-based methods for predicting polypeptide immunogenicity

Abstract

The presently disclosed subject matter provides methods for determining the propensity of a composition, e.g., a composition comprising an antibody or fragment thereof, to induce the production of anti-drug antibodies (ADA), and kits for carrying out such methods.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 062,991 filed on 7 August 2020 and U.S. Provisional Application No. 63 / 215,199 filed on 25 June 2021, the contents of each of these applications being invoked by reference in whole, and each of these applications claiming priority.

[0002] This disclosure relates to a method for determining the tendency of a composition to induce the production of anti-drug antibodies (ADAs), and a kit for carrying out such a method. [Background technology]

[0003] Therapeutic drugs (e.g., antibodies) have greatly improved the treatment of increasingly serious and difficult-to-treat diseases. Unfortunately, such drugs can induce the production of anti-drug antibodies (ADAs) when administered to patients. ADAs can have a neutralizing effect on the therapeutic drug. These neutralizing effects may include limiting the activity of the therapeutic drug, increasing its clearance, and a potential decrease in the overall clinical response resulting from the administration of the therapeutic drug. In certain cases, ADA production also occurs concurrently with the occurrence of serious adverse events in patients, including hypersensitivity reactions and anaphylaxis.

[0004] Understanding the immunogenicity of a therapeutic agent in the pre-clinical stage of drug development can improve the likelihood of success of the therapeutic agent in subsequent clinical stages. Immunogenic epitopes are generally predicted using in silico tools, although several cell-based techniques have been developed to determine the immunogenic potential of pre-clinical therapeutic agent candidates. One such technique is called major histocompatibility complex (MHC) class II-associated peptide proteomics (MAPP). MAPP involves incubating a population of antigen-presenting cells (APCs), such as dendritic cells, with a therapeutic agent of interest, such as a polypeptide-based therapeutic agent. The APCs internalize and process the therapeutic agent into short peptides. The peptides are loaded onto MHC class II molecules and presented on the surface of the APCs. By immunoprecipitating these MHC-peptide complexes and analyzing them via liquid chromatography mass spectrometry (LC / MS), potential immunogenic epitopes in the therapeutic agent can be identified. Another technique for determining the immunogenic potential of pre-clinical therapeutic agent candidates is the T cell proliferation assay, which involves detecting T cell proliferation after co-culture of APCs, such as dendritic cells, incubated with a polypeptide-based therapeutic agent of interest. However, these techniques are labor-intensive, time-consuming, and require a large number of expensive instruments. Therefore, there is a need in the art for a more time- and cost-efficient method for determining the tendency to induce the production of anti-drug antibodies (ADA) in therapeutic agents, such as polypeptide-based therapeutic agents. Summary of the Invention

[0005] This disclosure provides a method for determining the tendency of a composition to induce the production of antibodies specific to that composition, compared to a reference tendency. In certain embodiments, the method of this disclosure may include (a) culturing lymphocytes in the presence of the composition to generate stimulated lymphocytes; (b) culturing lymphocytes in the absence of the composition to generate unstimulated lymphocytes; (c) determining the proportion of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; (d) determining the proportion of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; and (e) calculating a stimulation index value. In certain embodiments, if the stimulation index value in (e) is greater than or equal to a reference stimulation index value, the composition has a greater tendency to induce antibodies specific to that composition. In certain embodiments, if the stimulation index value in (e) is less than the reference stimulation index value, the composition is less likely to induce antibodies specific to that composition. In certain embodiments, the stimulation index value can be determined by: (i) dividing the percentage of stimulated lymphocytes determined in (c) by the percentage of unstimulated lymphocytes determined in (d), (ii) outlier sum analysis and / or (iii) linear regression. In certain embodiments, lymphocytes are obtained from a single donor. In certain embodiments, lymphocytes are obtained from about 20 to about 50 donors, for example, about 35 to about 45 donors. In certain embodiments, lymphocytes are obtained from at least about 20 donors, at least about 25 donors, at least about 30 donors, at least about 35 donors, at least about 40 donors, or at least about 45 donors.

[0006] In certain embodiments, a method for determining the tendency of a composition to induce the production of antibodies specific to the composition comprises: (a) culturing lymphocytes from individual donors separately in the presence of the composition to generate stimulated lymphocytes; (b) culturing lymphocytes from individual donors separately in the absence of the composition to generate unstimulated lymphocytes; (c) determining the proportion of stimulated lymphocytes from individual donors that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137; (d) determining the proportion of unstimulated lymphocytes from donors that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137; (e) calculating a stimulation index value for each donor; and (f) calculating the number of reactive lymphocyte donors whose stimulation index value is greater than or equal to a reference stimulation index value and the number of unstimulated lymphocyte donors whose stimulation index value is less than the reference stimulation index value. In certain embodiments, the stimulation index value can be determined by: (i) dividing the proportion of stimulated lymphocytes of an individual donor determined in (c) by the proportion of unstimulated lymphocytes of the individual donor determined in (d); (ii) outlier sum analysis; and / or (iii) linear regression. In certain embodiments, if the number of stimulated donors exceeds 30% of the total number of donors, the composition has a high tendency to induce the production of antibodies specific to the composition. In certain embodiments, if the number of stimulated donors is less than 20% of the total number of donors, the composition has a low tendency to induce the production of antibodies specific to the composition. In certain embodiments, the lymphocytes are obtained from about 20 to about 50 donors, such as about 35 to about 45 donors. In certain embodiments, the lymphocytes are obtained from at least about 20 donors, at least about 25 donors, at least about 30 donors, at least about 35 donors, at least about 40 donors, or at least about 45 donors.

[0007] In certain embodiments, the composition comprises a neoantigen. In certain embodiments, the composition is a peptide, polypeptide, or small molecule compound. In certain embodiments, the polypeptide is an antibody or a fragment thereof, for example, the antibody or fragment is a human, humanized, or chimeric antibody. In certain embodiments, the composition is an antibody-drug conjugate (ADC). In certain embodiments, the antibody or fragment thereof is a bispecific antibody.

[0008] The disclosure further provides a method for determining the tendency of a neoantigen to induce a neoantigen-specific immune response compared to a reference antigen. For example, but not limited to, the method may include (a) culturing lymphocytes in the presence of a neoantigen to generate stimulated lymphocytes; (b) culturing lymphocytes in the absence of a neoantigen to generate unstimulated lymphocytes; (c) determining the proportion of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; (d) determining the proportion of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; and (e) calculating a stimulation index value. In certain embodiments, the stimulation index value can be determined by (i) dividing the percentage of stimulated lymphocytes determined in (c) by the percentage of unstimulated lymphocytes determined in (d), (ii) summation of outliers analysis and / or (iii) linear regression. In certain embodiments, if the stimulation index value in (e) is greater than or equal to the reference stimulation index value, the neoantigen is more likely to induce a neoantigen-specific immune response, and if the stimulation index value in (e) is less than the reference stimulation index value, the neoantigen is less likely to induce a neoantigen-specific immune response.

[0009] In certain embodiments, the reference stimulus index value is the stimulus index value of a reference composition, for example, a composition that does not induce ADA production in a clinical setting, or has a low tendency to induce ADA production in a clinical setting. In certain embodiments, the reference stimulus index value is about 1.0 to about 4.0, i.e., about 1.0 to about 2.0. In certain embodiments, the reference stimulus index value is about 1.6 or greater, about 1.7 or greater, or about 1.8 or greater.

[0010] In certain embodiments, lymphocytes include T cells. In certain embodiments, at least 30% of lymphocytes include T cells. In certain embodiments, T cells are CD8-. In certain embodiments, at least 10% of T cells include CD8- T cells. In certain embodiments, about 1 × 10 5 ~Approx. 1×10 7 Individual lymphocytes are cultured with the composition. In certain embodiments, the lymphocytes are cultured with the composition at a concentration of approximately 10 μg / µl to approximately 1,000 μg / ml. In certain embodiments, the lymphocytes are cultured with the composition for approximately 48 hours or less.

[0011] In certain embodiments, determining the percentage of stimulated or unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137 is performed by flow cytometry.

[0012] This disclosure provides a method for determining the tendency of a composition to induce the production of antibodies specific to that composition, compared to a reference tendency. In certain embodiments, the method may include (a) culturing antigen-presenting cells (APCs) in the presence of the composition to produce stimulated APCs; (b) culturing APCs in the absence of the composition to produce unstimulated APCs; (c) culturing stimulated APCs separately with CD4+ lymphocytes and unstimulated APCs separately with CD4+ lymphocytes; (d) determining the proportion of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; (e) determining the proportion of CD4+ lymphocytes cultured with unstimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; and (f) calculating a stimulation index value. In certain embodiments, if the stimulation index value in (f) is greater than or equal to the reference stimulation index value, the composition is more likely to induce antibodies specific to the composition, and if the stimulation index value in (f) is less than the reference stimulation index value, the composition is less likely to induce antibodies specific to the composition. In certain embodiments, the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes determined in (d) by the percentage of CD4+ lymphocytes determined in (e). In certain embodiments, the stimulation index value is determined by outlier sum analysis or by linear regression. In certain embodiments, APC is obtained from a single donor. In certain embodiments, APC is obtained from about 20 to about 50 donors. In certain embodiments, APC is obtained from about 35 to about 45 donors. In a particular embodiment, the APC is obtained from at least about 20 donors, at least about 25 donors, at least about 30 donors, at least about 35 donors, at least about 40 donors, or at least about 45 donors.

[0013] This disclosure provides a method for determining the tendency of a composition to induce the production of antibodies specific to that composition, the method comprising: (a) culturing APCs from individual donors separately in the presence of the composition to produce stimulated APCs; (b) culturing APCs from individual donors separately in the absence of the composition to produce unstimulated APCs; (c) culturing stimulated APCs separately with CD4+ lymphocytes and unstimulated APCs separately with CD4+ lymphocytes; and (d) (i) CD134, (ii) CD137, or (iii) CD134 and CD13 The method includes (e) determining the percentage of CD4+ lymphocytes cultured with stimulated APCs expressing 7, (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (f) calculating a stimulation index value for each donor, and (g) calculating the number of reactive lymphocyte donors whose donor stimulation index value is greater than or equal to a reference stimulation index value and the number of non-reactive lymphocyte donors whose donor stimulation index value is less than a reference stimulation index value. In a particular embodiment, if the number of reactive donors exceeds 30% of the total number of donors, the composition is more likely to induce the production of antibodies specific to the composition, and if the number of reactive donors is less than 20% of the total number of donors, the composition is less likely to induce the production of antibodies specific to the composition. In certain embodiments, the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes from individual donors determined in (d) by the percentage of CD4+ lymphocytes from individual donors determined in (e). In certain embodiments, the stimulation index value is determined by outlier sum analysis or by linear regression. In certain embodiments, the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes from individual donors determined in (d) by the percentage of CD4+ lymphocytes from individual donors determined in (e). In certain embodiments, the APC is obtained from about 20 to about 50 donors. In certain embodiments, the APC is obtained from about 35 to about 45 donors.In a particular embodiment, the APC is obtained from at least about 20 donors, at least about 25 donors, at least about 30 donors, at least about 35 donors, at least about 40 donors, or at least about 45 donors.

[0014] This disclosure provides a method for determining the tendency of a neoantigen to induce a neoantigen-specific immune response compared to a reference antigen. In certain embodiments, the method includes (a) culturing APCs in the presence of a neoantigen to produce stimulated APCs; (b) culturing APCs in the absence of a neoantigen to produce unstimulated APCs; (c) culturing stimulated APCs separately with CD4+ lymphocytes and unstimulated APCs separately with CD4+ lymphocytes; (d) determining the percentage of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; (e) determining the percentage of CD4+ lymphocytes cultured with unstimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; and (f) calculating a stimulation index value. In certain embodiments, if the stimulation index value in (f) is greater than or equal to the reference stimulation index value, the neoantigen is more likely to induce a neoantigen-specific immune response, and if the stimulation index value in (f) is less than the reference stimulation index value, the neoantigen is less likely to induce a neoantigen-specific immune response. In certain embodiments, the neoantigen exists in complex with an MHC class II molecule. In certain embodiments, the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes determined in (d) by the percentage of CD4+ lymphocytes determined in (e). In certain embodiments, the stimulation index value is determined by outlier sum analysis or by linear regression. In certain embodiments, APCs are obtained from approximately 20 to 50 donors. In certain embodiments, APCs are obtained from approximately 35 to 45 donors. In a particular embodiment, the APC is obtained from at least about 20 donors, at least about 25 donors, at least about 30 donors, at least about 35 donors, at least about 40 donors, or at least about 45 donors.

[0015] In certain embodiments, the reference stimulus index value is approximately 1.0 to approximately 4.0, approximately 1.0 to approximately 3.0, or approximately 1.8 to approximately 3.0. In certain embodiments, the reference stimulus index value is approximately 1.6 or greater, approximately 1.7 or greater, approximately 1.8 or greater, approximately 1.9 or greater, approximately 2.0 or greater, approximately 2.1 or greater, approximately 2.2 or greater, approximately 2.3 or greater, approximately 2.4 or greater, approximately 2.5 or greater, approximately 2.6 or greater, approximately 2.7 or greater, approximately 2.8 or greater, approximately 2.9 or greater, or approximately 3.0 or greater.

[0016] In certain embodiments, CD4+ lymphocytes include CD8-T cells. In certain embodiments, at least 10% of CD4+ lymphocytes include CD8-T cells.

[0017] In certain embodiments, the composition comprises a peptide, polypeptide, or small molecule compound. In certain embodiments, the peptide or polypeptide comprises a neoantigen. In certain embodiments, the polypeptide is an antibody or a fragment thereof. In certain embodiments, the antibody is a human, humanized, or chimeric antibody. In certain embodiments, the composition is an antibody-drug conjugate (ADC).

[0018] In one particular embodiment, approximately 1 × 10 5 ~Approx. 1×10 7 APCs are cultured with the composition and / or neoantigen. In certain embodiments, APCs are cultured with the composition and / or neoantigen at concentrations ranging from approximately 10 μg / µl to approximately 1,000 μg / ml. In certain embodiments, APCs are cultured with the composition and / or neoantigen for a period of approximately 48 hours or less.

[0019] In certain embodiments, determining the percentage of CD4+ lymphocytes expressing (i) CD134, (ii) CD137, or (iii) both CD134 and CD137 is performed by flow cytometry.

[0020] This disclosure further provides a kit for carrying out any one of the methods disclosed herein. [Brief explanation of the drawing]

[0021] [Figure 1] A schematic diagram of a non-limiting embodiment of a method for determining the tendency of compositions to induce ADA production is shown. [Figure 2] Displaying FACS analyses of two different antibodies, Avastin® and vococizumab. [Figure 3] This report displays analyses of six antibodies with different clinical ADA rates: Avastin®, GNE-αPCSK9 (also known as RG7652), alirocumab (PRALUENT®), evolocumab (REPATHA®), vococizumab, and HA33. [Figure 4A] This displays the number of donors expressing CD134 for Avastin (registered trademark), HA33, and KLH. [Figure 4B] This displays the number of donors expressing CD137 for Avastin (registered trademark), HA33, and KLH. [Figure 4C] This shows the number of donors expressing CD134 and CD137 for Avastin (registered trademark), HA33, and KLH. [Figure 4D] This displays the number of donors expressing CD134 and / or CD137 for Avastin®, HA33, and KLH. [Figure 5] This invention demonstrates a correlation between the predicted immunogenicity determined by the assay disclosed herein and the immunogenicity observed in the clinic. [Figure 6A] A schematic diagram showing the blockade of HLA-DR and HLA-II antibodies is displayed. [Figure 6B] This displays the number of positive donors when HLA-DR and HLA-II are blocked. [Figure 7] This indicates that there is no correlation between in vitro IL-2 secretion and clinical immunogenicity. [Figure 8] This indicates that there is no correlation between cytokine secretion in vitro and clinical immunogenicity. [Figure 9] A schematic diagram of a non-limiting embodiment of the method of the present disclosure for determining the tendency of a composition to induce ADA production is shown, in which isolated APCs are first cultured with the composition, and these APCs are subsequently co-cultured with T cells, and the tendency of the composition to induce ADA production is determined using T cell activation. [Figure 10] A schematic diagram of a non-limiting rapid embodiment of the method of the present disclosure for determining the tendency of a composition to induce ADA production is shown, in which APCs are first cultured with the composition and subsequently co-cultured with T cells, and the tendency of the composition to induce ADA production is determined using T cell activation. [Figure 11A] This shows the analysis of four bispecific antibodies that possess antigen-binding domains specific to T cells. The stimulation index (SI) value line indicates a value greater than 1.8. [Figure 11B] This shows the analysis of four bispecific antibodies that possess antigen-binding domains specific to T cells. The SI value line indicates a value greater than 3. [Figure 12A] This shows the analysis of two bispecific antibodies that have antigen-binding domains specific to T cells. The SI value line indicates a value greater than 1.8. [Figure 12B] This shows the analysis of two bispecific antibodies that have antigen-binding domains specific to T cells. The SI value line indicates a value greater than 3. [Figure 13A] To demonstrate that the proposed immunogenicity is due to therapeutic-specific activation of T cells, a schematic diagram of a T cell activation assay, including HLA blockade, is shown. [Figure 13B] Figure 13A shows the analysis of the bispecific antibody TDB2 using the assay depicted. [Figure 14] This section displays the analysis of bispecific antibodies (TDB4A) produced by the expression of their two antigen-binding domains in a single cell, or bispecific antibodies (TDB4B) produced by a two-cell system in which each cell expresses one of the two antigen-binding domains of the bispecific antibody. [Modes for carrying out the invention]

[0022] For clarity, but without limitation, a detailed description of the subject matter disclosed in this invention is divided into the following sections: I. Definition; II. Method; III. Composition; IV. Kits; and V. Exemplary embodiments.

[0023] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field to which this invention pertains. The following references provide general definitions of many terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, the following terms have the meanings defined below, unless otherwise specified.

[0024] As used herein, the use of the words “a” or “an” may mean “one” when used in combination with the term “comprising” in the claims and / or specification, but also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.” Furthermore, “having,” “including,” “containing,” and “comprising” are interchangeable, and those skilled in the art will recognize that these terms are open-ended terms.

[0025] As used herein, the terms “about” or “approximately” may mean that a particular value, as determined by those skilled in the art, is within an acceptable margin of error, but depends in part on how the value is measured or determined, for example, on the limitations of the measuring system. For example, “about” may mean a standard deviation of one or more than one, according to convention for a given value. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” may mean an acceptable margin of error for that particular value, such as ±10% of the value modified by the term “about.”

[0026] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0027] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0028] An antibody that "binds" to a target antigen is one that binds to the antigen with sufficient affinity so that it is useful as an assay reagent, for example, as a detection antibody. Typically, such antibodies do not significantly cross-react with other polypeptides. With regard to the binding of polypeptides to target molecules, the terms "specific binding," "specifically binding to," or "specific to" a particular polypeptide or epitope on a particular polypeptide target mean binding that is clearly different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of the target molecule by comparing it to the binding of a control molecule, which is a molecule of a similar structure that generally does not possess binding activity.

[0029] "Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). d ) can be expressed as follows. Affinity can be measured by common methods known in the art, including those described herein. Specific concrete and exemplary embodiments for measuring binding affinity are described below.

[0030] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a particular source or species, and the remainder of the heavy chain and / or light chain originates from a different source or species.

[0031] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0032] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with cell function and / or causes cell death or cell destruction. Cytotoxic agents include radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes such as nuclease and fragments thereof; antibiotics; low molecular weight toxins, or toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed hereinafter, but are not limited thereto.

[0033] As used herein, "detection antibody" refers to an antibody that specifically binds to a target molecule in a sample. Under certain conditions, the detection antibody forms a complex with the target molecule. The detection antibody is detectable either directly through a detectable label or indirectly through the use of another antibody that is labeled and binds to the detection antibody, for example. For direct labeling, the detection antibody is typically conjugated to a portion detectable by some means, for example, a fluorophore, but not limited to these.

[0034] The term “detection” is used herein to include both qualitative and quantitative measurements of a target molecule or its processed form. In certain embodiments, detection includes identifying the mere presence of a target molecule, as well as determining whether the target molecule is present at a detectable level.

[0035] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0036] The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the native sequence Fc region and the mutant Fc region. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0037] The term "framework" or "FR" refers to variable domain residues other than those in the hypervariable region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0038] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a naturally occurring antibody, or antibodies having a heavy chain containing an Fc region as defined herein.

[0039] A "human antibody" is one that is produced by a human or human cell, or has an amino acid sequence equivalent to the amino acid sequence of an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody-coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0040] The "Human Consensus Framework" is a framework representing the most commonly present amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences derives from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Vols. 1-3. In certain embodiments, for VL, the subgroup is subgroup Kappa I, as described by Kabat et al. In certain embodiments, for VH, the subgroup is subgroup III, as described by Kabat et al.

[0041] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human CDR and amino acid residues derived from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR (e.g., CDR) corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0042] The terms “hypervariable region” or “CDR,” as used herein, refer to each region of an antibody variable domain whose sequence is hypervariable (also referred herein as “complementarity-determining region” or “CDR”), and / or which forms a structurally defined loop (“hypervariable loop”), and / or which contains an antigen contact residue (“antigen contacts”). Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) within a variable domain are numbered herein according to Kabat et al. Generally, an antibody contains six CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary CDRs herein include: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact sites located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c) including CDR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

[0043] "Immune conjugates" refer to antibodies conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0044] As used herein, “individual,” “subject,” or “donor” refers to a human or a non-human animal, such as a vertebrate, including mammals. Mammals include, but are not limited to, humans, non-human primates, livestock, sports animals, rodents, and pets. Non-exclusive examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, and non-human primates such as apes and monkeys. In certain embodiments, the individual, subject, or donor is human.

[0045] As used herein, the term "in vitro" refers to an artificial environment and the processes or reactions that occur within it. Exemplary in vitro environments include, but are not limited to, test tubes and cell cultures.

[0046] As used herein, the term “in vivo” refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur within that natural environment, such as embryonic development, cell differentiation, and neural tube formation.

[0047] "Isolated" antibodies are those separated from components of their natural environment. In certain embodiments, antibodies are purified to a purity of over 95% or 99% when measured, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange HPLC or reverse-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79~87 (2007).

[0048] As used herein, the terms “label” or “detectable label” refer to any chemical group or moiety that can bind to a substance to be detected or quantified, such as an antibody. A label is a detectable label suitable for high-sensitivity detection or quantification of a substance. Non-limiting examples of detectable labels include, but are not limited to, luminescent labels, such as fluorescent, phosphorescent, chemiluminescent, bioluminescent, and electrochemiluminescent labels, radioactive labels, enzymes, particles, magnetic materials, and electroactive species. Alternatively, a detectable label may signal its presence by participating in a specific binding reaction. Non-limiting examples of such labels include haptens, antibodies, biotin, streptavidin, his tags, nitrilotriacetate, glutathione S-transferase, and glutathione.

[0049] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, excluding, for example, naturally occurring mutations or mutant antibodies that may arise during the production of a monoclonal antibody preparation (such mutants are generally present in trace amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the subject matter disclosed herein can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0050] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ).

[0051] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a sequence of bases, thereby representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain natural or non-natural nucleotides. Examples of non-natural nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. The nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for the direct expression of antibodies of this disclosure in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, by chemically modifying mRNA to enhance the stability of the RNA vector and / or the expression of the encoded molecule, mRNA can be injected into a target to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, doi:10.1038 / nm.4356, published online on June 12, 2017, or European Patent No. 2101823B1).

[0052] When used herein, a “purified” polypeptide (e.g., an antibody) refers to a polypeptide that has been purified to a purer form than it would be in its natural environment, and / or to a higher purity than it would be if it were first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0053] When used herein, the term “packaging instructions” refers to the instructions that are conventionally included with commercially available packaging, which contain information about the use of the components of the packaging.

[0054] The "amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after performing sequence alignment and, if necessary, introducing gaps to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the skill of the art, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for performing sequence alignment, including any algorithm necessary to achieve the maximum possible alignment for the full length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, was filed with the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program needs to be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0055] In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity % between a given amino acid sequence A and a given amino acid sequence B, between a given amino acid sequence A and a given amino acid sequence B, or between a given amino acid sequence A and a given amino acid sequence B (or, to put it another way, a given amino acid sequence A that has or contains a certain amino acid sequence identity % between a given amino acid sequence B, between a given amino acid sequence B and a given amino acid sequence B, or between a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y In the formula, X is the number of amino acid residues scored as a perfect match in the alignment of programs A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A. Unless otherwise explicitly stated, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0056] The terms “polypeptide” and “protein,” as used interchangeably herein, refer to polymers of amino acids of any length. These polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or conjugation with labeling components. Furthermore, polypeptides containing, for example, one or more analogues of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art, are also within the scope of this definition. The terms “polypeptide” and “protein,” as used herein, specifically encompass antibodies.

[0057] As used herein, the term “recombinant protein” generally refers to genetically modified peptides and proteins. In certain embodiments, such recombinant proteins are “heterogeneous,” i.e., exogenous to the cells in which they are utilized.

[0058] As used herein, “sample” refers to a small portion of a large quantity of material. In certain embodiments, samples include, but are not limited to, cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids (e.g., blood, plasma, serum, feces, urine, lymph, ascites, duct lavage fluid, saliva, and cerebrospinal fluid) and tissue samples. Sources of samples may include solid tissues (e.g., from fresh, frozen, and / or preserved organs, tissue samples, biopsies, or aspirates), blood or any blood component, bodily fluids (e.g., urine, lymph, cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid), or cells of an individual, including circulating cells.

[0059] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (CDRs). (e.g., Kindt et al., Kuby Immunology, 6) th See ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using the VH or VL domain from the antigen-binding antibody, and libraries of complementary VL or VH domains may be screened, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0060] II. Method The subject matter of this disclosure provides a method for determining the tendency of a therapeutic agent, such as a composition comprising a polypeptide or a fragment thereof, to induce the production of anti-drug antibodies (ADAs). In certain embodiments, the method of this disclosure can be used to determine the tendency of a composition comprising an antibody or a fragment thereof or an antibody-drug conjugate (ADC) to induce the production of ADAs. This disclosure also provides a kit for carrying out the method disclosed herein.

[0061] In certain embodiments, the methods of the present disclosure can be used to identify polypeptide variants, e.g., antibody variants, that have a reduced tendency to induce ADA production compared to a parent polypeptide, e.g., a parent antibody. In certain embodiments, the methods disclosed herein can be used to analyze newly developed polypeptides, e.g., antibodies. For example, but not limited to, the methods disclosed herein can be used to identify polypeptides, e.g., antibodies, that have a low tendency to induce ADA from a large repertoire of polypeptides that specifically bind to the same antigen. In certain embodiments, the methods of the present disclosure can be used to determine the potential immunogenicity of newly developed polypeptides, e.g., antibodies, before clinical trials. In certain embodiments, the methods disclosed herein can be used to determine the potential immunogenicity of polypeptides, e.g., antibody aggregates. In certain embodiments, the methods disclosed herein can be used to analyze the immunogenicity of antibody sequence variants, e.g., those that occur during antibody production and / or manufacturing. In certain embodiments, the methods disclosed herein can be used to analyze the immunogenicity of neoantigens. In certain embodiments, the methods disclosed herein can be used to analyze the immunogenicity of peptides.

[0062] In certain embodiments, the method of the present disclosure may include culturing lymphocytes in the presence of the composition to produce stimulated lymphocytes. In certain embodiments, the method may further include culturing lymphocytes in the absence of the composition to produce unstimulated lymphocytes. For example, but not limited to, lymphocytes may be cultured in the presence of the composition for, for example, about 24 to about 72 hours. In certain embodiments, lymphocytes may be cultured in the presence of polypeptide for about 12 to about 72 hours, about 12 to about 60 hours, about 12 to about 48 hours, about 12 to about 24 hours, about 24 to about 72 hours, about 24 to about 60 hours, about 24 to about 48 hours, about 48 to about 72 hours, or about 48 to about 60 hours. In certain embodiments, lymphocytes may be cultured in the presence of the composition for about 48 hours or less.

[0063] The concentration of lymphocytes used in the methods disclosed in this invention may depend on the size of the culture dish and / or plate used. For example, but not limited to, lymphocytes may be present in a concentration of approximately 1 × 10⁶ in a 24-well plate and / or a 96-well plate. 5 ~Approx. 1×10 7 Cells / ml, e.g., approximately 2 × 10⁻⁶ 6 It can be used at a concentration of cells / ml. In a particular embodiment, the number of lymphocytes used is approximately 1 × 10⁶ 5 ~Approx. 9×10 6 cells, approximately 3 x 10 5 ~Approx. 8×10 6 cells, approximately 3 x 10 5 ~Approx. 7×10 6 cells, approximately 4 x 10 5 ~about 6×10 6 cells, approximately 5 x 10 5 ~Approx. 5×10 6 cells, approximately 6 x 10 5 ~Approx. 4×10 6 cells, approximately 7 x 10 5 ~Approx. 3×10 6 cells, approximately 8 x 10 5 ~about 2×10 6 cells, approximately 9 x 10 5 ~about 2×10 6 Cells, or approximately 9 x 105 ~Approx. 1×10 6 It can be a cell. In a particular embodiment, about 1 × 10 6 Lymphocytes are used. In a particular embodiment, the number of lymphocytes used is approximately 1 × 10⁶ 5 cells ~ approx. 3 x 10 5 It could be a cell, for example, about 2 × 10⁻⁶ 5 Lymphocytes are used. In a particular embodiment, the lymphocytes are approximately 0.1 × 10⁻⁶ 6 cells / ml ~ approx. 1×10 6 cells / ml, e.g., approximately 0.2 × 10⁻⁶ 6 cells / ml ~ approx. 0.4×10 6 It can be used at a concentration of cells / ml.

[0064] Lymphocytes for use in the methods disclosed herein include any cells capable of interacting with antibodies that complex with the major histocompatibility complex (MHC) on the cell surface. For example, but not limited to, lymphocytes may include T cells. For example, but not limited to, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of lymphocytes are T cells. In certain embodiments, at least about 20%, for example, at least about 30%, of lymphocytes are T cells, for example, CD4+ T cells. In certain embodiments, the lymphocytes may further include antigen-presenting cells (APCs).

[0065] In certain embodiments, T cells are CD8-. In certain embodiments, T cells are CD4+. In certain embodiments, T cells are CD4+CD8-. For example, but not limited to, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of T cells are CD8-, CD4+, or CD4+CD8-. In certain embodiments, at least about 20%, for example, at least about 30%, of T cells are CD8-, CD4+, or CD4+CD8-.

[0066] A non-limited source of lymphocytes is peripheral blood mononuclear cells (PBMCs) isolated from a donor. In certain embodiments, PBMCs are isolated from a donor sample, for example, from a donor blood sample. PBMCs can be isolated from a donor sample by any method known in the art, for example, by density gradient centrifugation. In certain embodiments, PBMCs are first isolated from a donor sample and then subjected to CD8-negative selection to isolate and / or select CD8- cells, for example, CD8-T cells. In certain embodiments, PBMCs may be PBMC cell lines. In certain embodiments, lymphocytes may include T cells, for example, CD8-T cells, CD4+ T cells, or CD4+CD8-T cells. In certain embodiments, lymphocytes can be differentiated from stem cells or iPSC cells. For example, but not limited to, lymphocytes may be T cells, e.g., CD8-T cells, CD4+ T cells, or CD4+CD8-T cells, differentiated from stem cells or iPSC cells. In certain embodiments, but not limited to, APCs, including dendritic cells, macrophages, and B cells, can be isolated from PBMCs and used in a manner comprising culturing the APCs in the presence of the composition of interest and / or T cells, as discussed below. Alternatively and / or additionally, lymphocytes obtained from PBMCs may include T cells, e.g., CD8-T cells, CD4+ T cells, or CD4+CD8-T cells, and APCs for use in the manner disclosed herein.

[0067] In certain embodiments, the method of the present disclosure may include isolating CD14+ cells from PBMCs prior to exposure to the composition. For example, but not limited to, the isolated CD14+ cells can be induced to differentiate into APCs, e.g., dendritic cells, e.g., immature dendritic cells, by exposure to GM-CSF and / or IL-4, and then cultured in the presence of the composition to produce stimulated APCs, e.g., stimulated mature dendritic cells. In certain embodiments, the APCs, e.g., immature dendritic cells, can be cultured with the composition and in the presence of GM-CSF, IL4, TNF-α, IL-1β, IL6 and / or PGE2 to produce stimulated APCs, e.g., stimulated mature dendritic cells. The stimulated APCs, e.g., monocyte-derived dendritic cells, can then be cultured with T cells, e.g., CD4+ T cells and / or CD4+CD8- T cells, to assay T cell activation. In certain embodiments, T cells can be isolated from PBMCs as discussed below. In certain embodiments, APCs and T cells can be isolated from the same PBMC sample or population. In certain embodiments, APCs and T cells can be isolated from the same donor. In certain embodiments, the possibility of assay interference due to direct activation of T cells by the composition can be avoided by culturing APCs with the composition in the absence of T cells, and then culturing the APCs with T cells.

[0068] In certain embodiments, lymphocytes are cultured with compositions ranging from approximately 10 μg / ml to approximately 1,000 μg / ml, and, for example, with a composition of approximately 100 μg / ml. For example, but not limited to, compositions ranging from approximately 30 μg / ml to approximately 1,000 μg / ml, approximately 40 μg / ml to approximately 1,000 μg / ml, approximately 50 μg / ml to approximately 1,000 μg / ml, approximately 60 μg / ml to approximately 1,000 μg / ml, approximately 70 μg / ml to approximately 1,000 μg / ml, approximately 80 μg / ml to approximately 1,000 μg / ml, approximately 90 μg / ml to approximately 1,000 μg / ml, approximately 10 μg / ml to approximately 900 μg / ml, It can be used at concentrations of approximately 10 μg / ml to 800 μg / ml, approximately 10 μg / ml to 700 μg / ml, approximately 10 μg / ml to 600 μg / ml, approximately 10 μg / ml to 500 μg / ml, approximately 10 μg / ml to 400 μg / ml, approximately 10 μg / ml to 300 μg / ml, approximately 10 μg / ml to 200 μg / ml, approximately 50 μg / ml to 150 μg / ml, or approximately 75 μg / ml to 125 μg / ml. In certain embodiments, lymphocytes are cultured with a composition of approximately 100 μg / ml. In certain embodiments, lymphocytes may be T cells, e.g., CD4+ T cells and / or CD4+CD8- T cells, and these are cultured with the composition. In certain embodiments, lymphocytes may include T cells and APCs, and these are cultured with the composition. Alternatively and / or additionally, the method may include culturing APCs, e.g., dendritic cells, macrophages, and / or B cells, in the presence of the composition of interest and lymphocytes, e.g., T cells. In certain embodiments, the method may include culturing dendritic cells in the presence of the composition of interest and lymphocytes, e.g., T cells. In certain embodiments, the method may include culturing isolated APCs, e.g., dendritic cells, macrophages, and / or B cells, in the presence of the composition of interest but in the absence of T cells. In certain embodiments, the method may include culturing isolated dendritic cells in the presence of the composition of interest but in the absence of T cells.

[0069] In certain embodiments, the method may further include determining the percentage of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. In certain embodiments, the method may include determining the percentage of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. In certain embodiments, the method may include determining whether such cells are alive. In certain embodiments, determining the quantity of unstimulated and / or stimulated lymphocytes includes (i) contacting lymphocytes, e.g., T cells, with one or more detection agents that bind to CD4, CD134, and / or CD137, and (ii) determining the number of lymphocytes, e.g., T cells, that are bound to one or more detection agents.

[0070] In certain embodiments, the detection agent for use in the methods disclosed herein is an antibody (also referred to herein as “detection antibody”). In certain embodiments, the detection agent specifically binds to a polypeptide analyzed by the disclosed methods, for example, the detection agent specifically binds to an epitope present on the polypeptide or a fragment thereof. In certain embodiments, the detection agent is an antibody that binds to CD4. In certain embodiments, the detection agent is an antibody that binds to CD134. In certain embodiments, the detection agent is an antibody that binds to CD137. In certain embodiments, the detection agent used in the assay methods disclosed herein can be used at concentrations of about 0.05 μg / ml to about 5 μg / ml, for example, about 1 μg / ml.

[0071] In certain embodiments, a detection agent for use in the disclosed method, such as a detection antibody, may be labeled. Labels include, but are not limited to, directly detectable labels or moieties such as fluorescent labels, chromophore labels, electron density labels, chemiluminescent labels, and radioactive labels, as well as moieties such as enzymes or ligands that are indirectly detected, for example, through enzymatic reactions or molecular interactions. Non-limiting examples of labels include radioisotopes.32 P, 14 C, 125 I, 3 H and 131 Examples include fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (see U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated with enzymes that utilize hydrogen peroxide to oxidize pigment precursors such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin-labeled, bacteriophage-labeled, and stable free radicals. In certain embodiments, the detection agent, for example, an antibody, is labeled with a fluorophore.

[0072] In certain embodiments, determining the number of cells, such as lymphocytes, labeled with one or more detection agents is carried out by any method capable of detecting the detection agents. In certain embodiments, the detection agents can be detected by monitoring the labeling of the detection agents, such as fluorescent labeling. In certain embodiments, determining the number of lymphocytes labeled with one or more detection agents is carried out by flow cytometry.

[0073] In certain embodiments, the method further includes calculating a stimulation index value. In certain embodiments, the stimulation index value can be determined by dividing the proportion of stimulated lymphocytes by the proportion of unstimulated lymphocytes. Alternatively or additionally, the stimulation index value can be determined by outlier sum analysis or by linear regression. In certain embodiments, the stimulation index value can be determined by dividing the maximum or mean value of stimulated lymphocytes by the maximum or mean value of unstimulated lymphocytes.

[0074] In certain embodiments, the method may include comparing the stimulation index value with a reference stimulation index. In certain embodiments, if the stimulation index value exceeds the reference stimulation index value, the polypeptide is more likely to induce ADA production than the reference product. Alternatively, if the stimulation index value of the polypeptide is less than the reference stimulation index value, the polypeptide is less likely to induce ADA production than the reference product, for example, in a clinical setting.

[0075] In certain embodiments, the reference stimulus index exhibits a known tendency to induce ADA production, for example, in a clinical setting. In certain embodiments, the reference stimulus index is approximately 1.0 to approximately 4.0, for example, approximately 1.0 to approximately 3.0, approximately 1.1 to approximately 2.0, approximately 1.2 to approximately 2.0, approximately 1.3 to approximately 2.0, approximately 1.4 to approximately 2.0, approximately 1.5 to approximately 2.0, approximately 1.6 to approximately 2.0, approximately 1.7 to approximately 2.0, approximately 1.8 to approximately 2.0, or approximately 1.8 to approximately 3.0. In certain embodiments, the reference stimulus index is approximately 1.5 to approximately 2.0. In certain embodiments, the reference stimulus index is approximately 1.6 to approximately 1.8. In certain embodiments, the reference stimulus index value is approximately 1.6 or greater. In certain embodiments, the reference stimulus index value is approximately 1.7 or greater. In certain embodiments, the reference stimulus index value is approximately 1.8 or greater. In certain embodiments, the reference stimulus index value is approximately 1.9 or higher. In certain embodiments, the reference stimulus index value is approximately 2.0 or higher. In certain embodiments, the reference stimulus index value is approximately 2.1 or higher. In certain embodiments, the reference stimulus index value is approximately 2.2 or higher. In certain embodiments, the reference stimulus index value is approximately 2.3 or higher. In certain embodiments, the reference stimulus index value is approximately 2.4 or higher. In certain embodiments, the reference stimulus index value is approximately 2.5 or higher. In certain embodiments, the reference stimulus index value is approximately 2.6 or higher. In certain embodiments, the reference stimulus index value is approximately 2.7 or higher. In certain embodiments, the reference stimulus index value is approximately 2.8 or higher. In certain embodiments, the reference stimulus index value is approximately 2.9 or higher. In certain embodiments, the reference stimulus index value is approximately 3.0 or higher.

[0076] In certain embodiments, the reference stimulation index is a value produced by a composition (e.g., a reference composition) that is less likely to induce ADA production in a clinical setting. For example, the reference composition may be an antibody that has been shown not to induce ADA production in a clinical setting, or that is less likely to induce ADA production in a clinical setting. Alternatively or additionally, the reference stimulation index may be the stimulation index of a composition (e.g., a reference composition) that has been shown to induce ADA production. For example, the reference composition may be an antibody that has been shown to induce ADA production in a clinical setting. In certain embodiments, the reference composition may be an antibody disclosed in any one of the figures and / or examples. Alternatively or additionally, with respect to an antibody variant, the reference stimulation index may be the stimulation index of the parent antibody. In certain embodiments, with respect to a bispecific antibody, the reference stimulation index may be the stimulation index of one of the parent antibodies.

[0077] In certain embodiments, the method of the present disclosure may include (i) culturing lymphocytes in the presence of the composition to generate stimulated lymphocytes; (ii) culturing lymphocytes in the absence of the composition to generate unstimulated lymphocytes; (iii) determining the proportion of stimulated lymphocytes that are CD4+ and express (a) CD134, (b) CD137, or (c) CD134 and CD137; (iv) determining the proportion of unstimulated lymphocytes that are CD4+ and express (a) CD134, (b) CD137, or (c) CD134 and CD137; and (v) calculating a stimulation index value. In certain embodiments, if the stimulation index value in (v) is greater than or equal to a reference stimulation index value, the composition is more likely to induce antibodies specific to the composition. In certain embodiments, if the stimulation index value in (v) is less than a reference stimulation index value, the composition is less likely to induce antibodies specific to the composition.

[0078] In certain embodiments, the method of the present disclosure may include culturing APCs, e.g., CD14+ APCs, in the presence of the composition and in the absence of CD4+ lymphocytes, e.g., T cells. In certain embodiments, the APCs are isolated from PBMA prior to culturing such APCs with the composition. In certain embodiments, the method may also include culturing APCs, e.g., isolated APCs, in the absence of the composition and in the absence of CD4+ lymphocytes, e.g., T cells. In certain embodiments, the method may further include co-culturing CD4+ lymphocytes, e.g., T cells, with APCs previously cultured in the presence of the composition to produce stimulated CD4+ lymphocytes, e.g., stimulated T cells. In certain embodiments, the method may further include co-culturing CD4+ lymphocytes, e.g., T cells, with APCs previously cultured in the absence of the composition to produce unstimulated CD4+ lymphocytes, e.g., unstimulated T cells. In certain embodiments, CD4+ lymphocytes, e.g., CD4+ T cells and / or CD4+CD8- T cells, are isolated from PBMCs. In certain embodiments, T cells and APCs are isolated from the same PBMC population. In certain embodiments, the T cells, e.g., CD4+ T cells, and APCs, e.g., CD14+ APCs, are autologous. In certain embodiments, the APCs are dendritic cells.

[0079] In certain embodiments, the method of the present disclosure may include: (i) culturing APCs in the presence of a composition to produce APCs that display the antigen of the composition; (ii) culturing APCs in the absence of a composition to produce APCs that do not display the antigen of the composition; (iii) co-culturing the APCs from (i) with CD4+ lymphocytes; (iv) co-culturing the APCs from (ii) with CD4+ lymphocytes; (v) determining the proportion of CD4+ lymphocytes from the co-culture from (iii) that are CD4+ and express (a) CD134, (b) CD137, or (c) CD134 and CD137; (vi) determining the proportion of T cells from the co-culture from (iv) that are CD4+ and express (a) CD134, (b) CD137, or (c) CD134 and CD137; and (vii) calculating a stimulation index value. In certain embodiments, the method may further include comparing the stimulation index value in (vii) with a reference stimulation index value. In certain embodiments, if the stimulation index value in (vii) is greater than or equal to the reference stimulation index value, the composition is more likely to induce antibodies specific to the composition. In certain embodiments, if the stimulation index value in (vii) is less than the reference stimulation index value, the composition is less likely to induce antibodies specific to the composition.

[0080] In certain embodiments, the method may include analyzing a composition using lymphocytes obtained from two or more donors. In certain embodiments, the method of the disclosure may include analyzing the tendency of a composition to stimulate ADA production by (i) culturing lymphocytes derived from individual donors separately with a target composition to generate stimulated lymphocytes, and (ii) culturing lymphocytes derived from individual donors separately in the absence of the composition to generate unstimulated lymphocytes.

[0081] For example, but not limited to, lymphocytes (e.g., PBMCs, APCs, and / or T cells) used in conjunction with the methods of this disclosure may originate from at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 individual donors. In certain embodiments, such lymphocytes, e.g., PBMCs or APCs, may be cultured separately from the composition of interest. In certain embodiments, lymphocytes derived from about 20 to about 50 donors may be used and, for example, can be cultured separately from the composition of interest. In certain embodiments, lymphocytes derived from about 35 to about 45 donors may be used separately and, for example, can be cultured with the composition of interest. In certain embodiments, lymphocytes derived from individual donors may be T cells, e.g., CD4+ T cells and / or CD4+CD8- T cells, which are cultured together with the composition. In certain embodiments, lymphocytes derived from individual donors may include T cells and APCs, which are cultured together with the composition. Alternatively and / or additionally, the method may include culturing APCs, e.g., dendritic cells, macrophages and / or B cells, in the presence of the composition of interest and lymphocytes derived from individual donors, e.g., T cells.

[0082] In certain embodiments, the method may further include (a) determining the proportion of stimulated lymphocytes from individual donors that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137, and (b) determining the proportion of unstimulated lymphocytes from donors that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137.

[0083] In certain embodiments, the method may further include (a) determining the proportion of CD4+ lymphocytes cultured with stimulated APCs expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137, and (b) determining the proportion of CD4+ lymphocytes cultured with unstimulated APCs expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137.

[0084] In a particular embodiment, the stimulation index value for each individual donor can be determined, for example, by dividing the proportion of stimulated lymphocytes in that individual donor by the proportion of unstimulated lymphocytes in that individual donor.

[0085] In a particular embodiment, the stimulation index value for each individual donor can be determined, for example, by dividing the proportion of CD4+ cells cultured with a stimulated APC expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 for that individual donor by the proportion of CD4+ lymphocytes cultured with an unstimulated APC expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 for that individual donor.

[0086] In certain embodiments, the method also includes calculating the number of reactive lymphocyte donors whose donor stimulation index value is equal to or greater than a reference stimulation index value, and the number of non-reactive lymphocyte donors whose donor stimulation index value is less than a reference stimulation index value. In certain embodiments, if the number of reactive donors exceeds 30% of the total number of donors, the composition is more likely to induce antibody production. Alternatively, if the number of reactive donors is less than 20% of the total number of donors, the composition is less likely to induce antibody production.

[0087] This disclosure further provides a method for determining the tendency of a neoantigen to induce an immune response to that neoantigen. In certain embodiments, the method includes (a) culturing lymphocytes in the presence of a neoantigen to generate stimulated lymphocytes; (b) culturing lymphocytes in the absence of a neoantigen to generate unstimulated lymphocytes; (c) determining the proportion of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; (d) determining the proportion of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; and (e) calculating a stimulation index value (for example, by dividing the proportion of stimulated lymphocytes determined in (c) by the proportion of unstimulated lymphocytes determined in (d)). In certain embodiments, if the stimulation index value in (e) is greater than or equal to the reference stimulation index value, the neoantigen is more likely to induce a neoantigen-specific immune response, and if the stimulation index value in (e) is less than the reference stimulation index value, the neoantigen is less likely to induce a neoantigen-specific immune response. In certain embodiments, the neoantigen exists in complex with an MHC molecule, for example, an MHC class II molecule. For example, but not limited to, the neoantigen can form a complex with an MHC class II molecule.

[0088] This disclosure further provides a method for determining the tendency of a neoantigen to induce an immune response to that neoantigen. In a particular embodiment, the method includes (a) culturing APCs in the presence of a neoantigen to produce stimulated APCs; (b) culturing APCs in the absence of a neoantigen to produce unstimulated APCs; (c) culturing stimulated APCs separately with CD4+ lymphocytes and unstimulated APCs separately with CD4+ lymphocytes; (d) determining the proportion of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; (e) determining the proportion of CD4+ lymphocytes cultured with unstimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137; and (f) calculating a stimulation index value (for example, by dividing the proportion of stimulated lymphocytes determined in (d) by the proportion of unstimulated lymphocytes determined in (e)). In certain embodiments, if the stimulation index value in (f) is greater than or equal to the reference stimulation index value, the neoantigen is more likely to induce a neoantigen-specific immune response, and if the stimulation index value in (f) is less than the reference stimulation index value, the neoantigen is less likely to induce a neoantigen-specific immune response. In certain embodiments, the neoantigen exists in complex with an MHC molecule, for example, an MHC class II molecule. For example, but not limited to, the neoantigen can form a complex with an MHC class II molecule.

[0089] III. Composition This disclosure provides a method for determining the tendency of compositions to induce ADA production. Non-limiting examples of such compositions that can be analyzed by the disclosed method are provided below. For example, but not limited to, compositions assayed using any of the methods disclosed herein may include polypeptides or polypeptide fragments, e.g., peptides. In certain embodiments, the composition may include antibodies or fragments thereof, e.g., human, humanized, or chimeric antibodies. In certain embodiments, the composition may include antibody-drug conjugates (ADCs). In certain embodiments, the antibody may be a single-domain antibody. In certain embodiments, the composition may include neoantigens or neoantigen-containing complexes. In certain embodiments, the composition is an antibody that is specific to a neoantigen.

[0090] 1. Polypeptides and peptides In certain embodiments, the composition analyzed by the method disclosed herein may include peptides or proteins or fragments thereof.

[0091] In certain embodiments, the composition may be a protein or a fragment thereof. In certain embodiments, the protein may have a molecular weight of at least about 15–100 kD, for example, closer to about 15 kD. In certain embodiments, the protein may contain at least about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500 amino acids, about 1,000 amino acids, about 1,500 amino acids, about 2,000 amino acids, about 2,500 amino acids, about 3,000 amino acids, about 35,000 amino acids, or about 40,000 amino acids. Non-limiting examples of proteins include all proteins and generally include proteins containing one or more disulfide bonds, including multi-chain polypeptides containing one or more interchain and / or intrachain disulfide bonds. In certain embodiments, the protein, for example, an antibody, may contain other post-translational modifications, including but not limited to glycosylation and lipidization. For example, see Prabakaran et al., WIREs Syst Biol Med (2012), which is incorporated herein by reference in its entirety.

[0092] In certain embodiments, the composition may be a peptide. In certain embodiments, the peptide may consist of about 3 to 50 amino acid residues. In certain embodiments, the 3 to 50 amino acid residues may be consecutive within a larger polypeptide or protein, or they may be discontinuous within the primary sequence of a larger polypeptide or protein but spatially close in three-dimensional space. In certain embodiments, the peptide may be part of a peptide, part of a complete protein or polypeptide, and may be released from that protein or polypeptide by proteolytic treatment, or may remain part of the protein or polypeptide.

[0093] In a particular embodiment, the peptide may have a length of 3 or more residues, 4 or more residues, 5 or more residues, 6 or more residues, 7 or more residues, 8 or more residues, 9 or more residues, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, 14 or more residues, 15 or more residues, 16 or more residues, 17 or more residues, 18 or more residues, 19 or more residues, 20 or more residues, 21 or more residues, 22 or more residues, 23 or more residues, 24 or more residues, 25 or more residues, 26 or more residues, 27 or more residues, 28 or more residues, 29 or more residues, 30 or more residues, 31 or more residues, 32 or more residues, 33 or more residues, 34 or more residues, 35 or more residues, 36 or more residues, 37 or more residues, 38 or more residues, 39 or more residues, 40 or more residues, 41 or more residues, 42 or more residues, 43 or more residues, 44 or more residues, 45 or more residues, 46 or more residues, 47 or more residues, 48 ​​or more residues, 49 or more residues, or 50 or more residues. In certain embodiments, the peptide has a length of 3-50 residues, 5-50 residues, 3-45 residues, 5-45 residues, 3-40 residues, 5-40 residues, 3-35 residues, 5-35 residues, 3-30 residues, 5-30 residues, 3-25 residues, 5-25 residues, 3-20 residues, 5-20 residues, 3-15 residues, 5-15 residues, 3-10 residues, 3-10 residues, 5-10 residues, 10-15 residues, 15-20 residues, 20-25 residues, 25-30 residues, 30-35 residues, 35-40 residues, 40-45 residues, or 45-50 residues. In certain embodiments, the peptide has a length of about 5-30 residues.

[0094] In certain embodiments, the peptide has a length of 9 residues. In certain embodiments, the peptide has a length of 10 residues. In certain embodiments, the peptide has a length of 11 residues. In certain embodiments, the peptide has a length of 12 residues. In certain embodiments, the peptide has a length of 13 residues. In certain embodiments, the peptide has a length of 14 residues. In certain embodiments, the peptide has a length of 15 residues. In certain embodiments, the peptide has a length of 16 residues. In certain embodiments, the peptide has a length of 17 residues. In certain embodiments, the peptide has a length of 18 residues. In certain embodiments, the peptide has a length of 99 residues. In certain embodiments, the peptide has a length of 20 residues. In certain embodiments, the peptide has a length of 21 residues. In certain embodiments, the peptide has a length of 22 residues. In certain embodiments, the peptide has a length of 23 residues. In certain embodiments, the peptide has a length of 24 residues. In certain embodiments, the peptide has a length of 25 residues. In certain embodiments, the peptide has a length of 26 residues. In certain embodiments, the peptide has a length of 27 residues. In certain embodiments, the peptide has a length of 28 residues. In a particular embodiment, the peptide has a length of 29 residues. In a particular embodiment, the peptide, for example, has a length of 30 residues. In a particular embodiment, the peptide has a length of 31 residues. In a particular embodiment, the peptide has a length of 32 residues. In a particular embodiment, the peptide has a length of 33 residues. In a particular embodiment, the peptide has a length of 34 residues. In a particular embodiment, the peptide has a length of 35 residues. In a particular embodiment, the peptide has a length of 36 residues. In a particular embodiment, the peptide has a length of 37 residues. In a particular embodiment, the peptide has a length of 38 residues. In a particular embodiment, the peptide has a length of 39 residues. In a particular embodiment, the peptide has a length of 40 residues. In a particular embodiment, the peptide has a length of 41 residues. In a particular embodiment, the peptide has a length of 42 residues.In certain embodiments, the peptide has a length of 43 residues. In certain embodiments, the peptide has a length of 44 residues. In certain embodiments, the peptide has a length of 45 residues. In certain embodiments, the peptide has a length of 46 residues. In certain embodiments, the peptide has a length of 47 residues. In certain embodiments, the peptide has a length of 48 residues. In certain embodiments, the peptide has a length of 49 residues. In certain embodiments, the peptide has a length of 50 residues.

[0095] In certain embodiments, the protein or a fragment thereof may be an antibody or an antigen-binding fragment thereof, as disclosed herein.

[0096] In certain embodiments, the peptide may be a neoantigen, as disclosed herein.

[0097] 2. Antibodies or their fragments In certain embodiments, the composition analyzed by the methods disclosed herein includes an antibody or a fragment thereof, for example, a monoclonal antibody or a fragment thereof. For example, but not limited to, the methods disclosed herein can be used to determine the potential immunogenicity of a newly developed and / or identified antibody or a fragment thereof.

[0098] Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments listed below. For a review of a particular antibody fragment, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of the scFv fragment, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), as well as International Publication No. 93 / 16185, and U.S. Patents Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives, see U.S. Patent No. 5,869,046. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as described herein, and production by recombinant host cells (e.g., Escherichia coli (E. coli) or phages).

[0099] In certain embodiments, the composition analyzed by the methods disclosed herein may be a diabody. A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, European Patent No. 404097, International Publication No. 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003) and can be analyzed by the methods disclosed.

[0100] In certain embodiments, the antibody that can be analyzed by the disclosed method may be a single-domain antibody. A single-domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see, for example, U.S. Patent No. 6,248,516B1; Domantis, Inc., Waltham, MA). Additional non-limiting examples of single-domain antibodies are disclosed in Iezzi et al., Front Immunol. 9:273 (2018), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the antibody is a hybridoid (Hybrigenics Services, Cambridge, MA).

[0101] 3. Chimeras, humanization, and human antibodies In certain embodiments, the composition analyzed by the methods disclosed herein includes a chimeric antibody, for example, a humanized antibody. For example, but not limited to, the methods disclosed herein can be used to identify, for example, a chimeric variant of an antibody that is less likely to induce ADA production compared to the parent antibody or other chimeric variants of the antibody. Alternatively or additionally, the methods disclosed herein can be used to identify a chimeric antibody that is less likely to induce ADA production.

[0102] Certain chimeric antibodies are described in the art, for example, U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In certain embodiments, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody may be a “class-switched” antibody in which its class or subclass is changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0103] In certain embodiments, a chimeric antibody may be a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, e.g., a CDR, where the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody also optionally contains at least a portion of the human constant region. In certain embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which its CDR residues are derived) to repair or improve the specificity or affinity of the antibody, for example.

[0104] In certain embodiments, the composition analyzed by the methods disclosed herein may be a human antibody. For example, but not limited to, the methods disclosed herein can be used to identify chimeric variations of antibodies that are less likely to induce the production of human antibodies that are less likely to induce ADA production.

[0105] 4. Antibodies derived from the library In certain embodiments, the compositions analyzed by the methods disclosed herein may include antibodies or fragments thereof isolated by screening a combinatorial library for antibodies having a desired activity(s) or activity(s). For example, but not limited to, the methods disclosed herein can be used to identify, for example, library-derived antibodies that have a desired binding characteristic and / or a lower or lower tendency to induce ADA production compared to other library-derived antibodies that bind to the same antigen.

[0106] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0107] 5. Multispecific antibodies In certain embodiments, the compositions analyzed by the methods disclosed herein may include multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different epitopes. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. For example, but not limited to, the methods disclosed herein can be used to identify multispecific antibodies that have a lower or less likely tendency to induce ADA production compared to other multispecific antibodies that bind to the same epitope. Alternatively or additionally, the methods disclosed herein can be used to identify multispecific antibodies that have a lower tendency to induce ADA production.

[0108] In certain embodiments, the methods disclosed herein can be used, for example, to identify multispecific antibodies, such as bispecific antibodies, that have a lower or lower tendency to induce ADA production compared to other antibodies, such as monospecific or multispecific antibodies that bind to at least one of the same epitopes as multispecific antibodies.

[0109] In certain embodiments, the compositions analyzed by the methods disclosed herein may include bispecific antibodies having binding specificity to T cells. For example, but not limited to, the bispecific antibody may include a first antigen-binding domain that binds to T cells, and a second binding specificity to a second epitope, such as an epitope that is not present on T cells.

[0110] Modified antibodies having three or more functional antigen-binding sites, including the "octopus antibody," can also be analyzed by the disclosed method (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0111] 6. Immunoconjugate In certain embodiments, the compositions analyzed by the methods disclosed herein may include immunoconjugates comprising antibodies conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) or radioisotopes. For example, an antibody or antigen-binding moiety can be functionally linked (e.g., by chemical bonding, gene fusion, non-covalent bonding, etc.) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.

[0112] In certain embodiments, the immunoconjugate is an antibody such as meitansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP0425235); auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patents Nos. 5,635,483, 5,780,588, and 7,498,298); drastatin; calichemycin or its derivatives (see U.S. Patents Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296); Hinman See et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters See 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065, among others, which are antibody-drug conjugates (ADCs) conjugated to one or more drugs.

[0113] In certain embodiments, the immunoconjugate includes antibodies conjugated to enzymatically active toxins or fragments thereof, including but not limited to diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecenes.

[0114] In certain embodiments, the immunoconjugate includes an antibody that conjugates to a radioactive atom to form a radioconjugate. Various radioisotopes can be used to generate the radioconjugate. A non-limiting example is At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Examples include radioactive isotopes of Lu. When radioactive conjugates are used for detection, this can include radioactive atoms for scintigraphy studies, such as TC99M or I123, or spin-labeled iodine-123, iodine-131, as well as indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron for nuclear magnetic resonance (NMR) imaging (also known as MRI).

[0115] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Char et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0116] In certain embodiments, the immunoconjugate may include, but is not limited to, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) conjugates prepared with crosslinking reagents.

[0117] 7. Antibody variants In certain embodiments, the compositions analyzed by the methods disclosed herein may include antibody variants of previously disclosed antibodies. For example, the methods of this disclosure can be used to identify antibodies that are variants of previously disclosed antibodies that are less likely to induce ADA production than the parent antibody. In certain embodiments, amino acid substitutions can be introduced into the antibody of interest, and the antibody variants can be screened for immunogenicity using the methods disclosed.

[0118] In certain embodiments, antibody variants may be amino acid sequence variants of an antibody, prepared, for example, by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, but are not limited to, deletions from and / or insertions into the amino acid sequence of the antibody, and / or substitutions of residues within the amino acid sequence of the antibody. Target sites for such mutations include, but are not limited to, CDRs and FRs. Any combination of deletions, insertions, and substitutions can lead to a final construct, insofar as the final antibody, i.e., the modified antibody, possesses desirable properties, such as antigen binding.

[0119] In certain embodiments, an antibody variant may be an antibody modified to increase or decrease the degree to which the antibody is glycosylated. For example, but not limited to, the addition or deletion of glycosylation sites in an antibody can be achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0120] In certain embodiments, the antibody analyzed by the method disclosed herein is an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0121] In certain embodiments, an antibody variant may be a cysteine-modified antibody, e.g., "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site of the antibody, which can then be used to create an immunoconjugate by conjugating the antibody to a drug moiety or other moiety, such as a linker-drug moiety, as further described herein. In certain embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0122] 8. Neoantigens In certain embodiments, the composition analyzed by the methods disclosed herein may include neoantigens. Neoantigens are antigens not associated with normal tissue or organ and are generally obtained from gene mutations, e.g., insertions / deletions, gene fusions, frameshift mutations, single-nucleotide mutations, or combinations thereof. In certain embodiments, the neoantigen is a tumor neoantigen (also referred to as a tumor-specific antigen or TSA). Because tumor neoantigens are considered "non-self," they are processed and displayed via MHC molecules on antigen-presenting cells (APCs), such as T cells, e.g., CD8 + and / or CD4 + The binding of such tumor neoantigens presented on APCs by T cells is one way in which an immune response against tumors associated with tumor neoantigens is developed. See, for example, Jiang et al., J. of Hematology & Oncology 12(93)(2019) (their contents are incorporated herein by reference).

[0123] In certain embodiments, neoantigens can be analyzed in relation to complexes. For example, but not limited to, neoantigens can exist in complex with MHC molecules, such as MHC class I or II molecules. In certain embodiments, neoantigens can exist in complex with MHC class II molecules.

[0124] Neoantigens for use in this disclosure can be identified by any method known in the art. For example, but not limited to, neoantigens can be identified by next-generation sequencing and / or in silico modeling. See, for example, Garcia-Garijo et al., Front Immunol. 10:1392 (2019), the contents of which are incorporated herein by reference. In certain embodiments, neoantigens identified by such methods can be analyzed by methods disclosed herein to determine the tendency of the neoantigen to induce a neoantigen-specific immune response.

[0125] IV. Kit The subject matter disclosed herein further provides kits containing materials useful for carrying out the methods disclosed herein. In certain embodiments, the kit of this disclosure includes a container containing lymphocytes and / or a container containing one or more agents for detecting markers, such as CD4, CD134, and / or CD137 as described herein. Non-limiting examples of suitable containers include bottles, test tubes, vials, and microtiter plates. Containers can be formed from a variety of materials, such as glass or plastic.

[0126] In certain embodiments, the kit may comprise one or more containers containing one or more lymphocytes. In certain embodiments, the kit may comprise at least one container containing PBMCs, lymphocytes, APCs, and / or T cells. For example, but not limited to, the kit may comprise at least one container containing CD8-T cells, CD4+ T cells, and / or CD4+CD8-T cells. In certain embodiments, the kit of the Disclosure comprises lymphocytes derived from one or more donors in one or more containers. In certain embodiments, the kit of the Disclosure may further comprise one or more agents for detecting one or more markers disclosed herein, for example, CD134 and / or CD137 antibodies.

[0127] In certain embodiments, the kit further includes a package insert providing instructions for using the components provided in the kit. For example, the kit of this disclosure may include a package insert providing instructions for using lymphocytes and / or drugs in the disclosed manner.

[0128] Alternatively or additionally, the kit may include other materials desirable from a commercial and user perspective, including other buffers, diluents, and filters. In certain embodiments, the kit may include materials for collecting and / or processing blood samples, for example, to isolate lymphocytes from the sample.

[0129] V. Exemplary Embodiments A. The subject matter disclosed in the present invention is a method for determining the tendency of a composition to induce the production of antibodies specific to that composition, compared to the tendency of a reference. (a) Culturing lymphocytes in the presence of the composition to generate stimulated lymphocytes, (b) To culture lymphocytes in the absence of the composition to generate unstimulated lymphocytes, (c) Determine the percentage of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. (d) Determine the percentage of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. (e) including calculating the stimulus index value, The present invention provides a method in which, if the stimulation index value in (e) is equal to or greater than the reference stimulation index value, the composition has a greater tendency to induce antibodies specific to the composition, and if the stimulation index value in (e) is less than the reference stimulation index value, the composition has a less tendency to induce antibodies specific to the composition.

[0130] A1. The method described in A, wherein the reference stimulus index value is approximately 1.0 to approximately 2.0.

[0131] A2. The method according to A, wherein the reference stimulus index value is approximately 1.6 or higher, approximately 1.7 or higher, or approximately 1.8 or higher.

[0132] A3. The method according to any one of A to A2, wherein the stimulation index value is determined by dividing the percentage of stimulated lymphocytes determined in (c) by the percentage of unstimulated lymphocytes determined in (d).

[0133] A4. A method according to any one of A-A3, wherein the stimulus index value is determined by outlier sum analysis or by linear regression.

[0134] A5. The method described in any one of A to A4, wherein the lymphocytes include T cells.

[0135] A6. The method according to A5, wherein at least 30% of the lymphocytes are T cells.

[0136] A7. The method according to A5 or A6, wherein the T cells include CD8-T cells.

[0137] A8. The method according to A7, wherein at least 10% of the T cells are CD8-T cells.

[0138] A9. A method according to any one of A-A8, wherein lymphocytes are obtained from a single donor.

[0139] A10. A method using one of A to A8, wherein lymphocytes are obtained from approximately 20 to 50 donors.

[0140] A11. The method described in A10, in which lymphocytes are obtained from approximately 35 to 45 donors.

[0141] A12. The method of A10, wherein lymphocytes are obtained from at least approximately 20 donors, at least approximately 25 donors, at least approximately 30 donors, at least approximately 35 donors, at least approximately 40 donors, or at least approximately 45 donors.

[0142] A13. The method according to any one of A to A12, wherein approximately 1 × 10⁵ to approximately 1 × 10⁷ lymphocytes are cultured together with the composition.

[0143] A14. The method according to any one of A to A13, wherein lymphocytes are cultured with a composition of approximately 10 μg / ul to approximately 1,000 μg / ml.

[0144] A15. The method according to any one of A to A14, wherein the composition comprises a peptide, polypeptide, or small molecule compound.

[0145] A16. The method according to A15, wherein the peptide or polypeptide contains a neoantigen.

[0146] A17. The method according to A15, wherein the polypeptide is an antibody or a fragment thereof.

[0147] A18. The method according to any one of A17, wherein the antibody is human, humanized, or chimeric.

[0148] A19. The method according to any one of A to A14, wherein the composition is an antibody-drug conjugate (ADC).

[0149] A20. The method according to any one of A to A19, wherein lymphocytes are cultured with the composition for approximately 48 hours or less.

[0150] A21. The method according to any one of A to A20, wherein the percentage of stimulated or unstimulated lymphocytes expressing (i) CD134, (ii) CD137, or (iii) both CD134 and CD137 is determined by flow cytometry.

[0151] B. The subject matter disclosed in the present invention is a method for determining the tendency of a composition to induce the production of antibodies specific to the composition, (a) Generating stimulated lymphocytes by separately culturing lymphocytes from individual donors in the presence of the composition, (b) To culture lymphocytes from individual donors in the absence of the composition to generate unstimulated lymphocytes, (c) Determine the percentage of stimulated lymphocytes from individual donors that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. (d) Determine the percentage of unstimulated lymphocytes from a CD4+ donor expressing (i) CD134, (ii) CD137, or (iii) both CD134 and CD137, (e) Calculate the stimulation index value for each donor, (f) This includes calculating the number of reactive lymphocyte donors whose donor stimulation index value is equal to or greater than the reference stimulation index value, and the number of non-reactive lymphocyte donors whose donor stimulation index value is less than the reference stimulation index value, The present invention provides a method in which, when the number of reactive donors exceeds 30% of the total number of donors, the composition has a high tendency to induce the production of antibodies specific to the composition, and when the number of reactive donors is less than 20% of the total number of donors, the composition has a low tendency to induce the production of antibodies specific to the composition.

[0152] B1. The method described in B, wherein the reference stimulus index value is approximately 1.0 to approximately 2.0.

[0153] B2. The method according to B, wherein the reference stimulus index value is approximately 1.6 or higher, approximately 1.7 or higher, or approximately 1.8 or higher.

[0154] B3. The method according to any one of B to B2, wherein the stimulation index value is determined by dividing the percentage of stimulated lymphocytes of an individual donor determined in (c) by the percentage of unstimulated lymphocytes of that individual donor determined in (d).

[0155] B4. A method according to any one of B-B3, wherein the stimulus index value is determined by outlier sum analysis or by linear regression.

[0156] B5. The method according to any one of B to B4, wherein the lymphocytes include T cells.

[0157] B6. The method according to B5, wherein at least 30% of the lymphocytes include T cells.

[0158] B7. The method according to B5 or B6, wherein T cells include CD8-T cells.

[0159] B8. The method according to B7, wherein at least 10% of the T cells are CD8-T cells.

[0160] B9. A method using one of B to B8, in which lymphocytes are obtained from approximately 20 to 50 donors.

[0161] B10. Lymphocytes are obtained from approximately 35 to 45 donors, using the method described in B9.

[0162] B11. The method according to B9, wherein lymphocytes are obtained from at least approximately 20 donors, at least approximately 25 donors, at least approximately 30 donors, at least approximately 35 donors, at least approximately 40 donors, or at least approximately 45 donors.

[0163] B12. The method according to any one of B to B11, wherein the composition comprises a peptide, polypeptide, or small molecule compound.

[0164] B13. The method according to B12, wherein the polypeptide is an antibody or a fragment thereof.

[0165] B14. The method according to B13, wherein the antibody is human, humanized, or chimeric.

[0166] B15. The method according to B12, wherein the peptide or polypeptide contains a neoantigen.

[0167] B16. The method according to any one of B to B11, wherein the composition is an antibody-drug conjugate (ADC).

[0168] B17. The method according to any one of B to B16, wherein lymphocytes are cultured with the composition for approximately 48 hours or less.

[0169] B18. The method according to any one of B-B17, wherein the percentage of stimulated or unstimulated lymphocytes expressing (i) CD134, (ii) CD137, or (iii) both CD134 and CD137 is determined by flow cytometry.

[0170] C. Subject matter disclosed in the present invention is a method for determining the tendency of a neoantigen to induce a neoantigen-specific immune response compared to a reference antigen, (a) Culturing lymphocytes in the presence of neoantigens to generate stimulated lymphocytes, (b) To generate unstimulated lymphocytes by culturing lymphocytes in the absence of neoantigens, (c) Determine the percentage of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. (d) Determine the percentage of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) both CD134 and CD137. (e) including calculating the stimulus index value, The present invention provides a method in which, if the stimulation index value in (e) is greater than or equal to the reference stimulation index value, the neoantigen is more likely to induce an immune response specific to that neoantigen, and if the stimulation index value in (e) is less than the reference stimulation index value, the neoantigen is less likely to induce an immune response specific to that neoantigen.

[0171] C1. The method according to C, wherein the neoantigen exists in complex with an MHC class II molecule.

[0172] C2. The method described in C or C1, wherein the reference stimulus index value is approximately 1.0 to approximately 2.0.

[0173] C3. The method according to C or C1, wherein the reference stimulus index value is approximately 1.6 or higher, approximately 1.7 or higher, or approximately 1.8 or higher.

[0174] C4. The method according to any one of C to C3, wherein the stimulation index value is determined by dividing the percentage of stimulated lymphocytes determined in (c) by the percentage of unstimulated lymphocytes determined in (d).

[0175] C5. A method according to any one of C-C3, wherein the stimulus index value is determined by outlier sum analysis or by linear regression.

[0176] C6. A method for which lymphocytes include T cells, and which is one of the C-C5 methods.

[0177] C7. The method described in C6, wherein at least 30% of the lymphocytes include T cells.

[0178] The method of C6 or C7, wherein C8 T cells include CD8-T cells.

[0179] The method for C8, wherein at least 10% of the C9 T cells are CD8-T cells.

[0180] C10. A method using one of the C-C9 methods, in which lymphocytes are obtained from approximately 20 to 50 donors.

[0181] C11. Lymphocytes are obtained from approximately 35 to 45 donors, using the method described in C10.

[0182] C12. The method according to C10, wherein lymphocytes are obtained from at least approximately 20 donors, at least approximately 25 donors, at least approximately 30 donors, at least approximately 35 donors, at least approximately 40 donors, or at least approximately 45 donors.

[0183] C13. A method according to any one of C to C12, wherein lymphocytes are cultured with neoantigens for approximately 48 hours or less.

[0184] The method according to any one of C-C13, wherein C14 is CD4+ and the percentage of stimulated or unstimulated lymphocytes expressing (i) CD134, (ii) CD137, or (iii) both CD134 and CD137 is determined by flow cytometry.

[0185] D. The subject matter disclosed in the present invention provides a kit for carrying out any one of the methods described in any one of A to C14.

[0186] E. The subject matter disclosed in the present invention is a method for determining the tendency of a composition to induce the production of antibodies specific to that composition, compared to the tendency of a reference, (a) To culture antigen-presenting cells (APCs) in the presence of the composition to generate stimulated APCs, (b) Culturing APC in the absence of the composition to produce non-irritating APC, (c) Stimulated APCs are cultured separately with CD4+ lymphocytes, and unstimulated APCs are cultured separately with CD4+ lymphocytes. (d) Determine the percentage of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137. (e) Determine the percentage of CD4+ lymphocytes cultured with (i) CD134, (ii) CD137, or (iii) CD134 and CD137 expressing unstimulated APCs, (f) including calculating the stimulus index value, The present invention provides a method in which, when the stimulation index value in (f) is equal to or greater than the reference stimulation index value, the composition has a greater tendency to induce antibodies specific to the composition, and when the stimulation index value in (f) is less than the reference stimulation index value, the composition has a less tendency to induce antibodies specific to the composition.

[0187] E1. The method described in E, wherein the reference stimulus index value is approximately 1.0 to approximately 4.0, approximately 1.0 to approximately 3.0, or approximately 1.8 to approximately 3.0.

[0188] E2. The method according to E, wherein the reference stimulus index value is approximately 1.6 or higher, approximately 1.7 or higher, approximately 1.8 or higher, approximately 1.9 or higher, approximately 2.0 or higher, approximately 2.1 or higher, approximately 2.2 or higher, approximately 2.3 or higher, approximately 2.4 or higher, approximately 2.5 or higher, approximately 2.6 or higher, approximately 2.7 or higher, approximately 2.8 or higher, approximately 2.9 or higher, or approximately 3.0 or higher.

[0189] E3. The method according to any one of E to E2, wherein the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes determined in (d) by the percentage of CD4+ lymphocytes determined in (e).

[0190] E4. A method according to any one of E-E2, wherein the stimulus index value is determined by outlier sum analysis or by linear regression.

[0191] E5. A method according to any one of E to E4, wherein CD4+ lymphocytes include CD8-T cells.

[0192] E6. The method described in E5, wherein at least 10% of the CD4+ lymphocytes are CD8-T cells.

[0193] E7. A method of obtaining an APC from a single donor, according to one of the methods E-E6.

[0194] E8. APC is obtained from approximately 20 to 50 donors using one of the methods E to E6.

[0195] E9. APC is obtained from approximately 35 to 45 donors, using the method described in E8.

[0196] E10. The method in E8, wherein the APC is obtained from at least approximately 20 donors, at least approximately 25 donors, at least approximately 30 donors, at least approximately 35 donors, at least approximately 40 donors, or at least approximately 45 donors.

[0197] E11. Approximately 1×10 5 pieces~approx. 1×10 7 The method according to any one of E to E10, wherein individual APCs are cultured together with the composition.

[0198] E12. The method according to any one of E to E11, wherein APC is cultured with a composition at a concentration of approximately 10 μg / ul to approximately 1,000 μg / ml.

[0199] E13. The method according to any one of E to E12, wherein the composition comprises a peptide, polypeptide, or small molecule compound.

[0200] E14. The method according to E13, wherein the peptide or polypeptide contains a neoantigen.

[0201] E15. The method according to E13, wherein the polypeptide is an antibody or a fragment thereof.

[0202] E16. The method according to E15, wherein the antibody is human, humanized, or chimeric.

[0203] E17. The method according to any one of E to E12, wherein the composition is an antibody-drug conjugate (ADC).

[0204] The method according to any one of E to E17, wherein E18.APC is cultured with the composition for approximately 48 hours or less.

[0205] E19. The method according to any one of E-E18, wherein the percentage of CD4+ lymphocytes expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 is determined by flow cytometry.

[0206] E20. The method according to any one of E-E19, wherein the percentage of CD4+ lymphocytes expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 is determined by flow cytometry.

[0207] F. The subject matter disclosed in the present invention is a method for determining the tendency of a composition to induce the production of antibodies specific to the composition, (a) Generating stimulated APCs by separately culturing APCs from individual donors in the presence of the composition, (b) To produce non-stimulated APCs by culturing APCs from individual donors separately in the absence of the composition, (c) Stimulated APCs are cultured separately with CD4+ lymphocytes, and unstimulated APCs are cultured separately with CD4+ lymphocytes. (d) Determine the percentage of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137. (e) Determine the percentage of CD4+ lymphocytes cultured with unstimulated APCs expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (f) Calculate the stimulation index value for each donor, (g) This includes calculating the number of reactive lymphocyte donors whose donor stimulation index value is equal to or greater than the reference stimulation index value, and the number of non-reactive lymphocyte donors whose donor stimulation index value is less than the reference stimulation index value, The present invention provides a method in which, when the number of reactive donors exceeds 30% of the total number of donors, the composition has a high tendency to induce the production of antibodies specific to the composition, and when the number of reactive donors is less than 20% of the total number of donors, the composition has a low tendency to induce the production of antibodies specific to the composition.

[0208] F1. The method according to F, wherein the reference stimulus index value is approximately 1.0 to approximately 4.0, approximately 1.0 to approximately 3.0, or approximately 1.8 to approximately 3.0.

[0209] F2. The method according to F, wherein the reference stimulus index value is approximately 1.6 or higher, approximately 1.7 or higher, approximately 1.8 or higher, approximately 1.9 or higher, approximately 2.0 or higher, approximately 2.1 or higher, approximately 2.2 or higher, approximately 2.3 or higher, approximately 2.4 or higher, approximately 2.5 or higher, approximately 2.6 or higher, approximately 2.7 or higher, approximately 2.8 or higher, approximately 2.9 or higher, or approximately 3.0 or higher.

[0210] F3. A method according to any one of F to F2, wherein the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes of an individual donor determined in (d) by the percentage of CD4+ lymphocytes of that individual donor determined in (e).

[0211] F4. A method according to any one of F-F2, wherein the stimulus index value is determined by outlier sum analysis or by linear regression.

[0212] F5. A method according to any one of F to F4, wherein CD4+ lymphocytes include CD8-T cells.

[0213] The method described in F5, wherein at least 10% of the CD4+ lymphocytes are CD8-T cells.

[0214] F7. APC is obtained from approximately 20 to 50 donors using one of the methods F through F6.

[0215] F8. APC is obtained from approximately 35 to 45 donors, using the same method as in F7.

[0216] The method of F7, wherein F9.APC is obtained from at least approximately 20 donors, at least approximately 25 donors, at least approximately 30 donors, at least approximately 35 donors, at least approximately 40 donors, or at least approximately 45 donors.

[0217] F10. The method according to any one of F to F9, wherein the composition comprises a peptide, polypeptide, or small molecule compound.

[0218] The method according to F10, wherein the polypeptide is an antibody or a fragment thereof.

[0219] F12. The method according to F11, wherein the antibody is human, humanized, or chimeric.

[0220] F13. The method according to F10, wherein the peptide or polypeptide contains a neoantigen.

[0221] F14. The method according to any one of F to F9, wherein the composition is an antibody-drug conjugate (ADC).

[0222] The method according to any one of F to F14, wherein F15.APC is cultured with the composition for approximately 48 hours or less.

[0223] F16. The method according to any one of F-F15, wherein the percentage of CD4+ lymphocytes expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 is determined by flow cytometry.

[0224] G. The subject matter disclosed herein is a method for determining the tendency of a neoantigen to induce a neoantigen-specific immune response compared to a reference antigen, (a) Culturing APC in the presence of neoantigen to generate stimulated APC, (b) To produce non-stimulated APCs by culturing APCs in the absence of neoantigens, (c) Stimulated APCs are cultured separately with CD4+ lymphocytes, and unstimulated APCs are cultured separately with CD4+ lymphocytes. (d) Determine the percentage of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137. (e) Determine the percentage of CD4+ lymphocytes cultured with unstimulated APCs expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (f) including calculating the stimulus index value, The present invention provides a method in which, if the stimulation index value in (f) is greater than or equal to the reference stimulation index value, the neoantigen is more likely to induce an immune response specific to that neoantigen, and if the stimulation index value in (f) is less than the reference stimulation index value, the neoantigen is less likely to induce an immune response specific to that neoantigen.

[0225] G1. The method described in G, wherein the neoantigen exists in complex with an MHC class II molecule.

[0226] G2. The method according to G or G1, wherein the reference stimulus index value is approximately 1.0 to approximately 4.0, approximately 1.0 to approximately 3.0, or approximately 1.8 to approximately 3.0.

[0227] G3. The method according to G or G1, wherein the reference stimulus index value is approximately 1.6 or higher, approximately 1.7 or higher, approximately 1.8 or higher, approximately 1.9 or higher, approximately 2.0 or higher, approximately 2.1 or higher, approximately 2.2 or higher, approximately 2.3 or higher, approximately 2.4 or higher, approximately 2.5 or higher, approximately 2.6 or higher, approximately 2.7 or higher, approximately 2.8 or higher, approximately 2.9 or higher, or approximately 3.0 or higher.

[0228] G4. The method according to any one of G to G3, wherein the stimulation index value is determined by dividing the percentage of CD4+ lymphocytes determined in (d) by the percentage of CD4+ lymphocytes determined in (e).

[0229] G5. A method according to any one of G-G3, wherein the stimulus index value is determined by outlier sum analysis or by linear regression.

[0230] G6. A method according to any one of G to G5, wherein CD4+ lymphocytes include CD8-T cells.

[0231] G7. The method described in G6, wherein at least 10% of the CD4+ lymphocytes are CD8-T cells.

[0232] G8. The method described in any one of G1 to G7, wherein the APC is obtained from about 20 donors to about 50 donors.

[0233] G9. The method described in G8, wherein the APC is obtained from about 35 donors to about 45 donors.

[0234] G10. The method described in G8, wherein the APC is obtained from at least about 20 donors, at least about 25 donors, at least about 30 donors, at least about 35 donors, at least about 40 donors, or at least about 45 donors.

[0235] G11. The method described in any one of G1 to G10, wherein the APC is cultured for about 48 hours or less with a neoantigen.

[0236] G12. The method described in any one of G1 to G11, wherein the ratio of CD4+ lymphocytes expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 is determined by flow cytometry.

[0237] H. The present invention discloses a kit for performing any one of the methods described in any one of E to G12.

Examples

[0238] The following examples are merely illustrative of the subject matter disclosed in the present invention and should in no way be regarded as limiting.

[0239] Example 1 T cell CD4+ expression assay Polypeptide-based therapeutics have immunogenic potential to induce ADA production. In particular, such polypeptide-based therapeutics can be taken up and processed by antigen-presenting cells, such as dendritic cells, to present fragments of the polypeptide-based therapeutic on their surface in complex with class II MHC molecules. T cells then interact with the fragments presented on the surface of the antigen-presenting cells to trigger an immune response, leading to ADA production by B cells.

[0240] This specification describes a method developed to determine the tendency of antibodies to induce ADA production. Such a method can be an invaluable tool during drug development, as it can be used to predict the immunogenicity potential of newly developed drugs in the preclinical stage of development. Figure 1 provides a schematic diagram of the experimental details of this method. Peripheral blood mononuclear cells (PBMCs) were isolated from naive healthy donor blood by density gradient centrifugation using Uni-Sep blood separation tubes. In some experiments, CD8+ cells were depleted using CD8 dynabeads (Thermo Fisher, Waltham, MA, catalog number: 11147D). It should be noted that the assay also yielded good results when PBMCs were cultured without CD8 depletion. CD8- cells were then cultured with 10% human type AB serum (Sigma Aldrich, catalog number: H3667) in 24-well plates at a rate of 2 × 10⁶ cells. 6 At a concentration of cells / mL, or in a 96-well plate (Costar, catalog number 3526) at 0.2–0.4 × 10⁶ 6Cells were cultured in AIM-V medium (Thermo Fisher, Waltham, MA) at the specified cell concentration and challenged with a final concentration of test antibody at 100 μg / mL. All samples were tested in triplicates. For each donor, responses to a negative control consisting of cells treated with the medium (also called unstimulated cells) and a positive control using Imject Mariculture KLH (mcKLH) (100 μg / ml) were also included. Cells were incubated in a 5% CO2 incubator at 37°C for 42–48 hours. After 42–48 hours, the cells were gently resuspended, and 200 μl from each 24-well plate was transferred to a round-bottom 96-well plate. CD4 activation was measured using CD4, CD134, CD137 antibodies and living markers. Cells were analyzed by flow cytometry, and plots were analyzed using FlowJo FACS analysis software (Tree Star, Inc.; Ashland, OR). For data analysis, the Stimulation Index (SI) is calculated by dividing the average and / or maximum percentage of cells that were [live +CD4+CD134+CD137+ and live +CD4+CD134+CD137- and live +CD4+CD134-CD137+] for each treatment by the average percentage of cells that were [live +CD4+CD134+CD137+ and live +CD4+CD134+CD137- and live +CD4+CD134-CD137+] in wells treated only with the medium (unstimulated cells) for each treatment.

[0241] Figure 2 shows FACS analysis of two different antibodies, Avastin® and vococizumab, with different clinical ADA rates. Vococizumab, with its high ADA rate, resulted in a larger number of cells expressing the CD4 activation marker compared to Avastin®, with its low ADA rate.

[0242] Analysis of six antibodies with different clinical ADA rates confirmed that the disclosed assay results correlated with clinical ADA rates (Figure 3). Anti-PCSK9 antibody, Avastin®, GNE-αPCSK9, alirocumab (PRALUENT®), evolocumab (REPATHA®), vocizumab, and HA33 were analyzed using the method described above. The clinical ADA rates for Avastin®, GNE-αPCSK9, alirocumab (PRALUENT®), evolocumab (REPATHA®), vocizumab, and HA33A were 0.6%, 3.3%, 5.1%, 0.3%, 48%, and 73%, respectively (Figure 3). As shown in Figure 3, the number of positive donors for vocizumab or HA33 was significantly higher than for any of the other antibodies with low immunogenicity associated with the clinical ADA rates observed for each antibody.

[0243] In a clinic setting, T-cell responses to two known ADA-associated therapeutic agents, HA33 and Avastin®, were tested in 40 PBMCs derived from healthy donors. The majority of tested donors showed a positive response when stimulated with KLH. In addition, cell treatment with Avastin® had little effect on CD134 or CD137 expression, while HA33 treatment showed significant increases in CD134 (10 donors, Figure 4A) and CD137 (14 donors, Figure 4B), as well as in double positivity for CD134 and CD137 (13 donors, Figure 4C). Examination of CD134 and / or CD137 expression revealed that 14 donors were HA33-positive, while only one donor was Avastin®-positive (Figure 4D). These results correlate with observed clinical ADA.

[0244] Analysis of additional therapeutic agents confirmed a correlation between predicted immunogenicity and immunogenicity observed in the clinic. As shown in Figure 5, these therapeutic agents, which had a high proportion of positive donors in the assay, also showed higher clinical ADA rates in the clinic. For example, briakinumab, which resulted in positivity in 80% of donors, had a clinical ADA rate of 40–86%, while avelumab (Bavencio®), which resulted in positivity in 4.16% of donors, had a clinical ADA rate of 4.10%.

[0245] To confirm that T cell activation is dependent on the presentation of the biopharmaceutical antigen, assays were performed in the presence of HLA-DR and HLA-II blocking antibodies (Figure 6A). As shown in Figure 6A, the antibodies block the interaction of HLA-II with the TCR present on the surface of T cells, thereby blocking T cell activation. As shown in Figure 6B, blocking the HLA-DR and HLA-II proteins reduces the proportion of positive donors showing T cell activation, which is observed as increased expression of CD134 and / or CD137, and determining positive donors is dependent on antigen presentation. These data confirm that CD134 and / or CD137 can be used as markers of activation and that the disclosed assay can predict the immunogenicity of therapeutic agents.

[0246] We evaluated additional potential markers and determined whether the expression of such markers could also be used in this assay. In particular, cytokine secretion and expression were analyzed in this assay as potential markers of immunogenicity. As shown in Figure 7, there was no correlation between intracellular expression of IL-2 in vitro and clinical immunogenicity. Similarly, there was no correlation between the secretion of cytokines IL-4, TNFα, and INFγ in vitro and clinical immunogenicity (Figure 8).

[0247] As shown in Table 1, the method disclosed herein offers several advantages. In particular, known proliferation assays in the art take about 20 weeks to perform, require about two analysts, and cost about $30,000. In contrast, the assay disclosed herein takes only about two weeks to perform, requires one analyst, and costs about $1,000. JPEG0007834710000001.jpg45170

[0248] Example 2 T cell expression assay of bispecific antibodies that bind to T cells This specification describes a method for determining the tendency of antibodies to induce ADA production. A potential challenge of PBMC-based assays is interference by the bioactivity of the target biotherapy, such as immunomodulation through direct T cell involvement. To overcome this challenge, a second dendritic cell T cell assay platform was developed.

[0249] In this assay, PBMCs are separated from the blood of a naive healthy donor by density gradient centrifugation using Uni-Sep blood separation tubing, in at least two tubings (up to 30 × 10 per tubing). 6 Freeze the individual PBMCs. Figures 9 and 10 provide schematic diagrams of the experimental details of this method, with Figure 10 providing details on the conditions. On day 1, isolate CD14+ monocytes from at least one tube of PBMCs. Then, isolate the CD14+ monocytes at a rate of 1.0 × 10⁴ per ml in a 24-well plate containing DC medium (RPMI, 1% non-essential amino acids, 1% sodium pyruvate, 1% kanamycin, 10% AB serum) supplemented with IL4 (17.2 ng / mL) and GM-CSF (66.6 ng / mL). 6The cells were cultured at a density of 100,000 cells for 24 hours and placed in a 5% CO2 incubator. This culture of CD14+ monocytes enables differentiation of monocytes into dendritic cells (DCs). After 24 hours, monocyte-derived DCs were washed with sterile PBS and cultured in DC medium containing IL-4 (17.2 ng / mL), GM-CSF (66.6 ng / mL), TNF-α (5 ng / mL), IL-1β (5 ng / mL), IL-6 (150 ng / mL), PGE2 (1 μg / mL), and 100 μg / mL of the biopharmaceutical drug to be tested. The cells were placed in a 5% CO2 incubator. Subsequently, monocyte-derived DCs were cultured at concentrations of 100,000 cells / mL to 200 μL / well (20,000 cells / well in a 96-well plate) and allowed to mature for a further 24 hours.

[0250] At this stage, mature DCs were exposed to a biopharmaceutical for 24 hours to enable antigen uptake, processing, and presentation of the antigen peptide. On day 3, CD4+ cells were isolated from the same autologous PBMC population (from the aforementioned tubes). In parallel, mature DCs were washed three times with PBS. CD4+ T cells and mature DCs were co-cultured in a ratio of 5 T cells to 1 DC (200,000 T cells + 20,000 DCs). This method allows for precise control of the CD4+ T cell to APC ratio, which improves the sensitivity of the assay. The ratio of CD4+ T cells to DCs can be varied (5:1, 10:1, and 20:1) and can be further modified.

[0251] Cells were cultured in a 5% CO2 incubator for at least 19 hours (various durations including 24, 48, or 72 hours). All samples were tested in triplicates. For each donor, the response to a negative control consisting of cells treated in culture medium (also referred to as unstimulated cells) was analyzed. CD4 activation was measured using CD4, CD134, and CD137 antibodies and living markers. Cells were analyzed by flow cytometry, and plots were analyzed using FlowJo FACS analysis software (Tree Star, Inc.; Ashland, OR). For data analysis, the Stimulation Index (SI) was calculated by dividing the mean and / or maximum percentage of cells that are [Live +CD4+CD134+CD137+ and Live +CD4+CD134+CD137- and Live +CD4+CD134-CD137+] in each treatment by the mean and / or maximum percentage of cells that are [Live +CD4+CD134+CD137+ and Live +CD4+CD134+CD137- and Live +CD4+CD134-CD137+] in wells treated with medium only (unstimulated cells).

[0252] Five distinct bispecific antibodies, each possessing an antigen-binding domain that binds to T cells, namely TDB1, TDB2, TDB3, TDB4, and TDB5, were analyzed using the method described above. As shown in Figures 11A, 11B, 12A, and 12B, the stimulation indices of the bispecific antibodies were higher than those of Avastin®, which is known to have a low ADA rate. These data suggest that all five bispecific antibodies are more immunogenic than Avastin®.

[0253] To confirm that T cell activation is dependent on the presentation of the biopharmaceutical antigen, the assay was performed in the presence of an HLA-II blocking antibody (Figure 13A). As shown in Figure 13B, blocking the HLA-II protein reduced the stimulation index of the bispecific antibody to a level comparable to a reference known to have a low ADA rate.

[0254] Figure 14 provides an analysis of bispecific antibodies with binding specificity to T cells produced by two different methods. The first method involves the expression of both antigen-binding domains in a single cell and generates a bispecific antibody, shown as TDB4A in Figure 14. The second method involves the expression of each antigen-binding domain in separate cells, as well as the subsequent isolation and combination of the antigen-binding domains, and generates a bispecific antibody, shown as TDB4B in Figure 14. As shown in Figure 14, TDB4B resulted in a higher stimulation index than TDB4A.

[0255] In addition to the various embodiments depicted and claimed, the disclosed subject matter also covers other embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any preferred combination of the features disclosed herein. The foregoing descriptions of specific embodiments of the disclosed subject matter are presented for illustrative and explanatory purposes only. They are not intended to be exhaustive or to limit the disclosed subject matter to the disclosed embodiments.

[0256] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Accordingly, the disclosed subject matter is intended to include the modifications and variations in the appended claims and their equivalents.

[0257] Various publications, patents, and patent applications are cited herein, their contents incorporated herein by reference in their entirety.

Claims

1. A method for determining the tendency of a composition to induce the production of antibodies specific to the composition, compared to a reference trend, (a) Culturing lymphocytes in the presence of the composition to generate stimulated lymphocytes, (b) To culture lymphocytes in the absence of the composition to generate unstimulated lymphocytes, (c) Determine the percentage of stimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (d) Determine the percentage of unstimulated lymphocytes that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (e) Calculate the stimulus index value and Includes, A method wherein, if the stimulation index value in (e) is greater than or equal to the reference stimulation index value, the composition has a greater tendency to induce antibodies specific to the composition, and if the stimulation index value in (e) is less than the reference stimulation index value, the composition has a less tendency to induce antibodies specific to the composition.

2. The method according to claim 1, wherein the reference stimulus index value is 1.0 to 2.

0.

3. The method according to claim 1, wherein the reference stimulus index value is 1.6 or greater, 1.7 or greater, or 1.8 or greater.

4. (i) The stimulation index value is determined by dividing the percentage of stimulated lymphocytes determined in (c) by the percentage of unstimulated lymphocytes determined in (d); or (ii) The stimulus index value is determined by outlier sum analysis or by linear regression. The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein the lymphocytes include T cells.

6. The method according to claim 5, wherein at least 30% of the lymphocytes are T cells.

7. The method according to claim 5 or 6, wherein the T cells include CD8-T cells.

8. The method according to claim 7, wherein at least 10% of the T cells are CD8-T cells.

9. The method according to any one of claims 1 to 8, wherein the lymphocytes are obtained from a single donor.

10. The method according to any one of claims 1 to 8, wherein lymphocytes are obtained from 20 to 50 donors.

11. The method according to claim 10, wherein lymphocytes are obtained from 35 to 45 donors.

12. The method according to claim 10, wherein the lymphocytes are obtained from at least 20 donors, at least 25 donors, at least 30 donors, at least 35 donors, at least 40 donors, or at least 45 donors.

13. 1×10 ５ pieces ~ 1×10 ７ The method according to any one of claims 1 to 12, wherein individual lymphocytes are cultured together with the composition.

14. The method according to any one of claims 1 to 13, wherein lymphocytes are cultured with a composition containing 10 μg / μl to 1,000 μg / ml.

15. The method according to any one of claims 1 to 14, wherein the composition comprises a peptide, polypeptide, or small molecule compound.

16. The method according to claim 15, wherein the peptide or polypeptide comprises a neoantigen.

17. The method according to claim 15, wherein the polypeptide is an antibody or a fragment thereof.

18. The method according to any one of claims 1 to 14, wherein the composition is an antibody-drug conjugate (ADC).

19. The method according to any one of claims 1 to 18, wherein lymphocytes are cultured with the composition for 48 hours or less.

20. The method according to any one of claims 1 to 19, wherein the percentage of stimulated or unstimulated lymphocytes expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 is determined by flow cytometry.

21. A method for determining the tendency of a composition to induce the production of antibodies specific to that composition, (a) Generating stimulated lymphocytes by separately culturing lymphocytes from individual donors in the presence of the composition, (b) Generating unstimulated lymphocytes by separately culturing lymphocytes from individual donors in the absence of the composition, (c) Determine the percentage of stimulated lymphocytes from individual donors that are CD4+ and express (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (d) Determine the percentage of unstimulated lymphocytes from a CD4+ donor expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (e) Calculate the stimulation index value for each donor, (f) Calculate the number of reactive lymphocyte donors whose donor stimulation index value is equal to or greater than the reference stimulation index value, and the number of non-reactive lymphocyte donors whose donor stimulation index value is less than the reference stimulation index value. Includes, A method wherein, when the number of reactive donors exceeds 30% of the total number of donors, the composition has a high tendency to induce the production of antibodies specific to the composition, and when the number of reactive donors is less than 20% of the total number of donors, the composition has a low tendency to induce the production of antibodies specific to the composition.

22. A method for determining the tendency of a composition to induce the production of antibodies specific to the composition, compared to a reference trend, (a) Culture antigen-presenting cells (APCs) in the presence of the composition to generate stimulated APCs, (b) Culturing APC in the absence of the composition to produce non-irritated APC, (c) Stimulated APCs are cultured separately with CD4+ lymphocytes, and unstimulated APCs are cultured separately with CD4+ lymphocytes. (d) Determine the percentage of CD4+ lymphocytes cultured with stimulated APCs that express (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (e) Determine the percentage of CD4+ lymphocytes cultured with unstimulated APCs expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137, (f) Calculating the stimulus index value and Includes, A method wherein, if the stimulation index value in (f) is greater than or equal to the reference stimulation index value, the composition has a greater tendency to induce antibodies specific to the composition, and if the stimulation index value in (f) is less than the reference stimulation index value, the composition has a less tendency to induce antibodies specific to the composition.

23. The method according to claim 22, wherein the reference stimulus index value is 1.0 to 4.0, 1.0 to 3.0, or 1.8 to 3.

0.

24. (i) The stimulation index value is determined by dividing the percentage of CD4+ lymphocytes determined in (d) by the percentage of CD4+ lymphocytes determined in (e); or (ii) The stimulus index value is determined by outlier sum analysis or by linear regression. The method according to claim 22 or 23.

25. The method according to any one of claims 22 to 24, wherein the APC is obtained from a single donor.

26. The method according to any one of claims 22 to 24, wherein the APC is obtained from 20 to 50 donors.

27. (i) 1 × 10 ５ pieces ~ 1×10 ７ Each APC is cultured together with the composition; (ii) APC is cultured with a composition of 10 μg / μl to 1,000 μg / ml; and / or (iii) The APC is cultured with the composition for 48 hours or less. The method according to any one of claims 22 to 26.

28. The method according to any one of claims 22 to 27, wherein the composition comprises a peptide, polypeptide, or small molecule compound.

29. The method according to claim 28, wherein the peptide or polypeptide comprises a neoantigen.

30. The method according to claim 28, wherein the polypeptide is an antibody or a fragment thereof.

31. The method according to any one of claims 22 to 27, wherein the composition is an antibody-drug conjugate (ADC).

32. The method according to any one of claims 22 to 31, wherein the determination of the percentage of CD4+ lymphocytes expressing (i) CD134, (ii) CD137, or (iii) CD134 and CD137 is performed by flow cytometry.

33. The method according to any one of claims 21 to 32, wherein the lymphocytes include T cells.

34. The method according to any one of claims 21 to 32, wherein at least 30% of the lymphocytes are T cells.

35. The method according to claim 33 or 34, wherein the T cells include CD8-T cells.

36. The method according to claim 33 or 34, wherein at least 10% of the T cells are CD8-T cells.

37. A kit for carrying out any one of the methods described in any one of claims 1 to 36.

38. (a) One or more lymphocytes derived from one or more donors; and (b) One or more agents for detecting CD4, CD134 and / or CD137 The kit according to claim 37, including the following:

Images