WO2025076303A1 - Major histocompatibility complex (mhc)-associated peptide proteomics (mapps) assay for immunogenicity risk assessment of biotherapeutic attributes - Google Patents

Abstract

The disclosure provides a major histocompatibility complex II (MHC-II)-associated peptide proteomics (MAPPs) assay method that can identify immunogenic regions of therapeutic proteins having one or more molecular attributes (e.g., high molecular weight species, subvisible particle number, and/or aggregation).

Description

MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)-ASSOCIATED PEPTIDE PROTEOMICS (MAPPS) ASSAY FOR IMMUNOGENICITY RISK ASSESSMENT OF BIOTHERAPEUTIC ATTRIBUTES

FIELD

[0001] The disclosure relates to methods for performing a major histocompatibility complex II (MHC-II)-associated peptide proteomics (MAPPs) assay to assess immunogenicity of therapeutic proteins.

BACKGROUND

[0002] The immunogenicity of protein-based biotherapeutics remains a critical safety, efficacy, and regulatory concern during clinical development of these molecules. Immunogenicity typically refers to the ability of a therapeutic protein to provoke an immune response in a patient, such as the production of antidrug antibodies (AD As). Immunogenicity can be caused by both patient-related (e.g., patient genetic background and acquired immunity) or product-related factors (e.g., immunogenic sequences present in the product).

[0003] Interactions between antigen-presenting cells (APCs) and T cells initiate the immune response against a biotherapeutic. APCs like dendritic cells (DCs) degrade therapeutic proteins into individual peptide antigens which are presented at the cell surface as part of a complex with major histocompatibility complex (MHC) class II (MHCII) molecules. The resulting peptide- MHC II complexes are then recognized by specific helper (CD4+) T cells. The activation of helper T cells triggers the activation of B cells, eventually leading to the production of AD As. [0004] The presence of T cell epitopes (peptides presented on MHC II that are capable of activating T cells) in the amino acid sequence of a therapeutic protein plays a major role in determining the immunogenic potential of the therapeutic antibody. Potential T cell epitopes may be predicted using in silico tools. Several in vitro methods for assessing the immunogenicity risk of biotherapeutics also have been described. These methods typically investigate the effect of biotherapeutics on T cell proliferation or cytokine secretion from cultured peripheral blood mononuclear cells (PBMCs) from healthy donors. Mass spectrometrybased approaches, often called immunopeptidomics, have been developed to examine the repertoires of peptides presented by human leukocyte antigen (HLA) molecules of MHC class I or class II used for immunosurveillance by CD8+ or CD4+ T cells, respectively. For example, MAPPs assays are typically used to assess immunogenicity potential of biotherapeutics through the identification of immunogenic regions that are processed into peptide fragments by professional APCs in the immune system which are then presented via MHC class II receptors to CD4+ T cells.

[0005] Identification of potentially immunogenic amino acid sequences within protein-based biotherapeutics can lead to mitigation strategies through genetic engineering to remove and replace immunogenic regions/sequences, which will improve the overall quality, efficacy, and safety of biotherapeutic protein products. Thus, assessment of the potential for immunogenicity is essential to ensuring the safety of biotherapeutics.

[0006] MAPPs assays have been used to identify T cell epitopes in antigens, such as birch pollen allergen (Mutschlechner et al., J Allergy Clin Immunol., 125'. 711-718 (2010)), recombinant coagulation factor VIII product (Van Haren et al., PLoS One, S:e80239 (2013)), and SARS CoV-2 spike protein (Knierman et al., Cell Reports, 33: 108454 (2020)). MAPPs assays also have been developed to identify immunogenic epitopes on therapeutic antibodies (see, e.g., Sekiguchi et al., m^fe, 10(8): 1168-1181 (2018); Walsh

and Cohen et al., mAbs, 13(C): el898831 (2021)). While such methods can identify amino acid sequences of immunogenic epitopes, they have not been employed to identify immunogenic epitopes arising from structural changes of therapeutic proteins that can occur during the manufacturing, storage, and transportation process steps that lead up to administration to a patient. These process steps may include protein production (e.g., recombinant production), harvest, purification, formulation, filling, packaging, storage, delivery, and final preparation immediately prior to administration to the patient. During each of these steps, a therapeutic protein is placed in one or more environments that may or may not lead to a change in its structure. The change in structure, which is known as an “attribute,” can lead to the formation of different species of the therapeutic protein that results in a heterogeneous product. An attribute of a therapeutic protein that impacts its clinical efficacy or safety is referred to as a “critical quality attribute.”

[0007] Thus, there remains a need for methods to identify immunogenic epitopes of biotherapeutics in biologically relevant contexts that account for molecular attributes (e.g., high molecular weight (HMW) species, subvisible particle number, aggregation, chemical modifications, and conformational variants) that can impact function and bioavailability.

BRIEF SUMMARY

[0008] In some aspects, the disclosure provides a major histocompatibility complex II (MHC-II)-associated peptide proteomics (MAPPs) assay method, which method includes: (a) isolating monocytes from donor peripheral blood mononuclear cells (PBMCs); culturing the monocytes in a media including L-glutamine, streptomycin sulfate, gentamicin sulfate, GM-CSF, and IL-4 under conditions whereby the monocytes are differentiated into immature dendritic cells (DCs); (b) culturing the immature DCs in the media of (a) further including CD40L, TNF-a, IL-ip, IFN-y, and Poly-IC for up to five days, whereby mature DCs are produced; (c) incubating the mature DCs with a therapeutic protein under conditions whereby the mature DCs process the therapeutic protein, thereby producing one or more peptides, wherein each of the one or more peptides binds to an HLA molecule expressed by the mature DCs to form membranebound HLA-peptide complexes; (d) lysing the mature DCs to produce a membrane-bound protein lysate isolated from cytosolic proteins, wherein the membrane-bound protein lysate includes the membrane-bound HLA-peptide complexes; (e) isolating the membrane-bound HLA- peptide complexes from the membrane-bound protein lysate by immunoprecipitation, wherein the immunoprecipitation is performed with an antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules, optionally wherein the antibody is immobilized on a substrate; (f) eluting the peptides from the HLA-peptide complexes, and (g) identifying the eluted peptides by liquid chromatography-mass spectrometry (LC-MS).

[0009] In some aspects of the assay method, isolating monocytes includes culturing the PBMCs overnight.

[0010] In some aspects of the assay method, non-adherent PBMCs are removed from the media after culturing overnight.

[0011] In some aspects of the assay method, the monocytes are CD14+.

[0012] In some aspects of the assay method, the donor PBMCs are human. In some aspects of the assay method, the monocytes are human.

[0013] In some aspects of the assay method, the culture media of (a) includes 0.05-5 pg/mL GM-CSF, 100-300 ng/mL IL-4, 25-75 pg/mL streptomycin sulfate, 5-15 pg/mL gentamicin sulfate, and 0.15-0.4mg/mL L-glutamine. For example, the culture media of (a) may include 1 pg/mL GM-CSF, 200 ng/mL IL-4, 50 ug/mL streptomycin sulfate, 10 pg/mL gentamicin sulfate, and 0.29 mg/mL L-glutamine.

[0014] In some aspects of the assay method, wherein the culture media of (b) includes 0.05-5 pg/mL CD40L, 25-75 pg/mL TNF-a, 15-35 ng/mL IL-lb, 50-200 ng/mL IFN-y, and 10-30 ng/mL Poly-IC, such as 0.5 pg/mL CD40L, 50 pg/mL TNF-a, 25 ng/mL IL-ip, 100 ng/mL IFN-y, and 20 ng/mL Poly-IC.

[0015] In some aspects of the assay method, the immature DCs are cultured in a flask, such as a T175 flask.

[0016] In some aspects of the assay method, the HLA molecule is an HLA-DR molecule, an HLA-DP molecule, or an HLA-DQ molecule.

[0017] In some aspects of the assay method, the production of mature DCs is assessed by determining the expression of CD11c, CD40, CD80, CD83, CD86, CD209, and HLA-DR, and detecting loss of CD 14 expression during culture of the immature DCs.

[0018] In some aspects of the assay method, less than 10% of proteins in the membranebound protein lysate of (d) are cytosolic, optionally wherein said lysing and isolating is performed using a membrane protein isolation kit.

[0019] In some aspects of the assay method, said lysing and isolating is performed using a membrane protein isolation kit that isolates native proteins.

[0020] In some aspects of the assay method, the antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules is a monoclonal antibody. In some aspects of the assay method, the antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules comprises two or more different antibodies.

[0021] In some aspects of the assay method, the antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules is immobilized on a substrate.

[0022] In some aspects of the assay method, the substrate includes magnetic beads.

[0023] In some aspects of the assay method, the presence of membrane-bound HLA-peptide complexes is determined by a bicinchoninic acid (BCA) assay.

[0024] In some aspects, the assay method further includes performing an HLA moleculespecific enzyme linked immunosorbent assay (ELISA) before and after immunoprecipitation of membrane-bound HLA-peptide complexes. [0025] In some aspects of the assay method, the ELISA is performed with an antibody that specifically binds to HLA-DR.

[0026] In some aspects, the assay method includes two washes of the isolated membranebound HLA-peptide complexes prior to eluting the peptides from the HLA-peptide complexes. [0027] In some aspects, the assay method includes a first wash with 50 mM Tris and a second wash with 10 mM Tris.

[0028] In some aspects of the assay method, the peptides are eluted from the HLA-peptide complexes with 0.1% Trifluoroacetic acid.

[0029] In some aspects of the assay method, the therapeutic protein is an antibody, an antigen-binding fragment of an antibody, or a bispecific T cell engager (BiTE®) molecule. [0030] In some aspects, the assay method is performed on the therapeutic protein, said therapeutic protein including a molecular attribute, and wherein the method is further performed on therapeutic protein not including said molecular attribute, optionally wherein the molecular attribute includes one or more of: one of acidic species, basic species, HMW species, subvisible and visible particle number, aggregation, low molecular weight, middle molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), glycation, non-heavy chain and light chain, deamidation, deamination, cyclization, oxidation, sulfation, hydroxylysine, isomerization, fragmentation/clipping, N-terminal and C-terminal variants, a signal peptide, reduced and partial species, misassembled molecules, domain swapping, folded structure, surface hydrophobicity, chemical modification, covalent bond, thioether, trisulfide, mutations/misincorporations, a C-terminal amino acid motif PARG, or a C-terminal amino acid motif PAR- Amide.

[0031] In some aspects of the assay method, the donor PBMCs are pooled from multiple donors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram of an exemplary workflow of the disclosed MAPPs assay method.

[0033] Figure 2 is a series of flow cytometry plots showing loss of CD14 expression during monocyte maturation to dendritic cells. [0034] Figure 3 is a flow cytometry plot showing that monocyte population makes up about 10% of the full PBMC population.

[0035] Figure 4A is an image illustrating visual confirmation of mature DCs. Figure 4B is a series of graphs showing expression of cell surface markers validating maturation of iDCs into mDCs. The negative peak for each marker is shown on the left in light gray, and the positive peak for each marker is shown on the right in dark gray.

[0036] Figures 5A-5G are graphs of flow cytometry data showing validation of monocyte to DC differentiation as measured by upregulation of DC markers CD40 (5 A), CD80 (5B), CD83 (5C), CD11c (5D), CD86 (5E), CD209 (5F), and HLA-DR (5G).

[0037] Figure 6 is a series of flow cytometry plots confirming that monocytes can be differentiated into immature DCs and then further differentiated into mature DCs as measured by expression of cell surface markers.

[0038] Figure 7 is a graph showing the protein concentration of the membrane-bound protein lysate following isolation.

[0039] Figure 8 is an image of an ELISA assay plate showing reduction of HLA-DR following immunoprecipitation.

[0040] Figure 9 is a diagram showing detection of HLA peptides by LC-MS from Abl Aggregates, Ab2 Aggregates, and KLH aggregates.

[0041] Figures 10A and 10B contain graphs showing that a higher presentation of aggregate Abl peptides (Figure 10A) and aggregate Ab2 peptides (Figure 10B) were presented by HLA- molecules as compared to Abl and Ab2 monomers.

DETAILED DESCRIPTION

[0042] The present disclosure is predicated, at least in part, on the development of an in vitro assay for identifying immunogenic epitopes arising from structural changes of therapeutic proteins. The technology described herein assesses immunogenicity of therapeutic proteins under biologically relevant conditions. In some aspects, the disclosure provides a sensitive major histocompatibility complex II (MHC-II)-associated peptide proteomics (MAPPs) assay method that can identify immunogenic peptides of therapeutic antibodies having one or more molecular attributes (MAs). The technology described herein can assess the risk and impact of molecular attributes on the immunogenicity of therapeutic antibodies, attributes to which conventional immunogenicity assays do not consistently have sensitivity.

Definitions

[0043] To facilitate an understanding of the present technology, several terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0044] In some aspects, the term “assaying” means “measuring,” and may be used interchangeably with the terms “testing,” “analyzing,” or “determining.” The level of peptide, with or without a molecular attribute, which is assayed or determined by the presently disclosed methods can be a relative measurement, e.g., a determination that the level is higher, lower, or the same as a reference level. In some aspects, the “assaying” can yield a normalized measurement. For instance, the normalized measurement can be normalized to a reference protein, e.g., serum albumin. The “assaying” in certain instances yields an absolute measurement (e.g., neither normalized nor relative to a reference level).

[0045] In various aspects, a therapeutic protein is placed in a condition that leads to a change in its structure, for example, a change in the structure of an amino acid of the therapeutic protein, leading to the formation of a species of the therapeutic protein. In exemplary aspects, the changed structure of an amino acid is referred to as an “attribute” and may be characterized in terms of its chemical identity or attribute type and location within the amino acid sequence of the therapeutic protein, e.g., the position of the amino acid on which the attribute is present. Nonlimiting examples of molecular attributes for monoclonal antibodies, or antigen-binding fragments thereof, include high molecular weight (HMW) species, charge variants, oxidized species, deamidated species, and glycosylated species.

[0046] The term “critical quality attribute (CQA)” as used herein, refers to a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs are generally associated with a drug product, drug substance, excipients, and intermediates (in-process materials). For example, for large polypeptide therapeutic molecules, physical or molecular attributes and modifications of amino acids are important CQAs that are monitored during drug development, manufacture, and storage. [0047] In some aspects, the therapeutic protein may be an antigen-binding protein. The term “antigen-binding protein,” as used herein, refers to a proteinaceous molecule that specifically binds to an antigen. For example, an antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof, (such as a monoclonal antibody, for example an IgGl or IgG2 monoclonal antibody), an antibody protein product, a bi-specific T cell engager (BiTE®) molecule, a bispecific antibody, a trispecific antibody, or an Fc fusion protein.

[0048] An antigen-binding protein typically comprises the heavy chain variable region (VH) and/or the light chain variable region (VL) of an antibody, or comprises domains derived therefrom. In some embodiments, an antigen-binding protein comprises the structural requirements of an antibody which are sufficient for immunospecific target binding. This structural requirement may be defined by, for example, the presence of at least three light chain complementarity determining regions (CDRs) (i.e., CDR1, CDR2, and CDR3 of the VL region) and/or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), or of all six CDRs. It is within the knowledge of a skilled person where (and in which order) those CDRs are located in the antigen-binding protein.

[0049] As used herein, the term “antibody” refers to an immunoglobulin of any isotype with specific binding to the target antigen; an antibody may be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc. In a native antibody, a heavy chain comprises a variable region, VH, and three constant regions, CHI, CH2, and CH3. The VH domain is at the amino-terminus of the heavy chain, and the CH3 domain is at the carboxyterminus. In a native antibody, a light chain comprises a variable region, VL, and a constant region, CL. The variable region of the light chain is at the amino-terminus of the light chain. In a native antibody, the variable regions of each light/heavy chain pair typically form the antigenbinding site. The constant regions are typically responsible for effector function. A native antibody is a tetramer of two full-length heavy chains and two full-length light chains.

[0050] In a human antibody, CHI means a region having the amino acid sequence at positions 118 to 215 of the EU index or EU numbering system, which is based on the sequential numbering of the first human IgGl sequenced (i.e., the “EU antibody”) (Edelman et al., Proc Natl Acad Sci USA, 63(1)'. 78-85 (1969)). A highly flexible amino acid region called a “hinge region” exists between CHI and CH2. CH2 represents a region having the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents a region having the amino acid sequence at positions 341 to 446 of the EU index.

[0051] “CL” represents a constant region of a light chain. In the case of a kappa (K) chain of a human antibody, CL represents a region having the amino acid sequence at positions 108 to 214 of the EU index. In a lambda ( ) chain, CL represents a region having the amino acid sequence at positions 108 to 215.

[0052] In a native antibody, the variable regions typically exhibit the same general structure in which relatively conserved framework regions (FRs) are joined by three hypervariable CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Typically, CDR3 is the greatest source of molecular diversity within the antigen binding site. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Publication No. 91-3242, vols. 1-3, Bethesda, Md.); or Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol., 196 901-917. In some embodiments, the CDRs of an antigen binding protein are defined according to the definition of Kabat or Chothia. In the present application, the term “CDR” refers to a CDR from either the light or heavy chain, unless otherwise specified.

[0053] Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgMl and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. Accordingly, in exemplary embodiments, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgGl, IgG2, IgG3 or IgG4. [0054] The antibody can be a monoclonal antibody or a polyclonal antibody. The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352 : 624-628 (1991)); and Marks et al., J. Mol. Biol., 222'. 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., XENOMOUSE™ mouse, Green et al., Nature Genetics 7:13- 21 (1994), US 2003-0070185, WO 96/34096, and WO 96/33735). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0055] The term “chimeric antibody” refers to an antibody containing domains from two or more different antibodies. A chimeric antibody can, for example, contain the constant domains from one species and the variable domains from a second, or more generally, can contain stretches of amino acid sequence from at least two species. A chimeric antibody also can contain domains of two or more different antibodies within the same species. The term “humanized” when used in relation to antibodies refers to antibodies having at least CDR regions from a nonhuman source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies. For example, humanizing can involve grafting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence more similar to a human sequence.

[0056] An antibody can be cleaved into fragments by enzymes, e.g., papain, pepsin, or other engineered site-specific proteases (such as those commercially available from Genovis AB, Lund, Sweden). Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab’)2 fragment and a pFc’ fragment. In exemplary aspects, the antigen-binding protein of the present disclosure comprises an antigen binding antibody fragment. As used herein, the term “antigen binding antibody fragment” refers to a portion of an antibody molecule that is capable of binding to the antigen of the antibody and is also known as “antigen-binding fragment” or “antigen-binding portion.” Tn exemplary instances, an antigen binding antibody fragment is a Fab fragment, a F(ab’)2 fragment, or a single chain fragment variable (scFv).

[0057] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and has a valency (n) range from monomeric (n = 1), to dimeric (n = 2), to trimeric (n = 3), to tetrameric (n = 4), and potentially higher; such alternative formats are referred to herein as “antibody protein products.” Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs (e.g., Fab, Fab’, and F(ab’)2) and VHH/VH. The smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions of the light and heavy chains. A soluble, flexible amino acid peptide linker is used to connect the V regions in a scFv fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment. Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. A VHH/VH (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Heavy chain only antibodies (HcAb) are naturally produced by camelids and sharks. Other antibody protein products include bispecific T cell engager (BiTE®) molecules, disulfide-bond stabilized scFv (ds- scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. A peptibody or peptide-Fc fusion is yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain (see, e.g., Shimamoto et al., mAbs, 4(5): 586-591 (2012)).

[0058] The antigen-binding protein of the present disclosure may comprise any one of the antibody protein products described herein. In exemplary aspects, the antigen-binding protein of the present disclosure may comprise any one of an scFv, Fab VHH/VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, peptibody VHH/VH of camelid heavy chain antibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, bispecific T cell engager (BiTE®) molecule, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, or BsAb conjugate. [0059] In certain aspects, the antigen-binding proteins of the present disclosure may be “bispecific,” meaning that they are capable of specifically binding to two different antigens. In another aspect, the antigen-binding proteins of the present disclosure may be “trispecific,” meaning that they are capable of specifically binding to three different antigens. In another aspect, the antigen-binding proteins of the present disclosure may be “tetraspecific,” meaning that they are capable of specifically binding to four different antigens.

[0060] In some embodiments, the antigen-binding protein is a BiTE® molecule. BiTE® molecules are engineered bispecific antigen binding constructs which direct the cytotoxic activity of T cells against cancer cells. They are the fusion of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kDa. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule. Blinatumomab (BLINCYTO® product) is an example of a BiTE® molecule, specific for CD 19. BiTE® molecules that are modified, such as those modified to extend their half-lives, can also be used in the disclosed methods. By their design, BiTE® molecules are uniquely suited to transiently connect T cells with target cells and, at the same time, potently activate the inherent cytolytic potential of T cells against target cells. See e.g., WO 99/54440, WO 2005/040220, and WO 2008/119567.

[0061] In certain embodiments of the disclosure, the antigen-binding proteins may be multivalent. The valency of the binding protein denotes the number of individual antigen binding domains within the binding protein. In some embodiments, a bispecific antigen binding protein may be multivalent. For instance, in certain embodiments, a bispecific antigen binding protein may be tetravalent by comprising four antigen-binding domains: two antigen-binding domains binding to a first target antigen and two antigen-binding domains binding to a second target antigen.

[0062] As used herein, the terms “antigen binding domain” and “binding domain” may be used interchangeably to refer to the region of the antigen-binding protein that contains the amino acid residues that interact with the antigen and confer on the antigen-binding protein its specificity and affinity for the antigen. In some embodiments, the binding domain may be derived from the natural ligands of the target antigen(s). As used herein, the term “target antigen(s)” refers to a first target antigen and/or a second target antigen of a bispecific molecule and also refers to a first target antigen, a second target antigen, a third target antigen, and/or a fourth target antigen of a tetraspecific molecule.

An antigen-binding protein may comprise an immunoglobulin domain. The term “immunoglobulin domain,” as used herein, refers to a peptide comprising an amino acid sequence similar to that of immunoglobulin (i.e., antibody) and comprising approximately 100 amino acid residues including at least two cysteine residues. Examples of immunoglobulin domains include VH, CHI, CH2, and CH3 of an antibody heavy chain, and VL and CL of an antibody light chain. In addition, the immunoglobulin domain is found in proteins other than immunoglobulin. Examples of the immunoglobulin domain in proteins other than immunoglobulin include an immunoglobulin domain included in a protein belonging to an immunoglobulin super family, such as a major histocompatibility complex (MHC), CD1, B7, T cell receptor (TCR), and the like.

MAPPs Assay

[0063] As described above, a MAPPs assay is a liquid chromatography/mass spectrometry (LC-MS)-based technique used to identify peptide/amino acid sequences from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen- presenting cells, and therefore may induce immunogenicity. The MAPPs assay involves multiple steps, including human primary antigen presenting cell culture, peptide isolation, and peptide identification via liquid chromatography-mass spectrometry (LC-MS). Conventional MAPPs assays are described in Karie AC, Front Immunol., 11. 698 (2020); and Steiner et al., J. Proteome Res., 19, 9, 3792-3806 (2020).

[0064] The present disclosure provides a MAPPs assay method that can identify immunogenic peptides of a therapeutic protein having one or more molecular attributes, resulting in a more biologically relevant assay than conventional MAPPs assays. To this end, the MAPPs assay of the present disclosure differs from conventional MAPPs assays in one or more (or all) of the following aspects: (1) overnight culture of PBMCs, which may be performed in flasks and using pooled donor PBMCs (2) use of cell culture media containing L-glutamine, streptomycin sulfate, gentamicin sulfate, GM-CSF, and IL-4 for dendritic cell (DC) differentiation, (3) use of a cytokine cocktail for DC maturation, (4) isolation of membrane proteins and collection of membrane-bound protein lysate, and/or (5) HLA-DR-specific ELISA of membrane-bound protein lysate pre- and post-immunoprecipitation (IP) and two additional washes (50 mM and 10 mM Tris) prior to peptide elution. An exemplary workflow of the disclosed MAPPs assay method is shown schematically in Figure 1.

PBMC Culture and Monocyte Identification

[0065] In some aspects, the MAPPs assay provided herein comprises isolating monocytes from donor pooled peripheral blood mononuclear cells (PBMCs). It will be appreciated that human PBMCs are isolated from peripheral blood and identified as any blood cell with a round nucleus (e.g., lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells). “Monocytes” are innate immune cells with phagocytic and cytokine-producing functions that contribute to host defense and inflammation.

[0066] PBMCs may either be isolated from whole blood of human donors in a clinical setting using routine methods, or obtained from commercial sources. In some aspects, PBMCs may be freshly isolated or previously frozen/cryopreserved. The PBMCs may be “pooled” from donors in that several PBMC samples from multiple donors are combined together in a batch or pooled sample, on which an assay of interest is performed. Not to be bound by theory, it is believed that that a greater number and diversity of pooled donors elicits a greater immune response and a stronger LC-MS signal in the MAPPs assay described herein. In some aspects, PBMCs may be pooled in the cell culture before DC maturation. As noted above, the provided MAPPs assay method involves culturing the isolated donor PBMCs, such as pooled PBMCs, overnight. In some aspects, by “overnight” is meant at least about 6 hours but no more than about 25 hours (e g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In some aspects, an overnight culture comprises about 8-12 hours, about 12-16 hours, about 16-18 hours, about 18-20 hours, or about 20-24 hours. In some aspects, the PBMCs are cultured for about 24 hours. Without being limited by theory, it is contemplated that incubating donor PBMCs overnight permits higher numbers of monocytes to adhere, permitting greater recovery of monocytes for subsequent use. After overnight culture, any floating, non-adherent PBMCs may be removed from the culture media. Monocytes may be identified in the remaining adherent PBMCs based on expression of CD 14 and/or CD 16 expression. In some embodiments, the monocytes express CD14 (referred to as “CD14+” monocytes). Monocyte-Derived Dendritic Cell Differentiation

[0067] The MAPPs assay described herein further comprises culturing the aforementioned monocytes under conditions by which monocytes differentiate into immature dendritic cells (iDCs) and ultimately mature DCs. The term “dendritic cells (DCs),” as used herein, refers to a specialized type of antigen-presenting cells that mediate innate immunity and initiate adaptive immunity. DCs differentiate through immature and mature stages. Immature DCs (iDCs) are specialized phagocytic cells that differ morphologically and phenotypically from mature DCs. Immature DCs have a round and smooth surface, while mature DCs have a rough surface with multiple pseudopodia. Immature DCs express lower levels of costimulatory molecules, such as CD80, CD86, CD83, and MHC II and secrete lower levels of immunostimulatory cytokines, such as IL-12, IL-10, and TNF. In contrast, mature DCs express high levels of costimulatory molecules and immunostimulatory cytokines, which indicates that DCs are in a phenotypically and functionally mature state (see, e.g., M.K. Kim & J. Kim, RSC Adv., 9, 11230-11238 (2019)). [0068] Cell culture conditions and methods for generating monocyte-derived immature and mature DCs that can be used in the disclosed methods are known in the art. Exemplary conditions and methods are described in, e.g., Chometon et al., PLoS ONE, 15( ); e0231132 (2020); Posch et al., J. Vis. Exp., 118 54968 (2016); Bender et al., J Immunol Methods, 196

121-135 (1996); Romani et al., J. Exp. Med., 180; 83-93 (1994); and Romani et al., J Immunol Methods, 196: 137-151(1996). In addition, any suitable culture vessel may be used, including but not limited to, flasks, vials, dishes, multi-well plates, and roller bottles. In some aspects, a flask may be used, such as a T175 flask, which a has a surface area of about 175 cm². T175 flasks are commercially available from several sources (e.g., ThermoFisher Scientific, Waltham MA; and Corning Inc., Corning, NY). In exemplary aspects, the immature DCs are cultured in a T175 flask.

[0069] In some aspects, the disclosed MAPPS assay comprises culturing PBMC-derived monocytes in a medium comprising L-glutamine, streptomycin sulfate, gentamicin sulfate, GM- CSF, and IL-4 under conditions whereby the monocytes differentiate into immature dendritic cells (DCs). The cell culture medium can contain each of L-glutamine, streptomycin sulfate, gentamicin sulfate, GM-CSF, and IL-4 in any suitable amount. In some aspects, the cell culture media comprises 0.05-5 pg/mL GM-CSF, 100-300 ng/mL IL-4, 25-75 pg/mL streptomycin sulfate, 5-15 pg/mL gentamicin sulfate, and 0.15-0.4 mg/mL L-glutamine. For example, the cell culture medium may comprise 1 pg/mL GM-CSF, 200 ng/mL IL-4, 50 pg/mL streptomycin sulfate, 10 pg/mL gentamicin sulfate, and 0.29 mg/mL L-glutamine. Cell culture media comprising L-glutamine, streptomycin sulfate, and gentamicin sulfate is commercially available as AIM V™ (ThermoFisher Scientific, Waltham, MA) and may be used in the disclosed MAPPs assay method. In such cases, the AIM V™ media may supplemented with GM-CSF and IL -4 in suitable amounts as indicated above. Monocytes may be cultured for a duration of time suitable for generating iDCs. For example, the monocytes may be cultured for about 1 to about 6 days (e.g., 2, 3, 4, or 5 days) at 37 °C. In some aspects, the monocytes are cultured for about five days at 37 °C.

[0070] In further aspects, the immature DCs generated as described above may be differentiated into mature DCs by culturing iDCs in media containing a specialized cytokine cocktail. Not to be bound by theory, it is believed that this specialized “maturation cocktail” results in mature DCs with enhanced antigen processing and presentation capabilities. In some aspects, iDCs are cultured in the same media used for monocyte culture, but further comprising the cytokines CD40L, TNF-a, IL-ip, IFN-y, and Poly-IC. That is, the immature DCs can be cultured in a medium comprising L-glutamine, streptomycin sulfate, gentamicin sulfate, GM- CSF, and IL-4 (e.g., AIM V™ media) further comprising CD40L, TNF-a, IL-ip, IFN-y, and Poly-IC. As for monocyte culturing, the medium for iDC culture can contain each of L- glutamine, streptomycin sulfate, gentamicin sulfate, GM-CSF, IL-4, CD40L, TNF-a, IL-ip, IFN-y, and Poly-IC in any suitable amount. In some aspects, the cell culture media comprises 0.05-5 pg/mL GM-CSF, 100-300 ng/mL IL-4, 25-75 pg/mL streptomycin sulfate, 5-15 pg/mL gentamicin sulfate, 0.15-0.4 mg/mL L-glutamine, 0.05-5 pg/mL CD40L, 25-75 pg/mL TNF-a, 15-35 ng/mL IL-ip, 50-200 ng/mL IFN-y, and 10-30 ng/mL Poly-IC. For example, the cell culture medium may comprise 1 pg/mL GM-CSF, 200 ng/mL IL-4, 50 pg/mL streptomycin sulfate, 10 pg/mL gentamicin sulfate, 0.29 mg/mL L-glutamine, 0.5 pg/mL CD40L, 50 pg/mL TNF-a, 25 ng/mL IL-ip, 100 ng/mL IFN-y, and 20 ng/mL Poly-IC. The iDCs may be cultured for a duration of time suitable for generating mature DCs. For example, the iDCs may be cultured for about 1 to about 6 days (e.g., 2, 3, 4, or 5 days) at 37 °C. In some aspects, the iDCs are cultured for up to about five days (e.g., 5 days) at 37 °C.

[0071] The differentiation of monocytes to DCs may be assessed and confirmed by any suitable method. In some aspects, differentiation of monocytes to iDCs and iDCs to mature DCs may be assessed by measuring cell surface expression of one or more proteins that are characteristic of iDC and/or DC phenotypes. Expression of cell surface markers expression may be measured using flow cytometry. In some aspects, differentiation of iDCs and DM maturation may be assessed by measuring expression of one or more of the following markers: CD80, CD86, CD40, CD83, HLA-DR, CDl lc, and/or CD209. In some aspects, iDCs express or upregulate of all of CD11c, CD40, CD80, CD83, CD86, CD209, and expression of all of these markers may be increased in mature DCs. Other phenotypes indicative of iDCs and mature DCs that may be assessed include cell viability, visual confirmation, and/or loss of CD14 expression. Dendritic cell biology and function are reviewed in M.F. Lipscomb and B.J. Masten, Physiol Rev, 82: 97-130; 10.1152/physrev.00023.2001 (2002).

[0072] Following production of monocyte-derived DC, the mature DCs can be activated by exposure to antigen (in this case, for example, a therapeutic protein such as an antibody), whereupon antigen is internalized and degraded into short peptides. Some of these peptides are presented on major histocompatibility complex (MHC) class II (MHC-II) molecules of the DCs. The resulting peptide-MHC II complexes can then be recognized by specific helper T cells. Thus, in some aspects, the disclosed MAPPs assay method further comprises incubating the mature DCs with a therapeutic protein under conditions whereby the mature DCs process the therapeutic protein, thereby producing one or more peptides, wherein each of the one or more peptides binds to an HLA molecule expressed by the mature DCs to form membrane-bound HLA-peptide complexes.

[0073] MHC molecules are generally recognized as highly polymorphic glycoproteins encoded by MHC class I or MHC class II genes. MHC molecules in humans are also designated “human leukocyte antigens (HLAs).” There are two classes of MHC-molecules: MHC class I molecules and MHC class II molecules. MHC molecules are composed of an alpha heavy chain and beta-2-microglobulin (MHC class I receptors) or an alpha and a beta chain (MHC class II receptors), respectively. Their three-dimensional conformation results in a binding groove, which is used for non-covalent interaction with peptides. MHC class I molecules can be found on most cells having a nucleus, and present peptides that result from proteolytic cleavage of predominantly endogenous proteins and larger peptides. MHC class II molecules can be found predominantly on professional antigen presenting cells (APCs), such as dendritic cells, and primarily present peptides of exogenous or transmembrane proteins that are taken up by APCs during the course of endocytosis, and are subsequently processed. Complexes of peptide and MHC class I molecules are recognized by CD8+ cytotoxic T cells bearing the appropriate T cell receptor (TCR), whereas complexes of peptide and MHC class II molecules are recognized by CD4+helper-T cells bearing the appropriate TCR. In humans, the classical HLA loci include class la (HLA-A, -B, -C), class lb (HLA-E, -F, -G, -H), and class II (HLA-DR, -DQ, -DM, and - DP), which are involved in antigen presentation to CD8+ T cells, natural killer cells (NK cells), and CD4+ T cells, respectively. In some aspects, the one or more peptides of the therapeutic protein bind to an a HLA-DR molecule, an HLA-DP molecule, or an HLA-DQ molecule.

[0074] In some aspects, mature adherent DCs may be dissociated from the culture surface or vessel prior to DC lysis and protein isolation (described further herein). Mature adherent DCs may be dissociated from the culture surface by physical means (e.g., scraping) or enzymatic means (e.g., treatment with trypsin, collagenase, or ACCUTASE® (Innovative Cell Technologies, Inc., San Diego, CA)). It will be appreciated that enzymatic dissociation methods are suitable so long as the enzyme does not disrupt integrity of the receptors on the surface of the DCs. Non-enzymatic dissociation methods may also be used, including, for example, Cell Dissociation Buffer (Sigma Aldrich, Inc., St. Louis, MO) and refrigeration at a temperature between about 0 °C and 8 °C for about 10-30 minutes (e.g., about 15 minutes).

DC Lysis and Membrane Protein Isolation

[0075] A unique feature of the disclosed MAPPs assay method is the isolation of DC membrane-bound HLA-peptide complexes from cytosolic proteins. Not to be limited by theory, it is believed that the native conformation of the peptide is maintained when isolated in membrane-bound form. In contrast, other MAPPs assays employ protein isolation techniques that can unfold HLA-peptide complexes. Thus, the presently disclosed method allows for a more biologically-relevant analysis of potentially immunogenic epitopes of therapeutic proteins. In some aspects, the MAPPS assay described herein comprises lysing the mature DCs to produce a membrane-bound protein lysate isolated from cytosolic proteins, wherein the membrane-bound protein lysate comprises the membrane-bound HLA-peptide complexes. It will be appreciated that the membrane-bound protein lysate is not limited to proteins that are covalently bound to membrane molecules, but further encompasses proteins associated with or immobilized among membrane molecules, for example by non-covalent associations. The membrane-bound protein lysate is isolated from cytosolic proteins in that less than about 10% of proteins in the membrane-bound lysate are cytosolic (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0). [0076] Lysis of DCs and isolation of a membrane-bound protein lysate may be accomplished using any method, system, or kit known in the art, several of which are commercially available. Exemplary membrane protein isolation and purification protocols are described in Smith, SM, Methods Mol Biol 1485: 389-400 (2017). Doi: 10.1007/978-l-4939-6412-3_21; Lin, S-H and G. Guidotti, Methods Enzymol., 463: 619-29 (2009). Doi: 10.1016/S0076-6879(09)63035-4; Pandey et al., Biochemistry and Cell Biology, 94(6): 507-527 (2016). ; and Hunte et al. (eds.), Membrane Protein Purification and Crystallization: A Practical Guide, Elsevier, 2003.

[0077] In some aspects, lysing DCs and isolating of membrane-bound protein lysate may be performed using a membrane protein isolation kit, such as a membrane protein isolation kit that isolates native proteins. The term “native protein,” as used herein, refers to a protein in its properly folded and assembled form with operative structure and function. A native protein may possess primary, secondary, tertiary, and quaternary biomolecular structure, with the secondary through quaternary structures being formed from weak interactions along the covalently-bonded backbone. Native proteins typically are not altered by heat, chemicals, enzymes, or other denaturants. An exemplary membrane protein isolation kit that may be used in the context of the disclosure is the MEM-PER™ PLUS membrane extraction kit sold by ThermoFisher Scientific (Waltham, MA).

[0078] In some instances, the amount of proteins present in the membrane-bound protein lysate isolated may be quantified. Protein quantification methods that may be employed include, but are not limited to, turbidimetric assays, nephelometric assays, mass spectrometry assays, and colorimetric assays. In turbidimetric and nephelometric assays, a protein is quantified from the change in the turbidity of the reaction mixture based on the agglutination of the protein and a protein-specific binding partner. In colorimetric assays, a protein may be quantified with the aid of a color reagent. Colorimetric assays are characterized by formation, change, or depletion of color in the presence of the protein to be quantified. Exemplary colorimetric assays include the Coomassie blue G-250 dye-binding (Bradford), bicinchoninic acid (BCA), and Lowry assay. In some aspects, the presence and amount of proteins in the membrane-bound protein lysate, including HLA-peptide complexes, are determined by a BCA assay or by LC-MS. Immunoprecipitation of Membrane-Bound HLA-Peptide Complexes

[0079] Following production of the membrane-bound protein lysate, the membrane-bound HLA-peptide complexes formed upon DC activation are isolated from the lysate for analysis. The HLA-peptide complexes may be isolated from the lysate using any suitable method known in the art. Such methods include, for example, immunoprecipitation, SDS-PAGE, or chromatography methods. In some aspects, the disclosed MAPPs assay method comprises isolating the membrane-bound HLA-peptide complexes from the membrane-bound protein lysate by immunoprecipitation. It will be appreciated that immunoprecipitation (IP) is a technique for precipitating a target protein out of solution using an antibody that specifically binds to the target protein. By using an antibody that specifically recognizes the target protein, one of skill in the art can selectively “pull down” or precipitate the protein from the mixture, while leaving other molecules behind. Immunoprecipitation generally involves the following steps: incubation of target protein with antibody, antibody -target protein complex formation, precipitation and separation, washing, and elution.

[0080] Advantageously, immunoprecipitation of HLA-peptide complexes is performed with an antibody that specifically binds to multiple HLA alleles, so as to increase the repertoire of peptides detected and the scope of the allelic diversity in the global peptide population that is captured. In some aspects, immunoprecipitation is performed with an antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules. The antibody may be immobilized on a substrate, such as a solid phase support composed of polymeric materials in the form of planar substrates or beads. In some aspects, the substrate is a bead comprised of magnetic or nonmagnetic material. Exemplary non-magnetic beads include, but are not limited to, agarose beads and polystyrene beads. Agarose beads are sponge-like structures which may be of varying shapes and sizes (50 to 150 pm diameter). Polystyrene beads (or “microspheres”) may be coated with recognition molecules, such as antibodies, antigens, peptides, or nucleic acid probes, and can be loaded with hydrophobic dyes and other compounds. Magnetic beads are more widely used in the art for immunoprecipitation, due in part to their smaller size compared to agarose beads (1 to 4 pm in diameter), which collectively give them sufficient surface area for high- capacity antibody binding. Magnetic beads may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluidic. Magnetic beads suitable for immunoprecipitation in connection with the disclosure are known in the art and commercially available from several sources. In exemplary aspects, immunoprecipitation is carried out with FG beads (Tamagawa Seiki Co, Ltd.), which are magnetic beads with a diameter of about 0.2 pm, in which multiple magnetic materials (ferrites) are coated with a poly-glycidyl methacrylate polymer. In addition, immunoprecipitation may be carried out using standard protocols, such as those described in, e.g., Bonifacino, J. S., Dell ’Angelica, E. C. and Springer, T. A. (eds.), Immunoprecipitation. Current Protocols in Molecular Biology . 10.16.1-10.16.29 (2001), and Corthell, J.T. (ed.), Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls, Chapter 8: Immunoprecipitation, Academic Press, pp. 77-81 (2014), and in technical manuals available from commercial manufacturers such as Tamagawa Seiki Co, Ltd., ThermoFisher Scientific, Inc. (Waltham, MA), and Abeam (Cambridge, UK).

[0081] Desirably, immunoprecipitation is carried out under conditions that allow binding of HLA-DP, HLA-DQ, and/or HLA-DR peptide-complexes, if present in the lysate, to the anti- HLA-DP/DQ/DR antibody-conjugated beads. Following a sufficient incubation time, the immunoprecipitation process further comprises one or more washes with a suitable buffer in order to remove unwanted components of the lysate or other debris. In some aspects, the beads are washed at least once with a wash buffer prior to eluting peptides from the anti-HLA- DP/DQ/DR antibody-conjugated beads. In some embodiments, the beads are washed at least twice (e.g., 2, 3, 4, 5, or more times) prior to eluting the peptides from the anti-HLA-DP/DQ/DR antibody-conjugated beads, in which case the wash buffer may be the same for each wash step, or different wash buffers may be used for each wash step. In some aspects, two successive washes are employed. For example, the method may comprise two washes of the isolated membrane-bound HLA-peptide complexes prior to eluting the peptides from the HLA-peptide complexes. Any suitable wash buffer may be used, such as, for example, Tris-buffered saline (Tris), phosphate-buffered saline (PBS), Tween-20 and/or water. In some aspects, the method comprises a first wash with 50 mM Tris and a second wash with 10 mM Tris.

[0082] The one or more peptides may be removed, separated, or “eluted” from the HLA- peptide complexes with an elution buffer for further analysis. An “elution buffer” is a solution used to dissociate the affinity interactions between the peptide and HLA molecule. For protein extraction methods, an exemplary elution buffer may contain water, glycine, citric acid, trifluoroacetic acid (TFA), and/or Tris-HCl. In some aspects, the peptides are eluted from the HLA-peptide complexes with 0.1% tri fluoroacetic acid (TFA). In order to maximize peptide elution, in some aspects, the eluting can be repeated.

HLA-Specific ELISA and Peptide Analysis

[0083] It may be appropriate to confirm successful immunoprecipitation of HLA-peptide complexes from the membrane-bound protein lysate. To this end, the assay method may further comprise assessing the presence of HLA molecules in the membrane-bound protein lysate before and after immunoprecipitation (IP), with depletion of HLA molecules in the lysate post-IP indicative of successful immunoprecipitation of HLA-peptide complexes. The presence of HLA molecules (e.g., HLA-peptide complexes) in the membrane-bound protein lysate may be assessed by any suitable protein-specific detection method, such as those described herein or otherwise known in the art. In some aspects, HLA molecules are assessed by an immunoassay. The term “immunoassay,” as used herein, refers to a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody or an antigen. Any suitable immunoassay may be used, and a wide variety of immunoassay types, configurations, and formats are known in the art and within the scope of the present disclosure. Suitable types of immunoassays include, but are not limited to, enzyme- linked immunosorbent assay (ELISA), lateral flow assay (LFA) (also referred to as a “lateral flow immunoassay”), competitive inhibition immunoassay (e.g., forward and reverse), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), counting immunoassay (CIA), and enzyme multiplied immunoassay technique (EMIT). In some aspects, the disclosed MAPPs assay comprises performing an HLA molecule-specific ELISA before and after immunoprecipitation of membrane-bound HLA-peptide complexes. ELISA (also known in the art as enzyme immunoassay (EIA)) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. In an ELISA, a sample suspected of containing a target molecule (e.g., an HLA molecule) is immobilized on a solid surface (microplate) and then complexed with a target molecule-specific antibody that is linked to a reporter enzyme. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. The ELISA may be specific for any one or combination of the MHC II molecules HLA-DR, HLA-DQ, HLA-DM, and/or HLA-DP. In some aspects, the ELISA is specific for HLA-DR. An HLA molecule is “depleted” in a post-IP lysate sample if the level of HLA molecule is reduced by at least 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) as compared to the level of HLA molecule in the lysate prior to immunoprecipitation.

[0084] As noted above, ELISA-based determination of the presence of a particular HLA molecule in the pre-IP membrane-bound protein lysate and depletion of the HLA molecule in the post-IP lysate is indicative of successful IP. In such cases, the peptide eluted from the HLA- peptide complexes may be analyzed and identified. Peptide analysis and identification may be achieved using one or more one or more chromatography techniques, such as affinity chromatography, anion exchange chromatography, cation exchange chromatography, gelpermeation chromatography, paper chromatography, thin-layer chromatography, gas chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), reverse phase high performance liquid chromatography (RP-HPLC), ultracentrifugation (UC), etc. (see, e.g., Coskun, North Clin Istanb, 3(2): 156-160 (2016)). In other aspects, peptide analysis and identification, including detection of any molecular attributes, may be performed using one or more mass spectrometry (MS) techniques. Generally, mass spectrometry refers to an analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio. In this manner, an MS device can measure the mass of a molecule within a sample. In the case of polypeptide attributes, use of mass spectrometry allows for the assessment of more quality attributes using fewer analyses. In some aspects, eluted peptides may be separated prior to analysis by mass spectrometry. For example, peptides may be separated by liquid chromatography (LC) or gas chromatography (GC) techniques. Liquid chromatography-mass spectrometry (LC-MS) is typically used to analyze thermally unstable and nonvolatile molecules (e.g., biological fluids), while gas chromatography-mass spectrometry (GC-MS) is used for the analysis of volatile compounds (e.g., petrochemicals). Preferably, the assay method comprises identifying the eluted peptides by LC-MS, such as by using high performance liquid chromatography (HPLC). Following separation, peptides are ionized and sent to a mass spectrometer to measure the mass/charge ratio of each peptide. In some aspects, peptides may be further fragmented and analyzed by tandem mass spectrometry (also known as MS/MS or MS²), which produces a MS/MS spectrum containing a sequence of peaks that characterize the mass/charge ratio and intensity of an ion. The experimentally observed spectra obtained by the disclosed method may be compared to theoretical spectra predicted from sequences in protein databases (e.g., Peptide Atlas and National Institute of Standards and Technology (NIST) MS Search) to aid in identification. Mass spectrometry analysis of proteins is further described in, e.g., Hoffmann DE, Stroobant V: Mass spectrometry: principles and applications. 2^nd edition. John Wiley & Sons (2001); Finehout EJ, Lee KH , BiochemMol Biol Educ, 32: 93-100 (2004); and Bakhtiar R, Tse FL, Mutagenesis, 15: 415-30 (2000).

[0085] Other techniques that may be used for peptide analysis and identification include, but are not limited to, X-ray crystallography (Yee et al., J Am Chem Soc, 7^7(47): 16512-7. Doi: 10.102 l/jaO53565+ (2005)); nuclear magnetic resonance (NMR) spectroscopy (Yee et al., supra),' hydrogen deuterium exchange (HDX) mass spectrometry (Englander, The Official Journal of The American Society for Mass Spectrometry, 77(11): 1481-1489 (2006)); and fast photochemical oxidation of proteins (FPOP) (Jones et al., J Biol Chem., 294(32): 11969-11979. Doi: 10.1074/jbc.REVl 19.006218 (2011)).

Therapeutic Proteins

[0086] The MAPPS assay method provided herein can be used to identify potential immunogenic peptide sequences of therapeutic proteins. As used herein “therapeutic protein,” and variations of this root term, has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It refers to a polypeptide for medical use in a subject, typically a human subject. By way of example, a therapeutic protein may be a polypeptide approved for medical use by a government regulatory authority, such as the Food and Drug Administration or the European Medicines Agency. In some embodiments, a therapeutic protein is a therapeutic antigen-binding protein, such as a therapeutic antibody.

[0087] Therapeutic antigen-binding proteins, such as antibodies, encompassed by the present disclosure may include polypeptides that bind to one or more of the following: (i) CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; including those that interfere with receptor binding; (ii) HER receptor family proteins, including HER2, HER3, HER4, and the EGF receptor; (iii) cell adhesion molecules, for example, LFA-I, Mol, pl50, 95, VLA-4, ICAM- I, VCAM, and alpha v/beta 3 integrin; (iv) growth factors, such as vascular endothelial growth factor (“VEGF”), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian- inhibiting substance, human macrophage inflammatory protein (MIP-1 alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGF), including, among others, TGF-a and TGF-0, including TGF-01, TGF-02, TGF-03, TGF- 04, or TGF- 05, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors; (v) insulins and insulin-related proteins, including insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins; (vi) coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, and thrombopoietin; (vii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens; (viii) colony stimulating factors and receptors thereof, including the following, among others, M-CSF, GM-CSF, and G-CSF, and receptors thereof, such as CSF-1 receptor (c-fms); (ix) receptors and receptor-associated proteins, including, for example, flk2/flt3 receptor, CD112 receptor (CD112R), obesity (OB) receptor, LDL receptor, growth hormone receptors, thrombopoietin receptors (“TPO-R,” “c-mpl”), glucagon receptors, interleukin receptors, interferon receptors, T-cell receptors, stem cell factor receptors, such as c- Kit, and other receptors; (x) receptor ligands, including, for example, OX40L, the ligand for the 0X40 receptor; (xi) neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); (xii) relaxin A-chain, relaxin B chain, and prorelaxin; (xiii) interferons and interferon receptors, including for example, interferon-a, -0, and -y, and their receptors; (xiv) interleukins and interleukin receptors, including IL-1 to IL-33 and IL-1 to IL-33 receptors, such as the IL-8 receptor, among others; (xv) viral antigens, including an AIDS envelope viral antigen; (xvi) other proteins such as, e g., lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factoralpha and -beta, enkephalinase, Programmed Cell Death 1 (PD-1), Programmed Cell Death Ligand 1 (PD-L1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), RANTES (regulated on activation normally T cell expressed and secreted), mouse gonadotropin-associated peptide, DNAse, inhibin, activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, myostatins, TALL proteins, including TALL-I, amyloid proteins, including but not limited to amyloid-beta proteins, thymic stromal lymphopoietins (“TSLP”), RANK ligand (“RANKL” or “OPGL”), c-kit, TNF receptors, including TNF Receptor Type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing.

[0088] Examples of therapeutic antibodies suitable for the methods described herein include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, bemarituzumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbtaO5, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, namatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarlatamab, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, or variants of any of the foregoing.

[0089] In some aspects, the therapeutic protein comprises a molecular attribute, such as a critical quality attribute (CQA, defined herein). In such cases, the assay method may be further performed on a therapeutic protein not comprising the molecular attribute, the results of which can be compared to results obtained with the therapeutic protein comprising the molecular attribute to identify attribute-specific structural features that may affect immunogenicity of the therapeutic protein.

[0090] The therapeutic protein may comprise any one or combination of molecular attributes. Examples of molecular attributes include, but are not limited to, acidic species, basic species, HMW species, subvisible and visible particle number, aggregation, low molecular weight, middle molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), glycation, non-heavy chain and light chain, deamidation, deamination, cyclization, oxidation, sulfation, hydroxylysine, isomerization, fragmentation/clipping, N-terminal and C- terminal variants, a signal peptide, reduced and partial species, misassembled molecules, domain swapping, folded structure, surface hydrophobicity, chemical modification, covalent bond, thioether, trisulfide, mutations/misincorporations, a C-terminal amino acid motif PARG, or a C- terminal amino acid motif PAR- Amide. Critical quality attributes and their role in therapeutic antibody development are described in detail in, e.g., Xu et al., mAbs, 77(2): 239-264 (2019). [0091] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0092] This example describes a MAPPs assay method in accordance with the present disclosure. The MAPPs assay was used to assess immunogenic sequences in two monoclonal antibodies (Abl, Ab2) and aggregates thereof (Abl-AG, Ab2-AG), as well as keyhole limpet hemocyanin (KLH).

PBMC Generation of Adherent Monocytes; Monocytes Differentiation into Immature DCs (iDCs) and Maturation of iDCs into mDCs

[0093] 5xl0⁶- 20 xlO⁶ cryopreserved PBMCs (iXCells Biotechnologies, San Diego, CA) were thawed in a 37°C water bath and resuspended in AIM-V media. PBMCs were centrifuged at 1200 rpm for 5 minutes. Supernatant was removed and PBMCs were resuspended in AIM-V and transferred into T-175 flasks to allow for monocytes to adhere overnight. The presence of CD14 was assessed by flow cytometry, the results of which are shown in Figure 2. Figure 3 shows that the monocyte population made up about 10% of the full PBMC population, yielding approximately 18E6 monocytes per treatment.

Differentiation of CD 14 Adherent Monocytes into immature DCs (iDCs) [0094] After overnight incubation, all floating non-adherent cells were removed. IX PBS was added carefully over the adherent monocytes. The flask was then gently swirled to remove any non-adherent cells, which formed a thin film. These cells were removed using a pipette. AIM-V media was added along with GM-CSF and IL-4. The cells were allowed to differentiate for five days at 37°C. Fresh GM-CSF and IL-4 cytokines were added on day 3. Differentiation of monocytes into iDCs was assessed by flow cytometry on day five.

Maturation of iDCs into mDCs

[0095] After five days, all of the media was removed and new AIM-V media was added together with a maturation cocktail containing GM-CSF, IL-4, CD40L, TNF-oc, IL- 10, IFN-y, and Poly-IC. The cells were allowed to mature for 2 days at 37°C. Maturation of iDCs into mDCs was assessed using flow cytometry and by visual inspection of the cells, as shown in Figures 4A and 4B. The upregulation of DC markers showing validation of monocyte to DC differentiation is shown in Figures 5A-5G. Figure 6 shows a molecular assessment confirming that monocytes can be differentiated into immature DCs and then further differentiated into mature DCs according to the methods described herein.

Treatment of mDCs with Antibodies and Stirred Aggregate from Antibodies

[0096] After two days, antibody or antibody aggregate treatments (antigens) were added to AIM-V media with the mDCs. DCs challenged with tetanus toxin, diphtheria toxin, or culture media were used as controls. The cells were allowed to process the antigens for 2 days at 37°C.

Harvesting of mDCs After Two Days of Treatment and Preparation of Membrane-Bound Protein Lysates

[0097] After two days of antigen processing by the mDCs, all of the media was aspirated and the cells were washed with IX PBS. The adherent antigen-sensitized mDCs were dissociated with enzyme-free Cell Dissociation Solution for a total of 10 minutes at 37°C. The cells were centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and the cells were resuspended in 10 mL of IX PBS. The cells were centrifuged at 1200 rpm for 5 minutes. The pellets were then lysed according to the manufacturer’s instructions. Lysis occurred in the presence of DNAse 1 and a protease inhibitor cocktail. Membrane-bound proteins were isolated. Three different membrane protein isolation kits were evaluated (IP Lysis Buffer Kit (Pierce); Membrane-Per Plus Kit (Pierce); and Proteo-Extract Native Kit (Millipore), and HLA-DR was detected (by HLA-DR ELISA assay) in membrane lysates from all three kits. Upon generation of the membrane-bound protein lysates, protein levels were analyzed using the BCA assay. High levels of protein were obtained as shown in Figure 7. Pellets were then frozen at -80°C until immunoprecipitation.

Preparation of HLA-DP/DQ/DR NHS-Magnetic Bead Conjugates for Immunoprecipitation of Membrane-Bound Protein Lysates

[0098] Anti-human HLA-DP/DQ/DR antibody (MHC class II HLA-DR, DP, DQ) was covalently immobilized to FG NHS magnetic beads (Tamagawa Seiki Co, Ltd., TAS8848N1141) using Protein Immobilization Buffer (25 mM MES-NaOH (pH 6.0) for 30 minutes at 4°C. This was followed by further chemical reaction with 1.0 M Amino Ethanol solution (pH 8.0) overnight at 4°C while mixing. The HLA-DP/DQ/DR antibody-NHS bead conjugates were then washed with Protein Immobilized Bead Wash/Storage Buffer (10 mM HEPES-NaOH (pH 7.9), 50 mM KC1, 1 mM EDTA, 10% glycerol) until the immunoprecipitation.

Immunoprecipitation of HLA-DP/DQ/DR-Peptide Complexes, Washes and Elution of Peptides with 0,1% TFA

[0099] Membrane-bound protein lysates were thawed on ice. The Protein Immobilized Wash and Storage Buffer was removed from the NHS conjugated beads. The membrane-bound protein lysates were then added to the HLA-DP/DQ/DR-NHS magnetic bead conjugates and incubated between 16-24 hours at 4°C. The HLA-DP/DQ/DR-peptide-NHS magnetic bead pellets were then washed with a successive set of washes using the following washing buffers in the order shown: (1) 150 mM KCL, (2) 450 mM NaCl and 50 mM Tris, pH 7.4, (3) 150 mM NaCl and 50 mM Tris, pH 7.4, and (4.1) 50 mM Tris followed by (4.2) 10 mM Tris. After the final wash with 10 mM Tris, 0.1% trifluoroacetic acid (TFA) was used to elute the peptides from the HLA-DP/DQ/DR-peptide-NHS magnetic bead pellets for 5 minutes with shaking at room temperature (RT) two times with 50 LIL for a total of 100 pL. The TFA-eluted peptides were then stored at -80°C until confirmation of the presence of HLA-DR in pre- and postimmunoprecipitation (IP) lysate samples. HLA-DR ELISA to Confirm Depletion of HLA-DR Post-IP

[0100] HLA-DR ELISA (Cayman Chemicals, Ann Arbor, MI) was conducted according to the manufacturer’s recommendation on both pre-IP and post-IP samples. The goal was to assess if HLA-DR was present after membrane protein isolation and to assess if HLA-DR was depleted after IP with the HLA-DP/DQ/DR-peptide-NHS magnetic bead conjugate, confirming successful IP. If both were confirmed, the samples were sent for mass spectrometry. The HLA-DR ELISA showed reduction of HLA-DR following immunoprecipitation, as shown in Figure 8.

Liquid Chromatography Mass Spectrometry

[0101] Samples were lyophilized to dryness and evaporated by centrifugation, then dissolved in 0.1% formic acid (FA), 2% acetonitrile (aq). Nano LC-MS was then performed using (1) 20 mm X 75 pM loading column and 150 mm X 75 pM column rp C18 column, (2) 0.1% FA in water (A) or 90% Acetonitrile in water (B), (3) Loading at 300 bar for 6 pL, 300 nL/min, 4-40% B gradient, (4) MS 120K full scan, CID top speed ms². The data were processed with Mass Analyzer software (Z. Zhang, Anal. Chem. 2009, 81 : 8354-8364).

[0102] LC-MS identified HLA-presented peptides from Abl monomer, Abl Aggregates, Ab2 monomer, Ab2 Aggregates, and KLH, as shown in Figure 9. Molecular attributes showed higher presentation by HLA-molecules for Abl and Ab2 proteins comprised of aggregates, as compared to monomer Abl and Ab2 proteins lacking such attributes, as shown in Figures 10A and 10B.

[0103] Furthermore, the immunogenic peptides of Ab2 identified by the MAPPs assay described herein were compared to previously-reported immunogenic peptides of Ab2. Remarkably, the MAPPs assay described herein identified every previously reported high- confidence immunogenic peptide, as well as three new immunogenic amino acid sequences not previously reported. The newly-identified cluster of immunogenic peptides of Ab2 is summarized in Table 1 below.

Table 1

[0104] These results demonstrate that the MAPPs assays herein have superior sensitivity to conventional methods for identifying peptides with immunogenic properties under biological conditions.

EXAMPLE 2

[0105] This example demonstrates use of a MAPPs assay to identify immunogenic sequences in an antigen-binding protein and aggregates thereof.

[0106] Four different preparations of an antigen binding protein (ABP) were tested for immunogenicity using the MAPPs assay described herein: 1 aggregate (ABP-Ag), 2 nonaggregate preparations (ABP-1 and ABP-2), and blend of ABP-1 and ABP-2 (ABP-blend). An internal control antibody also was tested. Monocytes, iDCs, and mature DCs were prepared as described in Example 1. ABP-Ag, ABP-1, ABP-2, and ABP-blend treatments were added to AIM-V media with the mDCs. DCs challenged with tetanus toxin, diphtheria toxin, or culture media were used as controls. The cells were allowed to process the antigens for 2 days at 37°C. [0107] Using the procedures in Example 1, mDCs were harvested after two days of treatment with ABP preparations and membrane-bound protein lysates were prepared.

Immunoprecipitation of the membrane-bound protein lysates was performed with HLA- DP/DQ/DR NHS-magnetic bead conjugates as described in Example 1, followed by washing and elution of peptides with 0.1% TFA. HLA-DR ELISA confirmed depletion of HLA-DR postimmunoprecipitation, and Nano LC-MS was performed as described in Example 1.

[0108] LC-MS identified HLA-presented peptides within a 22 amino acid region of each of the ABP-Ag, ABP-1, ABP-2, and ABP-blend samples. The aggregate sample (ABP-Ag) exhibited the highest abundance of detected peptides. These results demonstrate that the disclosed MAPPs assay can identify immunogenic regions of a therapeutic protein having molecular attributes with high specificity. [0109] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0110] As used herein, the term “about” when used as a modifier to a specified numerical value (e.g., pH of “about” 7.0), indicates that variation around the numerical value can occur. These variations can occur by a variety of means, such as typical measuring and handling procedures, inadvertent errors, ingredient purity, and the like. If greater numerical precision is required, in some embodiments, “about” may refer to numerical values withing ± 5% of the specified numerical value.

[0U1] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0112] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A major histocompatibility complex II (MHC-II)-associated peptide proteomics (MAPPs) assay method, which method comprises: a. isolating monocytes from donor peripheral blood mononuclear cells (PBMCs); and culturing the monocytes in media comprising L-glutamine, streptomycin sulfate, gentamicin sulfate, GM-CSF, and IL-4 under conditions whereby the monocytes are differentiated into immature dendritic cells (DCs); b. culturing the immature DCs in the media of (a) further comprising CD40L, TNF-a, IL- 1 , IFN-y, and Poly-IC for up to five days, whereby mature DCs are produced; c. incubating the mature DCs with a therapeutic protein under conditions whereby the mature DCs process the therapeutic protein, thereby producing one or more peptides, wherein each of the one or more peptides binds to an HLA molecule expressed by the mature DCs to form membrane-bound HLA-peptide complexes; d. lysing the mature DCs to produce a membrane-bound protein lysate isolated from cytosolic proteins, wherein the membrane-bound protein lysate comprises the membrane-bound HLA-peptide complexes; e. isolating the membrane-bound HLA-peptide complexes from the membrane-bound protein lysate by immunoprecipitation, wherein the immunoprecipitation is performed with an antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules, optionally wherein the antibody is immobilized on a substrate; f. eluting the peptides from the HLA-peptide complexes, and g. identifying the eluted peptides by liquid chromatography-mass spectrometry (LC-MS).

2. The assay method of claim 1, wherein isolating monocytes comprises culturing the PBMCs overnight.

3. The assay method of claim 2, wherein non-adherent PBMCs are removed from the media after culturing overnight.

4. The assay method of any one of claims 1-3, wherein the monocytes are CD14+.

5. The assay method of any one of claims 1-4, wherein the media of (a) comprises 0.05-5 pg/mL GM-CSF, 100-300 ng/mL IL-4, 25-75 pg/mL streptomycin sulfate, 5-15 pg/mL gentamicin sulfate, and 0.15-0.4mg/mL L-glutamine, such as 1 pg/mL GM-CSF, 200 ng/mL IL- 4, 50 pg/mL streptomycin sulfate, 10 pg/mL gentamicin sulfate, and 0.29 mg/mL L-glutamine.

6. The assay method of any one of claims 1-5, wherein the media of (b) comprises 0.05-5 pg/mL CD40L, 25-75 pg/mL TNF-oc, 15-35 ng/mL IL- 10, 50-200 ng/mL IFN-y, and 10-30 ng/mL Poly-IC, such as 0.5 pg/mL CD40L, 50 pg/mL TNF-a, 25 ng/mL ZL-10, 100 ng/mL IFN-y, and 20 ng/mL Poly-IC.

7. The assay method of any one of claims 1-6, wherein the immature DCs are cultured in a flask.

8. The assay method of any one of claims 1-7, wherein the HLA molecule is an HLA-DR molecule, an HLA-DP molecule, or an HLA-DQ molecule.

9. The assay method of any one of claims 1-8, wherein the production of mature DCs is assessed by determining the expression of CD11c, CD40, CD80, CD83, CD86, CD209, and HLA-DR, and detecting loss of CD14 expression during culture of the immature DCs.

10. The assay method of any one of claims 1-9, wherein less than 10% of proteins in the membrane-bound protein lysate of (d) are cytosolic, optionally wherein said lysing and isolating is performed using a membrane protein isolation kit.

11. The assay method of claim 10, wherein said lysing and isolating is performed using a membrane protein isolation kit that isolates native proteins.

12. The assay method of any one of claims 1-11, wherein the antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules is a monoclonal antibody, or wherein the antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules comprises two or more different antibodies.

13. The assay method of any one of claims 1-12, wherein the antibody that specifically binds to HLA-DR, HLA-DP, and HLA-DQ molecules is immobilized on a substrate.

14. The assay method of claim 13, wherein the substrate comprises magnetic beads.

15. The assay method of any one of claims 1-14, wherein the presence of membranebound HLA-peptide complexes is determined by a bicinchoninic acid (BCA) assay.

16. The assay method of any one of claims 1-15, which further comprises performing an HLA molecule-specific enzyme linked immunosorbent assay (ELISA) before and after immunoprecipitation of membrane-bound HLA-peptide complexes.

17. The assay method of claim 16, wherein the ELISA is performed with an antibody that specifically binds to HLA-DR.

18. The assay method of any one of claims 1-17, which comprises two washes of the isolated membrane-bound HLA-peptide complexes prior to eluting the peptides from the HLA- peptide complexes.

19. The assay method of claim 18, which comprises a first wash with 50 mM Tris and a second wash with 10 mM Tris.

20. The assay method of any one of claims 1-19, wherein the peptides are eluted from the HLA-peptide complexes with 0.1% Trifluoroacetic acid.

21. The assay method of any one of claims 1-20, wherein the therapeutic protein is an antibody, an antigen-binding fragment of an antibody, or a bispecific T cell engager (BiTE®) molecule.

22. The assay method of any one of claims 1-21, wherein the method is performed on the therapeutic protein, said therapeutic protein comprising a molecular attribute, and wherein the method is further performed on therapeutic protein not comprising said molecular attribute, optionally wherein the molecular attribute comprises one or more of: acidic species, basic species, HMW species, subvisible and visible particle number, aggregation, low molecular weight, middle molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), glycation, non-heavy chain and light chain, deamidation, deamination, cyclization, oxidation, sulfation, hydroxy lysine, isomerization, fragmentation/clipping, N-terminal and C- terminal variants, a signal peptide, reduced and partial species, misassembled molecules, domain swapping, folded structure, surface hydrophobicity, chemical modification, covalent bond, thioether, trisulfide, mutations/misincorporations, a C-terminal amino acid motif PARG, or a C- terminal amino acid motif PAR- Ami de.

23. The assay method of any one of claims 1-22, wherein the donor PBMCs are pooled from multiple donors.