Artificial antigen-presenting molecules and their applications

Abstract

The present invention relates to artificial antigen-presenting cells (aAPCs) comprised of artificial antigen-presenting molecules (aAPMs), particularly dimers of aAPMs, and to methods for producing aAPCs. The invention further relates to compositions comprising aAPCs and vectors encoding the aAPMs of aAPCs. Embodiments of the invention have been specifically developed for use in assays for determining an antigen-specific T cell response or multiple antigen-specific T cell responses, and are described below with reference to the present specification. However, it will be understood that the invention is not limited to this particular field of use.

Description

[Technical Field]

[0001] The present invention relates to artificial antigen-presenting cells (aAPCs) comprising artificial antigen-presenting molecules (aAPMs), particularly dimers of aAPMs, and methods for producing aAPCs. The invention further relates to compositions comprising aAPCs and vectors encoding aAPMs for aAPCs. Embodiments of the invention have been specifically developed for use in assays for determining an antigen-specific T cell response or multiple antigen-specific T cell responses, and are described below with reference to the present specification. However, it will be understood that the invention is not limited to this particular field of use. [Background technology]

[0002] Any discussion of background art herein should in no way be taken as an admission that such art is well known or forms part of the common general knowledge in the art.

[0003] In an immune response, antigenic peptides are usually presented to antigen-specific T cells via antigen-presenting molecules (APMs), i.e., by complexing the antigenic peptide with major histocompatibility complexes (MHC) located on the outer membrane of antigen-presenting cells (APCs). APCs are distinguished by the class of MHC they use to present the antigenic peptide. Most cells in the body present the antigenic peptide to CD8 T cells via MHC class I molecules. + They can present antigenic peptides to cytotoxic T cells. Antigenic peptides presented by MHC class I molecules are usually derived from cytoplasmic proteins. However, "specialized" or "professional" APCs can present antigenic peptides to cytotoxic T cells or CD4 T cells via MHC class I or MHC class II molecules, respectively. + They present antigenic peptides to helper T cells. The antigenic peptides presented by MHC class II molecules are usually derived from extracellular proteins.

[0004] Artificial antigen-presenting cells (aAPCs) have been developed with the goal of generating large numbers of functional antigen-specific T cells in vitro for subsequent therapeutic use. Furthermore, aAPCs can be used to study antigen-specific T cell responses and in assays to evaluate T cell recognition of disease-associated antigens before and during APC-based immunotherapy. Various types of aAPCs are known that utilize membrane-bound APMs, such as MHC class I molecules. Some aAPCs utilize synthetic vesicles or liposomes containing lipid bilayers in combination with membrane-bound APMs. Recombinantly modified aAPCs are also known; for example, mouse fibroblasts transfected with vector constructs for the expression of MHC loaded with specific peptides have been described. Furthermore, microparticle and nanoparticle systems in which APMs are loaded onto the particles have also been developed. However, in such systems, the choice of APM is limited by structural compatibility with the transmembrane domain, which allows stable attachment to the particle, while also enabling specific and stable binding to target T cells to reproducibly elicit antigen-specific T cell responses.

[0005] Additionally, artificial APMs (aAPMs) have been developed as soluble analogs of proteins involved in the induction and / or suppression of immune responses and for use in aAPCs.

[0006] For example, US Pat. No. 6,268,411 B1 describes the use of soluble multivalent peptide-loaded MHC / Ig molecules to detect, activate, and suppress antigen-specific cell-dependent immune responses. These molecules are chimeric molecules in which an MHC class I molecule portion is fused to an immunoglobulin heavy chain. Furthermore, it also describes the covalent attachment of an antigen peptide to the MHC class I molecule portion via a peptide tether. Thus, immunoglobulins containing heavy and light chains are described in which antigen-presenting MHC class I molecule portions are fused to the N-terminus of both heavy chains. While these chimeric molecules are soluble because the entire MHC class I molecule portion is fused to an immunoglobulin containing two heavy chains and two light chains, they are certainly complex molecules, and their suitability for use in aAPCs is questionable.

[0007] There is a need in the art for improved and versatile tools for use in assays to determine antigen-specific T cell responses, including innovative aAPCs that contain specially designed aAPMs that stably bind antigenic peptides while attached to the surface of the aAPCs, so that they reproducibly induce measurable T cell responses when contacted with a T cell population.

[0008] It is an object of the present invention to overcome or ameliorate at least one of the drawbacks of the prior art, or to provide a useful alternative. In particular, it is an object of the present invention to provide improved aAPCs, including aAPMs specifically designed to stably bind antigenic peptides while attached to the surface of the aAPCs, so as to induce a measurable T cell response when contacted with a population of T cells. In particular, it is an object of the present invention to provide improved aAPCs and aAPMs that improve the versatility of assays for determining disease-associated, antigen-induced T cell responses. Summary of the Invention

[0009] As described above, the present invention aims to provide aAPCs for use in versatile assays to induce and determine T cell immune responses to selected antigens. In particular, assessing the recognition of disease-associated antigens by patient T cells can enable clinically relevant personalized therapeutic approaches. Given the limited availability of patient samples in clinical settings, aAPCs suitable for assessing combined immune responses are preferably configured not only to present antigens to induce T cell-mediated immune responses, but also to detect and capture effector molecules specifically released by T cells in response to antigen presentation.

[0010] Thus, in a first aspect, the present invention relates to an artificial antigen-presenting cell (aAPC) for detecting effector molecules of T cells in response to presentation of an antigenic peptide sequence, said aAPC comprising: (a) a surface, wherein the surface is a surface of a particle, and optionally the surface is a surface of a bead; (b) one or more artificial antigen-presenting molecules (aAPMs) attached to a surface via an attachment sequence or via direct chemical conjugation; and (c) one or more capture molecules attached to the surface; wherein each of the one or more aAPMs and / or each of the one or more dimers comprises the same antigenic peptide sequence.

[0011] Different types of particles can serve to provide the surface of the aAPCs of the present invention, i.e., to provide a surface onto which one or more aAPMs and one or more capture molecules are attached. In particular, rigid spherical particles, non-spherical particles, and / or fluid lipid bilayer-containing systems are all suitable particles for providing the surface of the aAPCs of the present invention. For example, polystyrene latex microbeads, magnetic nano- and microparticles or beads, nano-sized quantum dots, and poly(lactic-co-glycolic acid) (PLGA) microspheres are known as rigid spherical particles suitable for use in the aAPCs of the present invention. Carbon nanotube bundles, ellipsoidal PLGA microparticles, and nanoworms are known as known non-spherical particles suitable for use in the aAPCs of the present invention. 2D-supported lipid bilayers (2D-SLBs), liposomes, and RAFTsomes / microdomain liposomes and SLB particles are known as known fluid lipid bilayer-containing systems suitable for use in the aAPCs of the present invention.

[0012] The aAPCs of the present invention provide a robust, easy-to-use, cost- and time-efficient multiplex assessment platform for detecting a large number of different antigen-specific T cells from a sample by utilizing a custom-made aAPC composition tailored for multiplex assessment of T cell responses.

[0013] The aAPMs of the present invention, i.e., the aAPMs that must be attachable to the surface of aAPCs, can be any monomeric, dimeric, or multimeric molecule containing an MHC portion configured to elicit a T cell response, i.e., activate T cells. For example, the aAPMs can be: monomeric, dimeric, or multimeric molecules containing a soluble antigenic peptide-loadable MHC class I or soluble antigenic peptide-loadable MHC class II portion; or molecules containing an antigenic peptide covalently linked to an antigen-presenting domain, preferably co-entrapped. Thus, the aAPMs can be tagged recombinant soluble MHC-I molecules assembled with β2 microglobulin and a synthetic peptide, or tagged recombinant soluble MHC-I molecules covalently linked to β2 microglobulin and assembled with a synthetic peptide. Nevertheless, the antigen-presenting domain of the aAPMs of the present invention can contain any human leukocyte antigen (HLA) allele known to those skilled in the art. A database listing virtually all known HLA alleles, as described in the World Health Organization Nomenclature Commission Report, is available at https: / / www.ebi.ac.uk / ipd / imgt / hla / allele.html. Furthermore, the aAPMs of the present invention are versatile in terms of their potential attachment to the surface of aAPCs. However, the aAPMs of the present invention are not limited to CD8 + and / or CD4 + The ability to stimulate any T cell immune response in an antigen-specific manner particularly enhances versatility.

[0014] Preferably, the aAPM of the aAPCs of the invention comprises (a) a single polypeptide sequence comprising, from amino to carboxyl terminus, an antigen-presenting domain, a dimerization domain, an immunoglobulin (Ig) Fc domain, and an attachment sequence, wherein the antigen-presenting domain sequence comprises an N-terminal antigenic peptide sequence; or (b) a single polypeptide sequence comprising, from amino to carboxyl terminus, an antigen-presenting domain and an attachment sequence, wherein the antigen-presenting domain sequence comprises an N-terminal antigenic peptide sequence. Thus, the aAPMs described herein are not assembled from various separately produced polypeptide chains, but are produced as a single polypeptide chain that already contains all domains and sequences necessary for the aAPM to function in the aAPCs of the invention. In particular, the single polypeptide chain thus produced also contains the antigenic peptide sequence to be presented. Therefore, subsequent loading or conjugation with the antigenic peptide sequence is not required. This avoids the potential inefficiencies and inconsistencies between aAPM production batches that arise from previously described antigenic peptide loading and conjugation procedures.

[0015] Furthermore, the aAPCs of the present invention may contain a dimer of aAPM. Specifically, the aAPCs of the present invention may contain a dimer containing aAPM, wherein (d) the dimer is a homodimer or heterodimer of two aAPMs; or (e) The dimer is a heterodimer of aAPM and a second molecule.

[0016] Thus, in a second aspect, the present invention relates to a composition comprising: (a) a plurality of aAPCs according to the first aspect, wherein the composition comprises a plurality of identical aAPCs, optionally wherein the identical aAPCs comprise a single capture molecule specific for each single effector molecule, or wherein the identical aAPCs comprise several capture molecules specific for several respective effector molecules; or (b) Multiple groups of aAPCs according to the first aspect, wherein all aAPCs in each group are identically coded, the antigenic peptide sequences presented by the aAPCs in each group are identical within the group but different between each group, and optionally the aAPCs in each group comprise a single capture molecule specific for a respective single effector molecule, or the aAPCs in each group comprise several capture molecules specific for several respective effector molecules.

[0017] The aAPMs, aAPCs, and compositions comprising the same of the present invention are particularly useful in various embodiments of multiplexed assays for evaluating antigen-specific T cell responses. Specifically, the aAPMs, aAPCs, and compositions comprising the same of the present invention can be configured to provide various levels of complexity, i.e., various degrees of "multiplexity."

[0018] Thus, in a third aspect, the present invention relates to an assay for determining an antigen-specific T cell response, said assay comprising the steps of:

[0019] (a) contacting a T cell with a composition according to the second aspect under conditions and for a time suitable to elicit a response from the T cell upon presentation of the antigenic peptide sequence by the same aAPC; (b) Isolating aAPCs from T cells; (c) contacting the aAPC with a detection antibody against one or more effector molecules for which one or more capture molecules of the aAPC are specific; (d) analyzing the release of effector molecules from T cells by detecting effector molecules captured by one or more capture molecules of the aAPC; This determines the T cell response specific to the antigen peptide sequence presented by the same aAPC.

[0020] In a fourth aspect, the present invention relates to an assay for determining multiple antigen-specific T cell responses, the assay comprising the steps of: (a) contacting T cells with a composition according to the second aspect under conditions and for a time suitable to elicit a response from the T cells upon presentation of each of the different antigenic peptide sequences presented by the aAPCs of each group; (b) Isolating aAPCs from T cells; (c) contacting the aAPCs with a detection antibody against one or more effector molecules for which one or more capture molecules of the aAPCs in each population of aAPCs are specific; (d) identifying and isolating aAPCs from each of said populations of aAPCs; (e) analyzing the release of effector molecules from T cells by detecting effector molecules captured by one or more capture molecules of aAPCs within each population of aAPCs; This determines multiple antigen-specific T cell responses specific for each antigenic peptide sequence presented by each population of aAPCs.

[0021] In a fifth aspect, the present invention relates to a vector comprising:

[0022] (a) a polynucleotide sequence encoding a single polypeptide sequence of an aAPM as defined in the first aspect; or (b) a polynucleotide sequence for polycistron expression of both peptide chains of a dimer according to the first aspect.

[0023] In a further aspect, the present invention relates to a method for producing aAPCs according to the first aspect, the method comprising covalently bonding (a) an aAPM, preferably an aAPM, which is a single polypeptide sequence comprising, in order from amino terminus to carboxyl terminus, an antigen-presenting domain, a dimerization domain, an immunoglobulin (Ig) Fc domain, and an attachment sequence, wherein the sequence of the antigen-presenting domain includes an N-terminal antigenic peptide sequence; and (b) a capture molecule, preferably a capture antibody; to the surface of a microsphere, preferably a color-coded microsphere. Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows the domain structure and assembly of an exemplary MHC-I dimer (aAPM) for use in aAPCs of the invention.

[0025] [Figure 2] FIG. 1 shows the domain structure and assembly of an exemplary MHC-II dimer (aAPM) for use in aAPCs of the invention.

[0026] [Figure 3] FIG. 1 shows the domain structure of an exemplary polycistronic MHC-I aAPM construct and its assembly into an MHC-I aAPM for use in aAPCs of the invention.

[0027] [Figure 4] FIG. 1 shows the domain structure of additional MHC-I aAPM constructs and their assembly into MHC-I aAPMs for use in aAPCs of the invention.

[0028] [Figure 5] 1 shows the assembly of aAPCs of the invention for use in multiplex assays to determine antigen-specific T cell responses according to the described assays and methods: (a) aAPC concept; (b) architecture and assembly of exemplary aAPCs of the invention; (c) aAPC cooperation system; (d) basic principles of multiplex assays.

[0029] [Figure 6] Two different T-Plex assay workflows are illustrated: (a) the T-Plex assay workflow based on bead spotting and orbital shaking; (b) the T-Plex "rotation-one-tube reaction" principle.

[0030] [Figure 7] FIG. 1 shows the assembly of further aAPCs of the invention for use in multiplex assays for determining antigen-specific T cell responses according to the assays and methods described.

[0031] [Figure 8] FIG. 1 shows an exemplary assembly concept for designing CD4+ and / or CD8+ T-Plex2 assays according to the described assays and methods.

[0032] [Figure 9] FIG. 1 shows proof of concept of the T-Plex assay.

[0033] [Figure 10] FIG. 1 shows that bystander T cells do not reduce the sensitivity of the T-Plex assay, as further described in Example 2 below.

[0034] [Figure 11] 1 shows a comparison of pMHC-I multimer staining with the assay of the present invention according to the experiment of Example 3.

[0035] [Figure 12] FIG. 1 shows the first set of experiments performed to optimize T-Plex assay parameters, as further described in Example 4 below.

[0036] [Figure 13] FIG. 1 shows a second set of experiments performed to optimize T-Plex assay parameters, as described in Example 4 below.

[0037] [Figure 14] FIG. 10 illustrates a number of T-Plex bead assembly variations and their effect on the performance of the T-Plex assay, as further described in Example 5 below.

[0038] [Figure 15] FIG. 1 shows antigen-specific detection of MTB / DR3 CD4+ T cell clone RP15.1.1 by pMHC-II-Fc-loaded T-Plex beads, as further described in Example 6 below.

[0039] [Figure 16] FIG. 1 shows a proof-of-principle experiment of the T-Plex2 assay for antigen-specific detection of CD4+ T cells, as further described in Example 7 below.

[0040] [Figure 17] FIG. 1 shows that antigen-specific T cell detection using the T-Plex assay, as further described in Example 8 below, does not alter the phenotype of the original sample.

[0041] [Figure 18] FIG. 1 shows the successful eukaryotic cell-based production and antigen-specific binding of soluble pMHC-I-FcaAPM, as further described in Example 9 below.

[0042] [Figure 19] FIG. 1 shows the successful production and validation of antigen-specific binding of soluble pMHC-II-Fc aAPM, as further described in Example 10 below. DETAILED DESCRIPTION OF THE INVENTION

[0043] In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given such terms, the following definitions are provided.

[0044] definition In the context of the present application, the terms defined below shall be understood to have the following meanings.

[0045] "Artificial antigen-presenting cell (aAPC)"—an artificially generated surface, including a surface of a biological cell or synthetic material, configured to bind artificial antigen-presenting molecules (aAPMs; defined directly below), including peptide-loaded MHC molecules, and present antigens suitable for stimulating respective antigen-specific T cells, resulting in stimulation similar to that induced by naturally occurring antigen-presenting cells. For example, a particle having multiple defined aAPMs attached to its surface constitutes an aAPC according to the present disclosure. Furthermore, in the context of this application, an aAPC can further be configured, i.e., has the ability, to capture one or more effector molecules secreted by activated T cells. Different types of particles can serve to provide the surface of an aAPC of the present invention, i.e., to which one or more aAPMs and one or more capture molecules are attached. In particular, rigid spherical particles, non-spherical particles, and / or fluid lipid bilayer-containing systems are all suitable particles for providing the surface of an aAPC of the present invention. For example, polystyrene latex microbeads, magnetic nano- and microparticles or beads, nano-sized quantum dots, and poly(lactic-co-glycolic acid) (PLGA) microspheres are known as rigid spherical particles suitable for use in the aAPCs of the present invention; carbon nanotube bundles, ellipsoidal PLGA microparticles, and nanoworms are known as non-spherical particles suitable for use in the aAPCs of the present invention; and 2D-supported lipid bilayers (2D-SLBs), liposomes, and RAFTsomes / microdomain liposomes and SLB particles are known as fluid lipid bilayer-containing systems suitable for use in the aAPCs of the present invention. In certain embodiments, bead-based aAPCs as defined herein are hereinafter referred to as "T-Plex beads."

[0046] "Artificial antigen-presenting molecule (aAPM)" - a soluble or membrane-bound recombinantly produced MHC protein that contains an antigen-presenting domain and a specific, well-defined attachment sequence. For example, the MHC protein can be loaded or can already be loaded with an antigenic peptide so that the aAPM can mediate binding of the aAPM to a T cell receptor with matching (cognate) antigen specificity, while the attachment sequence is configured to immobilize the aAPM on the surface of a biological or synthetic material, such as, for example, a polystyrene-based microsphere or bead.

[0047] "Antigen-presenting domain" - a protein domain of an aAPM (as defined directly above) configured or loaded with an antigenic peptide to be presented, the presentation of which can mediate the binding of a T cell receptor with matching (cognate) antigen specificity. In some embodiments described herein, the antigen-presenting domain of an aAPM is or is derived from: a peptide-loaded β2-microglobulin-associated MHC-I ectodomain encoded by an allelic variant of the HLA-A, B, C, E, or F gene (or the respective polymorphic MHC-I gene from a non-human species); or a peptide-loaded MHC-II ectodomain encoded by an allelic variant of the HLA-DRB gene and HLA-DRA, HLA-DQA / HLA-DQB genes, or HLA-DPA / DPB genes (or the respective polymorphic MHC-II gene from a non-human species). "Derived" in this context means that a domain "derived" from a given first domain must have at least 80% sequence identity with this first domain and, importantly, still be able to mediate binding of a T cell receptor with the matching (cognate) antigen specificity.

[0048] "Dimerization domain" - a protein domain in a monomeric peptide chain that is configured to bind to a corresponding protein domain in another monomeric peptide chain, such that binding of the two corresponding protein domains forms a dimeric molecule consisting of both peptide chains. For example, the hinge domain of immunoglobulin G (IgG), or a heterophilic parallel coiled-coil leucine zipper sequence, or a combination of both, can function as a dimerization domain in the aAPMs described herein.

[0049] An "Ig Fc domain" is a protein sequence consisting of, or closely derived from, constant heavy chain (CH) domains 2 and 3 (CH2 and CH3) of IgG. "Derived" in this context means that a sequence "derived" from a first sequence consisting of constant heavy chain (CH) domains 2 and 3 (CH2 and CH3) of IgG must have at least 80% sequence identity with this first sequence and, importantly, must remain functionally equivalent to the first sequence.

[0050] "Attachment sequence" - a peptide sequence of an aAPM configured to attach the aAPM to the surface of a biological cell or synthetic material (e.g., polystyrene-based microspheres or beads). In certain embodiments, the attachment sequence may be a portion of an Ig Fc domain or a short peptide sequence such as a polyhistidine tag, Strep tag, or biotinylated Avi tag, which facilitates affinity chromatography-based protein purification as well as binding of soluble aAPM to the surface of an aAPC.

[0051] "MHC class I portion" - peptide-loadable MHC-I heavy chain ectodomain (α1-α3) associated with β2-microglobulin.

[0052] "MHC class II portion" - a heterodimeric peptide-loadable MHC-II α chain (α1-α2) ectodomain associated with an MHC-II β chain (β1-β2) ectodomain.

[0053] "Effector molecules" refer to molecules secreted by T cells that have stimulatory or inhibitory immune functions, such as cytokines, perforin, and enzymes such as granzyme B that induce cytotoxicity.

[0054] "Capture molecule" - an antibody or recombinant receptor protein that specifically binds to and thereby captures a defined effector molecule.

[0055] "Costimulatory molecule" - an agonist antibody or recombinant soluble ligand that engages a T cell costimulatory receptor (such as CD28 or 4-1BB).

[0056] "Multiplexing" - refers to a methodological process that allows for the simultaneous and differential detection of multiple analytes in a single reaction; therefore, experimental assays or methods that rely on this process may be referred to in this disclosure as "multiplex assays." In the context of this disclosure, the term "multiplexing" is typically used to describe the simultaneous detection of multiple antigen-specific T cell populations (analytes) through the use of distinguishable aAPCs (such as fluorescent color-coded "barcode" aAPCs) with effector molecule capture capabilities.

[0057] "T-Plex assay" - an assay that can detect multiple antigen-specific T cell populations based on separately distinguishable populations of T-Plex beads used (one-dimensional multiplexing). For example, a T-Plex assay can be performed using separately distinguishable populations of T-Plex beads, with each population displaying a separate aAPM. In such a T-Plex assay, if all T-Plex beads have the ability to detect at least one effector molecule (e.g., interferon-γ), at least two-dimensional multiplex evaluation can be achieved, i.e., (1) detection of multiple antigen-specific T cell populations and (2) simultaneous evaluation of whether the antigen-specific T cell populations secrete effector molecules (here, interferon-γ). A T-Plex assay that can simultaneously detect multiple antigen-specific T cell populations and their secreted effector molecules using T-Plex beads capable of capturing and evaluating multiple effector molecules is called a "T-Plex assay." 2 It's called an assay.

[0058] In addition to the above definitions, unless the context clearly requires otherwise, throughout this specification and claims, the words "comprise", "comprising", and the like shall be construed in an inclusive sense, i.e., "including but not limited to," rather than in an exclusive or exhaustive sense.

[0059] Furthermore, throughout this specification, references to "one embodiment," "some embodiments," or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "some embodiments," or "in an embodiment" in various places throughout this specification do not necessarily, and may, all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure.

[0060] As used herein, unless otherwise specified, describes a common object. Therefore, using ordinal adjectives such as "first," "second," and "third" is simply Indicates that different instances of a similar object are being referenced, and explains why. The objects described are located either temporally, spatially, sequentially, or in some other way. It is not intended to imply that the steps must be in a specific order.

[0061] The term "exemplary" is used herein in the sense of giving an example rather than denoting quality; that is, an "exemplary embodiment" is an embodiment provided by way of example, without necessarily being an embodiment of exemplary quality.

[0062] (Detailed explanation) According to a first aspect of the present invention, and as shown in the Figures, there is provided an artificial antigen presenting cell (aAPC) comprising an artificial antigen presenting molecule (aAPM).

[0063] The aAPM to be used with the aAPC of the present invention, i.e., the aAPM that must be capable of being attached to the surface of the aAPC, can be any monomeric, dimeric, or multimeric molecule that contains an MHC portion configured to induce a T cell response, i.e., activate T cells.

[0064] For example, an aAPM can be: a monomeric, dimeric, or multimeric molecule comprising a soluble antigenic peptide-loadable MHC class I or soluble antigenic peptide-loaded MHC class II moiety; or a molecule comprising a soluble MHC class I or MHC class II moiety covalently linked to an antigen-presenting domain, preferably containing a co-entrapped antigenic peptide.

[0065] Any MHC complex loaded with an antigenic peptide configured to elicit a T cell response and activate T cells can be used as an aAPM after being attached to the surface of an aAPC of the present invention. To be configured to elicit a T cell response and activate T cells, such an MHC complex must meet at least the following criteria: (1) peptide-loaded MHC complexes have the appropriate tertiary peptide / protein structure; and (2) The antigen peptide is correctly presented on the antigen-presenting domain of the MHC complex.

[0066] Of course, in order to be activated by an antigenic peptide, the T cells exposed to the aAPCs of the invention must have previously been exposed to the antigenic peptide, i.e., the T cells are not naive T cells.

[0067] Several different soluble MHC class I and class II molecules that are loaded or can be loaded with antigenic peptides and methods for their production, i.e., aAPMs suitable for use with the aAPCs of the present invention, have previously been described, and some of these aAPMs are commercially available.

[0068] Known aAPMs consisting of such MHC class I or MHC class II moieties include aAPMs in which an antigenic peptide is covalently linked to the aAPM (in the case of MHC class I, these are often called single-chain trimers (SCT aAPMs)), and aAPMs in which the antigenic peptide is not covalently linked to the aAPM (non-SCT aAPMs). Most commercially available aAPMs are non-SCT aAPMs. However, known non-SCT aAPMs can be further classified into (a) those already loaded with the antigenic peptide of interest and (b) those that must subsequently be loaded with the antigenic peptide of interest, i.e., MHC class I or class II aAPMs that are loaded with or can be loaded with the antigenic peptide.

[0069] Monomeric non-SCT MHC class I aAPMs are typically obtained in a denatured form from bacterial lysates and refolded in vitro into MHC complexes with antigenic peptides of interest and β2-microglobulin, as described by Altman et al. (US Pat. No. 5,635,363A). Because each aAPM must be refolded in the presence of the antigenic peptide of interest, generating a complete set of aAPMs covering an entire antigenic peptide library is tedious. To address this issue, aAPMs that refold in the presence of a photocleavable placeholder peptide have been described (Toebe et al., Nat. Med. 12:246-251, 2006). In these molecules, the photocleavable placeholder peptide can then be replaced with any antigenic peptide using ultraviolet light. More recently, methods for producing stable, refolded, but "empty" MHC class I molecules that can be loaded with antigenic peptides of interest through the use of peptide exchange catalysts have been described (Saini et al., Proc. Natl. Acad. Sci. 112:202-207, 2015) and Hein et al., J. Cell. Sci. 127:2885-2897, 2014). However, most MHC class I aAPMs can only be produced in eukaryotic cells with minimal efficiency and extremely low yields. The antigenic peptide-loadable aAPM described by Schneck et al. (US Pat. No. 6,268,411B1) is produced as a recombinant dimeric MHC-mIgG1 fusion construct (full-length mouse IgG1) in a eukaryotic cell line, but is loaded with an unrelated endogenous cellular peptide during the production process. These irrelevant peptides must then be replaced with the antigenic peptide of interest through a subsequent inefficient and incomplete passive peptide exchange reaction, again leading to a reduced yield of fully functional antigenic peptide-loaded aAPM. Furthermore, the manufacturing process proposed by Schneck et al. requires the establishment of a murine B myeloma (J558L)-based producer cell line that provides the missing mouse immunoglobulin (Ig) light chain and must be stably transfected with an MHC-mIgG1 construct, resulting in an overall low efficiency.Furthermore, Schneck et al. (US Pat. No. 6,268,411B1) and Greten et al., J. Immunol. Methods 271:125-135, 2002, have reported the production of soluble dimeric SCT-mIgG1 molecules using the J558L cell line.

[0070] Soluble monomeric murine MHC-I SCT secreted from CHO cells has been previously described (Mottez et al., J. Exp. Med. 181:493-502, 1995). Cell membrane-bound SCT aAPM, specifically those that use an additional disulfide bond, termed "disulfide trap" (dt), to anchor covalently linked antigens in the SCT construct design, have been previously described by Hansen et al. (US20090117153A1). Furthermore, Hansen et al. reported the production of soluble, monomeric disulfide-trapped SCT from bacterial lysates in a denatured form, requiring an in vitro refolding procedure prone to interference before its use.

[0071] Similar to the MHC class I aAPMs described above, slightly different formats for the production of monomeric MHC class II aAPMs have been described; Vollers et al., Immunology 123:305-313, 2008, provides a useful overview. Additionally, MHC class II aAPMs with C-terminal dimerization domains, such as leucine or Jun / Fos zippers, have been reported to stabilize them as heterodimers.

[0072] The aAPMs of the present invention can be easily produced in eukaryotic cells without the need for in vitro refolding and already contain the antigenic peptide sequence during recombinant production, particularly using transient mammalian expression systems such as suspension-grown FreeStyle CHO-S or FreeStyle 293-F cell lines, thereby avoiding these disadvantages of the prior art. In some embodiments, the aAPMs of the present invention can be used as either dimeric or monomeric aAPMs, but must, of course, contain an attachment sequence for attachment to the surface of aAPCs to be suitable for use in the assays of the present invention. A major advantage of the dtSCT-Fc construct design proposed in this application is the ability to efficiently produce correctly folded protein in transiently transfected mammalian expression systems such as suspension-grown FreeStyle CHO-S or FreeStyle 293-F cells.

[0073] In some embodiments of the invention, an aAPM is a single polypeptide sequence comprising, in amino- to carboxyl-terminal order, an antigen-presenting domain, a dimerization domain, an immunoglobulin (Ig) Fc domain, and an attachment sequence, wherein the antigen-presenting domain sequence comprises an N-terminal antigen peptide sequence. The antigen-presenting domain can comprise any human leukocyte antigen (HLA) allele known to those skilled in the art. A database listing known HLA alleles as described in the World Health Organization Nomenclature Commission Report is available at https: / / www.ebi.ac.uk / ipd / imgt / hla / allele.html.

[0074] In such aAPMs, the dimerization domain can comprise an IgG hinge region. Typically, the IgG hinge region comprises SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 is derived from a mouse IgG2a sequence and contains a C224S mutation, while SEQ ID NO: 2 is derived from a human IgG1 sequence and contains a C220S mutation. aAPMs comprising the hinge regions of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, are complementary such that two aAPMs having a dimerization domain comprising SEQ ID NO: 1 or two aAPMs having a dimerization domain comprising SEQ ID NO: 2 dimerize by forming disulfide bridges between the respective cysteine ​​residues in the dimerization domain. Because disulfide bonds are formed only between cysteines, the C224S and C220S mutations were introduced to avoid aberrant disulfidation of these cysteines in the absence of Ig light chains, which normally form intermolecular disulfide bridges.

[0075] In some versions of these aAPMs, a cleavage sequence, such as a His8-tag, biotinylated AviTag, or thrombin cleavage site, is present between the antigen-presenting domain and the dimerization domain (from the amino terminus to the carboxyl terminus), providing additional diversity to the resulting aAPM. For example, as shown in Figure 4, by incorporating such a combination of additional attachment sequences and cleavage sites, a dimeric aAPM can be used as shown in Figure 1, or as an aAPM consisting solely of the antigen-presenting domain and attachment sequence, separated by cleavage, such as thrombin cleavage, to separate the antigen-presenting domain, dimerization domain, and Ig Fc domain. Such cleaved aAPMs can no longer dimerize and therefore function as monomeric aAPMs in the aAPCs of the present invention. For example, the tetrameric aAPC shown in Figure 4 can be assembled using such an aAPM.

[0076] Therefore, the aAPM used in the aAPC of the present invention consists of a single polypeptide sequence comprising, in order from the amino terminus to the carboxyl terminus, an antigen-presenting domain and an attachment sequence, and the sequence of the antigen-presenting domain may include an N-terminal antigen peptide sequence.

[0077] Nevertheless, in embodiments in which the aAPM comprises an Ig Fc domain, the Ig Fc domain of the aAPM is typically a murine IgG2a Fc region comprising constant heavy chain regions 2 and 3 (CH2-CH3) or a human IgG1 Fc (CH2-CH3) region, respectively, that contains the aglycan mutation N297Q or N297A. In particular, the IgG Fc region comprises SEQ ID NO:3 or SEQ ID NO:4.

[0078] In the aAPMs of the present invention, particularly in the embodiments already described in detail above, the attachment sequence is a peptide tag for attaching the aAPM to a surface, particularly a peptide tag for attaching the aAPM to a surface by affinity-based binding and / or conjugation to the surface.

[0079] In some embodiments, the attachment sequence comprises a His-tag (SEQ ID NO: 5), a Strep-tag II (SEQ ID NO: 6), two Strep-tag II sequences flanking a glycine-serine-rich spacer sequence (SEQ ID NO: 7), a Strep-tag II and a C-terminal cysteine ​​residue (SEQ ID NO: 8), and / or a biotinylation attachment site (AviTag) (SEQ ID NO: 9).

[0080] In some embodiments, the above peptide tag sequences can be combined to form attachment sequences with dual specificities.

[0081] The aAPMs of the present invention comprise an antigenic peptide sequence. Typically, the antigenic peptide sequence is an antigen selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, parasitic antigens, autoimmune antigens, allergy-related antigens, and tumor antigens. Generally, the aAPMs are intended to elicit antigen-specific T cell responses, and the antigenic peptide sequence is a sequence of a disease-associated antigen, for example, to identify T cells in a patient sample that have previously been exposed to the antigen. However, those skilled in the art will understand that the clinical relevance of uncharacterized peptide sequences can also be determined by incorporating them as antigenic peptide sequences in the aAPMs of the present invention, since, depending on the T cell response elicited and analyzed, such peptide antigens can be correlated with the disease phenotype attributable to the patient from whom the T cell sample was obtained.

[0082] Some exemplary antigenic peptide sequences are listed below, but those skilled in the art will be aware of antigenic peptide sequences available through established, art-recognized peptide epitope databases such as the Immune Epitope Database (IEDB; accessible online at http: / / www.iedb.org) or the SYFPEITHI database of MHC ligand databases (accessible online at http: / / syfpeithi.de / ). The IEDB lists over 500,000 peptide epitopes, and SYFPEITHI lists over 7,000 peptide sequences known to bind to class I and class II MHC molecules.

[0083] Examples of MHC-I antigen peptide sequences suitable for use with the aAPMs of the invention are the sequences set forth in SEQ ID NOs: 10-29.

[0084] As already indicated, the above candidate antigenic peptide sequences are by no means exhaustive. In certain embodiments, the antigenic peptide sequence is selected from the group consisting of human cytomegalovirus pp65 495-503 (SEQ ID NO: 10); Epstein-Barr virus BMLF-1 259-267 (SEQ ID NO: 13); influenza virus matrix protein 58-66 (SEQ ID NO: 14), NY-ESO-1 157-165 / 165V (SEQ ID NO: 11); and Survivin 96-104 / 97M (SEQ ID NO: 12).

[0085] The aAPM of the present invention advantageously comprises a CD8 + or alternatively, a CD4 + The antigen-presenting domain can be configured to include an antigen-presenting domain from a T cell. Thus, in some embodiments, the antigen-presenting domain of a single polypeptide sequence includes, in amino- to carboxyl-terminal order, an antigenic peptide sequence, a first linker sequence, and an MHC class I portion. Specifically, in such embodiments, the MHC class I portion, in amino- to carboxyl-terminal order, includes a β2-microglobulin sequence, a second linker sequence, an MHC class I HLA-A2 α1 sequence, an MHC class I HLA-A2 α2 sequence, and an MHC class I HLA-A2 α3 sequence. The first linker sequence preferably includes a first cysteine ​​residue, and the MHC class I HLA-A2 α1 sequence includes a second cysteine ​​residue, with the first and second cysteine ​​residues forming a disulfide trap that enhances binding of the antigenic peptide sequence to the MHC class I portion of the antigen-presenting domain via a covalent bond. Preferably, the first linker sequence includes SEQ ID NO: 30.

[0086] The second cysteine ​​residue of the MHC class I HLA-A2 α1 sequence is the result of a tyrosine to cysteine ​​mutation at position 84 of the MHC class I HLA-A*02:01 α1 sequence. Preferably, the MHC class I portion comprises SEQ ID NO:31.

[0087] Additionally, in some embodiments, glutamine residue 115 of the MHC class I HLA-A2 α2 sequence is mutated to glutamic acid to enhance CD8 binding. Preferably, the MHC class I portion comprises SEQ ID NO:32.

[0088] In a preferred embodiment, the aAPM comprises, in order from amino terminus to carboxyl terminus: an antigenic peptide sequence and SEQ ID NO: 33, which comprises a β2-microglobulin sequence (SEQ ID NO: 34), a second linker sequence (SEQ ID NO: 35), an HLA-A*02.A, HLA-A*02:01[Y84C] ectodomain sequence (SEQ ID NO: 36), a third linker sequence (SEQ ID NO: 37), mouse IgG2a-Fc[C224S,N297Q] (SEQ ID NO: 38) and a Strep-tag II (SEQ ID NO: 6) connected to the linker sequence.

[0089] In alternative embodiments, the antigen-presenting domain of the single polypeptide sequence comprises, in amino- to carboxyl-terminal order, an antigenic peptide sequence, a first linker sequence, and an MHC class II moiety. Specifically, in such embodiments, the MHC class II moiety, in amino- to carboxyl-terminal order, comprises an MHC class II HLA-DRβ1 sequence and an MHC class II HLA-DRβ2 sequence.

[0090] Note that the endoplasmic reticulum (ER) leader sequences of SEQ ID NO:39 (derived / modified from influenza virus HA1 protein) and SEQ ID NO:40 (derived from human serum albumin protein) are present in the cDNAs generated for MHC class I and MHC class II β chain aAPMs, respectively, and affect the efficiency of expression / secretion, but are cleaved from the mature proteins. This applies to both MHC-I and MHC-II aAPMs.

[0091] In some preferred embodiments, the DRB1*03:01 / DRA binding peptide ligand may be selected from the peptides of SEQ ID NOs: 41-46.

[0092] The first linker sequence connecting each peptide to the DRB1*03:01 (or other MHC class II alloform) ectodomain may comprise a glycine-serine rich sequence, SEQ ID NO: 47. CLIP (class II-linked invariant chain peptide, human Invariant Chain 103-117 ) can be used as a universal placeholder peptide, such as SEQ ID NO: 48, along with a sequence containing a thrombin cleavage site linking CLIP to various MHC class II DRB alloforms, such as SEQ ID NO: 49.

[0093] In some embodiments, the MHC class II portion comprises the DRB1*03:01 ectodomain sequence of SEQ ID NO:50, and / or comprises, in amino- to carboxyl-terminal order, an MHC class II HLA-DRα1 sequence and an MHC class II HLA-DRα2 sequence, wherein the MHC class II portion comprises the DRA*01:01 sequence of SEQ ID NO:51.

[0094] Again, the HLA-DRA ER leader sequence of SEQ ID NO:52, encoded by the cDNA encoding aAPM, is beneficial for the expression / secretion of aAPM as a soluble protein, but is cleaved off during processing of the mature protein. Thus, the ER leader peptide of the DRA chain (SEQ ID NO:52) is not present in the mature protein of SEQ ID NO:51.

[0095] In aAPMs having an MHC class II antigen-presenting domain, the dimerization domain further comprises an acidic / basic zipper motif, such as the parallel coiled-coiled basic zipper motif comprising SEQ ID NO: 53, or alternatively, an acidic zipper motif, such as the parallel coiled-coiled acidic zipper motif comprising SEQ ID NO: 54. For example, when SEQ ID NO: 53 is present on one chain of an MHC class II heterodimer, SEQ ID NO: 54 is present on the other chain of the MHC class II heterodimer.

[0096] Preferably, the aAPM comprises, in order from amino terminus to carboxyl terminus, the antigenic peptide sequence SEQ ID NO:55, which is forced to be co-expressed with SEQ ID NO:56.

[0097] As already mentioned, the present invention relates to specially designed aAPMs that are configured to form dimers, either homodimers with another aAPM of the same type, or dimers with another molecule to form heterodimers in which only the aAPM contains an antigenic peptide sequence.

[0098] The aAPM of the present invention is specially designed to dimerize. Therefore, the present invention also relates to an aAPC consisting of a dimer comprising the above-mentioned artificial antigen-presenting molecule (aAPM).

[0099] The dimers are either homo- or heterodimers of two aAPMs containing an MHC class I antigen-presenting domain, or heterodimers of one containing an MHC class II antigen-presenting domain and a second molecule.

[0100] In MHC class II aAPM heterodimers, the second molecule contains a single polypeptide sequence consisting, from amino terminus to carboxyl terminus, of an MHC class II portion corresponding to the MHC class II portion of the aAPM but lacking the N-terminal antigenic peptide sequence, a dimerization domain complementary to the dimerization domain of the aAPM, an immunoglobulin (Ig) Fc domain, and an attachment sequence.

[0101] In the heterodimer, the dimerization domain of the second molecule contains an IgG hinge region complementary to the IgG hinge region of aAPM and a parallel coiled-coil acidic / basic zipper motif complementary to the parallel coiled-coil acidic / basic zipper motif of aAPM.

[0102] The attachment sequence of the second molecule of the heterodimer may be either the same as or a different sequence from the attachment sequence of the aAPM, for example, an MHC class II dimer may comprise SEQ ID NO: 55 and SEQ ID NO: 56, which have different attachment sequences.

[0103] Exemplary attachment sequences that allow attachment of aAPM have already been listed and described above. These attachment sequences can also be used as attachment sequences for second molecules. Thus, attachment sequences such as Strep-tag can be used to attach aAPM dimers to beads composed of specifically engineered streptavidin, i.e., Strep-Tactin. Similarly, polyhistidine (His-tag) attachment sequences can be used to attach dimers to nickel nitrilotriacetic acid (Ni-NTA)-coupled beads. Furthermore, site-specific, enzymatically biotinylatable tag sequences (i.e., AviTag) can be used to bind dimers to streptavidin-coupled beads. Furthermore, attachment to surfaces can be mediated by directly linking the N-terminus of aAPM and / or second molecules to the surface of carboxybeads via N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (NHS / EDC) crosslinking. Additionally, the IgG2a-Fc domain can be captured onto beads coated with anti-IgG2a antibodies or Protein A / G, thereby attaching the aAPM and / or a second molecule to the surface of the beads. Similarly, if the attachment sequence is a peptide tag, such a dimer can be attached to beads coated with an antibody specific for the peptide sequence of the tag, such as an anti-His-tag or anti-Strep-tag antibody.

[0104] Where the attachment sequences are different, one skilled in the art will be able to readily select appropriate variations and combinations of attachment sequences, such as a combination of conjugation via a peptide tag and affinity-based attachment.

[0105] Different attachment sequences can be used to purify the heterodimer (DRA / DRB) and remove the homodimer (DRA / DRA+DRB / DRB). However, we have found that the combination of an IgG hinge region and a parallel coiled-coil acidic / basic zipper motif favors the formation of the desired heterodimer. In such embodiments, sequential / differential purification may therefore be ignored.

[0106] However, when using biotinylated constructs, purification via the Strep-tag is not possible, so purification is via the histidine tag. The Strep-tag II / Strep-Tactin purification system contains the Strep-tag II sequence (WSHPQFEK), which binds with high selectivity to Strep-Tactin, a modified / mutated streptavidin. Thus, the Strep-tag allows affinity purification of target proteins fused to the Strep-tag via Strep-Tactin resin. Here, desthiobiotin or biotin, which have high affinity for Strep-Tactin as Strep-tags, are used to elute tagged proteins from Strep-Tactin. Biotin binds almost irreversibly to Strep-Tactin and can only be eluted with NaOH. Therefore, if the target protein is biotinylated, it will bind irreversibly to Strep-Tactin and be virtually impossible to elute. Therefore, a separate purification system is required.

[0107] Thus, the peptide tag of the second molecule allows the dimer to be attached to a surface by affinity-based attachment and / or direct conjugation.

[0108] The attachment sequence of the second molecule can be selected from peptide tags including SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0109] Figure 1 illustrates the domain structure and assembly of an exemplary MHC-I dimer (aAPM) for use in the aAPCs of the present invention. The exemplary disulfide-trapped (dt) peptide-MHC-class I (pMHC-I) immunoglobulin Fc (Fc) aAPM dimer shown contains two single polypeptide chains containing covalently linked T cell epitope peptide ligands (8-11 amino acids), the ectodomain of human β2-microglobulin (β2m), an HLA class I allele, and constant heavy chain (CH) domains 2 and 3 of mouse immunoglobulin isotype IgG2a. The dotted line indicates a flexible glycine-serine linker. An intramolecular disulfide trap between the C-terminal peptide extension and a cysteine ​​(C) residue in place of the MHC-I tyrosine (Y) 84 residue further stabilizes the pMHC complex. The C-terminal Strep-Tag II (STag) sequence allows for affinity purification under neutral conditions.

[0110] Figure 2 shows the domain structure and assembly of an exemplary MHC-II dimer (aAPM) for use in aAPCs of the invention. The exemplary peptide-MHC-class II (pMHC-II) monomeric immunoglobulin Fc fusion aAPM dimer shown comprises an MHC-II aAPM polypeptide chain and a second polypeptide chain (second molecule). The MHC-II aAPM polypeptide chain comprises an antigenic peptide fused to the N-terminus of the MHC-II β chain via a flexible glycine-serine linker. The C-terminus of the β chain ectodomain (β1-β2) is fused to a parallel coiled-coil (pCC) basic zipper, and the hinge domain and CH2 and CH3 of mIgG2a (Fc) are enzymatically biotinylated with a C-terminal hexahydrolase (His6) tag and an AviTag. The second molecule is a polypeptide chain consisting of the ectodomain of the monomorphic MHC-II α chain (α1-α2) fused to a C-terminal complementary acidic pCC-Fc and a C-terminal Strep-Tag II.

[0111] In a first aspect, the present invention relates to an artificial antigen-presenting cell (aAPC) for detecting effector molecules of T cells in response to presentation of an antigenic peptide sequence, the aAPC comprising: (a) a surface, the surface being a surface of a particle, optionally a bead; (b) one or more artificial antigen-presenting molecules (aAPMs), the aAPMs attached to the surface via their attachment sequences or via direct chemical bonds; (c) one or more capture molecules attached to the surface; wherein each of the one or more aAPMs and / or each of the one or more dimers comprises the same antigenic peptide sequence.

[0112] Suitable particles or beads to which aAPMs can be attached have a diameter of 0.5 to 50 μm, particularly 0.5 to 40 μm, particularly 0.5 to 30 μm, particularly 0.5 to 20 μm, particularly 0.5 to 10 μm, particularly 2.5 to 7.5 μm, particularly 3 to 7 μm, particularly 4 to 7 μm, particularly 5 to 7 μm, for example, 6.5 μm. In a specific embodiment of the aAPCs of the present invention, the particles are magnetic MagPlex® microspheres (hereinafter sometimes referred to as "Luminex beads") developed and provided by Luminex Corporation and having a diameter of approximately 6.5 μm.

[0113] The surface of the particle or bead to which the aAPMs are attached to form the aAPCs of the invention must be suitable for attachment of the aAPMs via their respective attachment sequences. In particular, the particle or bead can include sites on its surface that match corresponding sites on the attachment sequences of the aAPMs. For example, (a) The attachment sequence of the aAPM contains a biotin sequence and the particle or bead is coated with streptavidin; (b) the attachment sequence of the aAPM contains a Strep-tag sequence and the particle or bead is coated with Strep-Tactin; (c) The attachment sequence of the aAPM contains an Ig Fc sequence, and the particles or beads are coated with anti-Fc antibodies or protein A / G.

[0114] The capture molecules of aAPCs allow them to capture effector molecules released by T cells in response to antigen presentation by aAPCs. Depending on the aAPMs, and particularly depending on the combination of MHC class I and / or class II aAPMs on the aAPC, effector molecules may be CD4 + and / or CD8 + The one or more capture molecules may be released by T cells in response to presentation of an antigenic peptide sequence. Thus, the one or more capture antibodies may be one or more capture antibodies specific for one or more effector molecules released, or optionally secreted, by T cells. For example, the one or more capture antibodies may be specific for the same effector molecule, or may comprise one or more groups of capture antibodies, each group specific for a different effector molecule. The one or more capture molecules may be capture antibodies specific for one or more effector molecules selected from the group consisting of interferon gamma (IFN-γ), interleukin 2 (IL-2), IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17, IL-21, IL-35, granzyme B, tumor necrosis factor alpha (TNF-α), lymphotoxin alpha (LT-α), and transforming growth factor beta (TGF-β). This list is illustrative and not exhaustive. However, as will be apparent to those skilled in the art, the quality / affinity of the capture antibody has a significant impact on the efficient capture of the effector molecule.

[0115] Contacting T cells with costimulatory molecules during antigen presentation can enhance the T cell response to the presented antigen. Many such costimulatory molecules have been described, some of which are particularly useful in the present invention. Exposing T cells to costimulatory molecules to enhance their response to presentation of antigenic peptide sequences can be achieved either by (a) using soluble costimulatory molecules or (b) using aAPCs of the present invention that further comprise one or more immobilized costimulatory molecules attached to their surface.

[0116] Option (a) may be preferable in some cases compared to option (b), because the attachment of costimulatory molecules does not reduce the capacity of capture molecules on the aAPC surface, maximizing the sensitivity of the aAPCs and, therefore, the assays in which they are used. However, when option (b) is performed, costimulatory molecules can be attached to aAPCs in the same manner as aAPCs themselves. For example, costimulatory molecules can be fusion proteins consisting of an N-terminal stimulatory domain and an immunoglobulin (Ig) Fc domain, and optionally, a C-terminal attachment sequence. In particular, costimulatory molecules are suitable for attachment to aAPCs by affinity-based attachment and / or direct conjugation. Exemplary costimulatory molecules are fusions of the stimulatory domains of ICAM1 (CD54) or LFA-3 (CD58) with the mIgG2a-Fc portion.

[0117] When a pure and pre-characterized T cell population, i.e., a T cell line with only one antigen specificity and "bystander T cell-free," was exposed to the aAPCs of the present invention in combination with specific costimulatory molecules previously described to enhance T cell activation, the inventors found that T cell activation was not additionally enhanced, indicating that T cells are highly efficiently stimulated by the aAPCs of the present invention even in the absence of costimulatory molecules. In particular, it was noted that anti-CD28 / anti-4-1BB or anti-CD2 antibodies did not improve the T cell responses determinable in T cell response assays utilizing the aAPCs of the present invention. For example, the inventors have demonstrated that human cytomegalovirus (HCMV) pp65 495-503 Pure CD8 from various healthy donors specific for the / HLA-A*02:01 pMHC-I complex + A T cell line was used. The virus-specific T cells were identified by (1) pMHC multimer staining for HLA-A*02:01 allele expression (HLA-A2 +) identified in peripheral blood mononuclear cells (PBMCs) from healthy donors, (2) expanded with cognate peptide, (3) isolated using pMHC-Fc (aAPM described in this invention) and magnetic beads, and (4) irradiated cognate peptide-loaded HLA-A2 + The cells were expanded again in PBMC (feeder cells) to obtain a pure T cell line.

[0118] However, it is still considered beneficial to administer costimulatory molecules to T cell samples obtained from tumor patients to enhance the sensitivity of such assays.In particular, the assay using aAPCs of the present invention is particularly suitable for determining patient-specific T cell responses to tumor-specific antigens, i.e., enabling immunological characterization of the patient's T cell population, and thus enabling characterization of the antigenic composition of the patient's tumor.Therefore, the present invention enables the determination of tumor-specific antigens, i.e., immunological characterization of the patient's tumor.

[0119] Furthermore, recombinant human 4-1BBL (CD137L), human CD70, human B7-1 (CD80), or B7-2 (CD86), particularly when fused to IgFc, can function as stimulatory domains in costimulatory molecules suitable for use with the aAPCs of the present invention. Such recombinantly produced costimulatory IgFc fusion molecules may have increased costimulatory activity on T cells compared to corresponding anti-CD28 or anti-4-1BB antibodies that bind to the same costimulatory receptor. In some embodiments, the costimulatory molecule may be attached to the same aAPC as the aAPM. Alternatively, the costimulatory molecule may be attached to a different substrate so that it is available to T cells simultaneously with, or at least in such close time sequence to, exposure of the T cells to the aAPC, so that the stimulatory effect on the T cells is still ongoing when the T cells are presented with antigen by the aAPC. In some embodiments, the costimulatory molecule need not be attached to a surface or substrate, but may simply be available for T cell stimulation as a soluble component of the T cell culture medium.

[0120] The one or more costimulatory molecules are optionally one or more costimulatory antibodies selected from anti-CD2, anti-CD28, anti-CD27, anti-CD134, anti-CD137, or antibodies specific for one or more cell surface-expressed costimulatory T cell receptors selected from the group consisting of B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), LFA-3 (CD58), 4-1BBL (CD137L), and recombinant costimulatory molecules such as OX40L (CD252), CD40L (CD154), and CD70.

[0121] In one or more preferred embodiments, the one or more costimulatory antibodies are specific for the same costimulatory receptor, or the one or more costimulatory antibodies comprise one or more groups of costimulatory antibodies, each group being specific for a different group of the same costimulatory receptor.

[0122] The aAPCs of the present invention provide various levels and / or degrees of multiplexing. For example, aAPC particles may be coded to be distinguishable and separable from other particles, and optionally, the particles may be color-coded. In such embodiments, the color code indicates the antigenic peptide sequence of the aAPM attached to the aAPC. Thus, color-coded particles can be separated by flow cytometry analysis. Additionally or alternatively, the particles may be magnetic. By sorting and separating aAPCs based on the specific antigen or combination of antigens presented to T cells, the effector molecules captured by the capture molecules of the aAPCs can be analyzed. T cells can be captured by aAPCs by considering that individual T cells interact with individual aAPCs, resulting in physical proximity between the T cells and the aAPCs, allowing the effector molecules released by the T cells to be captured by the aAPCs presenting specific antigens to the cells. In this way, aAPCs can be separated based on the identity of the antigen they present or the capture molecules they bind, allowing us to dissect the combination (profile) of effector molecules released by T cells in response to a particular antigen.

[0123] A second aspect of the present invention provides a composition comprising a plurality of aAPCs according to the first aspect, for use in the assay of the present invention, wherein the composition comprises a plurality of identical aAPCs. The identical aAPCs comprise a single capture molecule specific for each single effector molecule, or the identical aAPCs comprise multiple capture molecules specific for each of multiple effector molecules. Such a composition is particularly useful for assessing the profile of effector molecules released in response to the presentation of a specific antigen in the assay of the third aspect of the present invention, as further described below.

[0124] Alternatively, in the composition of the second aspect, all aAPCs in each group are identically coded, and the antigenic peptide sequences presented by the aAPCs in each group are identical within the group but different between the groups. Preferably, each group of aAPCs contains a single capture molecule specific for a single respective effector molecule, or each group of aAPCs contains multiple capture molecules specific for several respective effector molecules.

[0125] Such compositions provide additional degrees and / or levels of multiplexing, and are particularly useful for assessing multiple T cell responses of T cell populations against different aAPCs, i.e., different antigens presented thereon, or for including multiple capture antibodies on aAPCs to detect multiple effector molecules in assays according to the fourth aspect of the invention, as described further below.

[0126] Figure 3 shows the domain structure of an exemplary polycistronic MHC-I aAPM construct and its assembly into an MHC-I aAPM for use in aAPCs of the invention. The exemplary polycistronic MHC-I aAPM construct shown is a pMHC-I heterodimer biotin-tagged Fc construct (pMHC-I-pCC-Fc), consisting of two separate polypeptide chains coexpressed in a single vector via a T2A sequence. The first aAPM polypeptide chain contains the pMHC-I portion as a single-chain trimer (SCT) (disulfide-trapped peptide ligand, β2m, HLA class I allele ectodomain) fused to a parallel coiled-coil (pCC) basic zipper, followed by the hinge domain and CH2 and CH3 of mIgG2a (Fc), and a combined C-terminal attachment sequence containing a His6 tag and AviTag for site-specific biotinylation. The second aAPM polypeptide chain contains the same pMHC-I portion but includes complementary acidic pCC and Fc domains and a C-terminal Strep-Tag II attachment sequence. Coexpression of a BirA ligase molecule fused to the ER retention signal sequence KDEL (BirA-KDEL) results in site-specific biotinylation of the indicated pMHC-I-pCC-Fc aAPM in vivo. The resulting biotinylated pMHC-I-pCC-Fc-Bio aAPM can be multimerized to pMHC-I octamer on a streptavidin surface or immobilized on streptavidin-coated beads to generate aAPCs of the invention, as shown.

[0127]

[0023] Figure 1 shows the domain structure of additional MHC-I aAPM constructs for use in aAPCs of the invention, and their assembly into MHC-I aAPMs. An exemplary pMHC-I-homodimer biotin-tagged aAPM construct has a cleavable Fc region (pMHC-I-AviTag-TCS-Fc). In particular, the construct shown contains a single polypeptide chain comprising the pMHC-I complex as a single-chain trimer (single-chain-timer, SCT, described for Figure 3 above) fused, in order, to an octahistidine (His8)-tag and AviTag, followed by a thrombin cleavage site (TCS) sequence. The cleavage site is fused at its C-terminus to the hinge domain and CH2 and CH3 of mIgG2a (Fc), and optionally to Strep-Tag II (C-terminus of Fc). The pMHC-I-AviTag-TCS-Fc aAPM construct is site-specifically biotinylated in vivo by coexpression of BirA ligase fused to the ER retention signal sequence KDEL (BirA-KDEL). The Fc portion is cleaved by thrombin to generate biotinylated pMHC-I aAPM monomers. The pMHC-I-biotin aAPM monomers can be multimerized into pMHC-I multimers using soluble streptavidin or immobilized on the surface of streptavidin-coated beads to generate aAPCs of the invention as shown.

[0128] In a third aspect, the present invention relates to an assay for determining an antigen-specific T cell response, said assay comprising the steps of: (a) contacting a T cell with a composition according to the second aspect under conditions and for a time suitable to elicit a response from the T cell upon presentation of the antigenic peptide sequence by the same aAPC; (b) Isolating aAPCs from T cells; (c) contacting the aAPC with a detection antibody against one or more effector molecules for which one or more capture molecules of the aAPC are specific; (d) analyzing the release of effector molecules from T cells by detecting effector molecules captured by one or more capture molecules of the aAPC; This determines the T cell response specific to the antigen peptide sequence presented by the same aAPC.

[0129] In a fourth aspect, the present invention relates to an assay for determining multiple antigen-specific T cell responses, said assay comprising the steps of: (a) contacting T cells with a composition according to the second aspect under conditions and for a time suitable to elicit a response from the T cells upon presentation of each of the different antigenic peptide sequences presented by the aAPCs of each group; (b) Isolating aAPCs from T cells; (c) contacting the aAPCs with a detection antibody against one or more effector molecules for which one or more capture molecules of the aAPCs in each population of aAPCs are specific; (d) identifying and isolating aAPCs from each of said populations of aAPCs; (e) analyzing the release of effector molecules from T cells by detecting effector molecules captured by one or more capture molecules of aAPCs within each population of aAPCs; This determines multiple antigen-specific T cell responses specific for each antigenic peptide sequence presented by each population of aAPCs.

[0130] Regarding the conditions and time suitable for eliciting a response from T cells in the assays according to the third and fourth aspects of the invention, certain important parameters have been identified, as shown in the Examples below, but the skilled artisan will be able to determine the most suitable conditions for success based on general knowledge of the culture conditions and parameters necessary to ensure proper maintenance of T cells used in T-Plex assays and by employing routine experimentation.

[0131] For the assay to be successful, it is important to keep the sample moving during the step of contacting the T cells with the composition containing aAPCs so as to produce a uniform mixed suspension of aAPCs and T cells. Such motion can be generated by gently but constantly rolling the reaction vessel on a laboratory roller ("One-tube reaction"), as shown in Figure 6(b). For example, an assay tube (approximately 1 cm in diameter) is placed horizontally between two rollers (approximately 3 cm in diameter) of a mixing / rolling device, and the assay tube is rotated along its longitudinal axis. Because the rotations per minute of the device closely match the rotations per minute of the assay tube, appropriate reaction conditions can be achieved while minimizing sample cross-contamination. In particular, without being bound by theory, constant motion is thought to limit cross-contamination (or "cross-bleeding") of soluble effector cytokines, namely IFN-γ, on bystander aAPCs linked to mismatched aAPMs. This is thought to occur under static conditions, where aAPMs are in close proximity to activated T cells in contact with matched aAPCs. In other words, constantly rolling the reaction vessel prevents cross-contamination / cross-bleeding of effector molecules captured on aAPCs that have not yet initiated a T cell response. Alternatively, in non-rolling reaction vessels such as 12-, 24-, 48-, and 96-well plates, the test sample can be moved by 3D / orbital movement of the vessel as shown in FIG. 6(a).

[0132] Alternatively, different colored beads can be spatially separated and immobilized in a shared assay room (spotting beads on a 6-well plate with a magnet underneath), followed by orbital / 3D mixing of the test sample. Furthermore, when using reaction vessels that facilitate rolling, centrifugation prior to rolling to compact the cells and aAPCs has proven effective in improving assay sensitivity. Without being bound by theory, the beneficial effect of this initial centrifugation is likely to maximize the chances that aAPCs and cognate T cells, due to their similar physical properties such as shape (diameter) and weight, will be positioned in close proximity within the pellet, i.e., positioned close to each other from the start of the assay. For example, human T cells have diameters in the 7-9 μm range, while Luminex beads used in some embodiments of the aAPCs of the present invention have diameters of approximately 6.5 μm. Thus, T cells and aAPCs are likely to reside in similar volumes in the three-dimensional space of the reaction volume, i.e., in close proximity within the pellet. This increases the likelihood of direct interaction between aAPCs and T cells.

[0133] The sensitivity of the assay also depends on the specificity and affinity of the capture molecule used in the aAPC. In some embodiments, the sensitivity of the assay can be improved by approximately two-fold by using a "better" anti-IFN-γ antibody clone as the capture molecule. Furthermore, when IFN-γ, IL-2, or TNF-α are the effector molecules to be captured, it is believed that 30-50% of the binding molecules on the aAPC must be pMHC (aAPM) to elicit an appropriate T cell response when a composition containing aAPC is contacted with T cells under appropriate conditions for approximately 4-6 hours.

[0134] A time period of 4 to 6 hours seems optimal, and activated CD8 +Consistent with reported observations that IFN-γ secretion by T cells has a lag phase of approximately 90 minutes (Dushek et al., Sci. Signal. 4(176):ra39, 2011), IFN-γ secretion peaks 4 hours after the initial stimulation of T cells (Betts et al., J. Immunol. Methods. 281(1-2):65-78, 2003), whereas longer exposure times increase the background signal caused by bystander effector molecule capture on aAPCs that did not elicit a T cell response.

[0135] In the assays of the third and fourth aspects, the T cells are purified T cells.

[0136] Figure 5 illustrates the assembly of aAPCs of the invention for use in multiplex assays to determine antigen-specific T cell responses according to the described assays and methods. In particular, the use of aAPCs of the invention to bind T cell specificities to defined bead colors is shown to illustrate the first level of multiplexing possible through aAPCs of the invention. (a) Concept of the aAPC. An exemplary aAPC for use in the illustrated assays of the invention is a bead-based, color-coded aAPC with T cell effector molecule capture capabilities, specifically the ability to capture the cytokine interferon-γ (IFN-γ). The aAPC is assembled by binding defined pMHC-I or pMHC-II and any other costimulatory molecules and effector cytokine capture antibodies to color-coded beads. (b) Construction and assembly of an exemplary aAPC of the invention. In the first step, an IFN-γ capture antibody (mouse IgG1 isotype) and a monoclonal rat anti-murine IgG2a antibody were covalently coupled in a 3:2 ratio to colored carboxylated magnetic polystyrene microparticles (MagPlex® Microspheres, Luminex Corp., hereafter referred to as "Luminex beads"). In the second step, the beads were loaded with saturating amounts of aAPM using either crude supernatant from CHO-S cells expressing aAPM or pMHC-I-mIgG2a-Fc purified by affinity chromatography. (c) Coordination system of aAPCs. A two-dimensional dot plot of 30 aAPC pools using the above Luminex beads and their respective regions (positions) measured by flow cytometry is shown. Each aAPC pool can be easily conjugated to a defined T cell epitope by conjugation with the respective pMHC-I or pMHC-II aAPM. (d) Basic principle of the multiplex assay: Color-coded aAPCs (T-Plex beads) conjugated with pMHC-Fc and anti-IFN-γ capture antibodies activate T cells in an antigen-specific manner, promoting IFN-γ secretion from the activated cognate T cells. The secreted IFN-γ is captured by the same beads and can be detected by a fluorescent dye-labeled αIFN-γ detection antibody.T-Plex beads can be analyzed with an appropriate bead analyzer (FACS) based on their inherent color (bead sorting) and IFN-γ loading. dt-pMHC-I-Fc dimer: disulfide-trapped (dt) peptide-MHC-class I (pMHC-I) immunoglobulin Fc (Fc) dimer; mAb: monoclonal antibody.

[0137] Figure 6 illustrates two exemplary T-Plex assay workflows. (a) T-Plex assay workflow involving bead spotting followed by orbital shaking. To enable multiplexing and avoid IFN-γ cross-bleeding, color-coded T-Plex beads loaded with different pMHC-Fc aAPMs are spotted onto individual magnetic reaction fields generated by a 96-well magnet placed under an appropriate reaction chamber (6-well plate or plate lid). T cells are added to the medium-filled chamber, and the entire reaction is incubated at 37°C under gentle, constant 3D orbital stirring for 4–6 hours. In the final step, T cells and beads can be magnetically separated. All beads are then pooled and their IFN-γ loading is analyzed. (b) T-Plex "rotation one-tube reaction" principle. Color-coded T-Plex beads loaded with different pMHC-Fc aAPMs are loaded into a conical skirted tube along with CO2-saturated assay medium and the T cell sample to be analyzed. The assay tube is closed and rotated at 37°C for 4-6 hours as shown. Finally, the magnetic beads are collected with a magnet and washed. The T-Plex beads are then analyzed for IFN-γ loading.

[0138] Figure 7 illustrates the assembly of additional aAPCs of the invention for use in multiplex assays to determine antigen-specific T cell responses according to the assays and methods described. In particular, antigen-specific CD4 +The use of aAPCs of the invention for T cell detection and parallel functional profiling is shown to illustrate some of the levels of multiplexing possible through aAPCs of the invention. + T cell detection and parallel functional profiling. Illustrating the workflow of two different T-Plex assays: (a) CD4 + For multiplex-based antigen-specific and functional phenotypic profiling of T cell populations, aAPCs consisting of a variety of different capture antibodies are used to bind specific aAPMs, such as pMHC-II-pCC-Fc and other optional costimulatory molecules, together with several different effector cytokine capture antibodies on color-coded beads (T-Plex). 2 (b) T-Plex 2 The beads (aAPC) bind to cognate CD4 + Activates T cells and promotes cytokine secretion depending on the functional phenotype and activated T cells. Th1CD4 + The decisive cytokine for T cells is IFN-γ, but Th2 differentiation leads to the secretion of IL4, Th17 differentiation leads to the secretion of IL17, and Treg differentiation leads to the secretion of IL10. The secreted cytokines are captured in close proximity to the same beads and can be detected with a panel of detection antibodies labeled with different fluorescent dyes (dyes A to D) depending on the cytokine.

[0139] Figure 8 shows CD4 + and / or CD8 + T-Plex 2 Figure 1 shows an exemplary assembly concept for designing an assay. Combining different cytokine capture antibodies with cytokine detection antibodies conjugated to different fluorescent dyes allows for two-step multiplexing, i.e., two-dimensional detection (T-Plex). 2The first dimension of the multiplex assay is coded by the internal color aAPC and reflects T cell antigen specificity, while the second dimension is based on detecting multiple different cytokines on the same T-Plex beads upon cognate T cell stimulation. This allows for the detection of multiple cytokines, including CD8+, CD8+, and CD8+ T cells, within a single assay reaction. + Not only the T cell pool, but also antigen-specific CD4 + This allows differentiation and analysis of the functional profile of the T cell pool.

[0140] Figure 9 shows the proof-of-concept of the T-Plex assay. In particular, the results of the experiment described in Example 1 below demonstrate the efficacy of three different pure antigen-specific CD8 + This highlights the multiplex detection capabilities of the T-Plex assay using T cell lines (model systems) / pMHC-I.

[0141] In particular, Examples 1-7 describe assays according to the third and fourth aspects of the present invention. The assays of the third and fourth aspects may include the additional steps of separating aAPCs from T cells, washing the aAPCs under conditions suitable to maintain T cell viability, and then recovering the separated T cells for further in vitro cell culture. Such T cell recovery is described in detail in Example 8 below.

[0142] In a fifth aspect, the present invention relates to a vector comprising: (a) a polynucleotide sequence encoding a single polypeptide sequence of an aAPM as defined in the first aspect; or (b) A polynucleotide sequence for polycistronic expression of both peptide chains of a dimer as defined for the first aspect.

[0143] Sequence information for a polynucleotide vector sequence suitable for encoding a single polypeptide sequence of an aAPM of the first aspect of the invention, and for polycistronic expression of both peptide chains of a dimer of the second aspect, is provided as SEQ ID NOs: 57 and 58, respectively.

[0144] In a further aspect, the present invention also relates to a method of producing an aAPC according to the first aspect, the method comprising covalently bonding: (a) an aAPM, preferably an aAPM, which is a single polypeptide sequence comprising, in order from amino terminus to carboxyl terminus, an antigen-presenting domain, a dimerization domain, an immunoglobulin (Ig) Fc domain, and an attachment sequence, wherein the sequence of the antigen-presenting domain includes an N-terminal antigenic peptide sequence; and (b) a capture molecule, preferably a capture antibody; to the surface of a microsphere, preferably a color-coded microsphere. [Example]

[0145] The present invention is further illustrated by the following non-limiting examples.

[0146] Example 1: Detection of a defined set of T cell lines by antigen-specific multiplex detection using the T-Plex assay In this experiment, three different pure antigen-specific CD8 + This demonstrates the multiplex detection capability of the T-Plex assay using a T cell line (model system) / pMHC-I.

[0147] Precursor T-Plex beads (anti-INFγ mAb and anti-mIgG2a-Fc (Fc) mAb-conjugated Luminex beads) of four different colors (Luminex bead ID / region: 012, 013, 014, 018) were loaded with a defined set of pHLA-A2-Fc aAPMs. 10,000 beads per aAPM-loaded T-Plex bead ID / T cell epitope were combined with the indicated amount of antigen-specific T cell line in a single 500 μl conical tube filled with approximately 500 μl of CO2-saturated cell culture medium. T-Plex assays were performed at 37°C for 4 hours at 40 rpm. The presence of T cell lines was indicated by the appearance of a subpopulation of IFN-γ-loaded (IFN-γ+ beads) cognate T-Plex beads over control beads, as shown in Figure 9. EBV BMLF-1 259-267 / HLA-A2(A2)-specific CD8 + T cell line #0144 (EBV / A2 T cell), HCMV pp65 495-503 / A2-specific CD8 + T cell line #416 (CMV / A2 T cells), Survivin 96-106 / A2-specific CD8 + A T cell line (Sur / A2 T cells) was used. The T-Plex data rows in Figure 9 are from the same reaction / bead mix (multiplex detection). pHLA-A2-Fc aAPMs were used for T-Plex bead assembly. Survivin 96-104 / HLA-A2-Fc(Survivin / A2-Fc), Influenza MP-1 58-66 / A2-Fc (Flu / A2-Fc), HCMV pp65 495-503 / A2-Fc (CMV / A2-Fc) and EBV BMLF-1 259-267 / A2-Fc (EBV / A2-Fc).

[0148] Example 2: Bystander T cells do not reduce the sensitivity of the T-Plex assay To analyze whether a certain amount of bystander T cells impairs the detection sensitivity of the T-Plex assay, we spiked bystander (control) T cells with the target T cell line. However, as shown in Figure 10, this was not the case.

[0149] aAPCs loaded with pMHC-I-Fc aAPM (i.e., 10,000 beads per T-Plex bead ID / T cell epitope) were incubated with 1,000 HCMVpp65 495-503 / A2-specific CD8 + T cell line #416 (CMV / A2 T cells), or 1,000 CMV / A2 T cells and 300,000 bystander Survivin 96-106 / A2-specific CD8 + T-Plex assays were performed in 500 μl tubes at 37°C, rotating at 40 rpm for 4 hours. The presence of T cell lines was confirmed by the expression of IFN-γ on recognized cognate T-Plex beads, as shown in Figure 10. + The emergence of subpopulations was indicated. Functionality of Sur / A2 T cells and all T-Plex beads used was confirmed by phorbol 12-myristate 13-acetate (PMA) / ionomycin (ionomycin). In this experiment, four colored T-Plex beads (bead IDs: 012, 013, 014, and 018) were loaded with defined pHLA-A2-Fc aAPMs. In Figure 10, only the IFN-γ signal from the cognate CMV / A2 T-Plex bead, ID 14 (blue / light gray), and one representative corresponding Ctrl-T-Plex bead, ID 018 (dark gray) are shown. The top and bottom FACS plots represent data analysis from the same reaction / bead mix (multiplex detection). In this experiment, Survivin was involved in the assembly of T-Plex beads. 96-104 / pHLA-A2-Fc aAPM NY-ESO-1 instead of HLA-A2-Fc 157-165 / HLA-A2-Fc was used.

[0150] Example 3: pMHC-I multimer staining of T cell line spiked samples compared to T-Plex assay A comparison was made using two T cell lines (model systems) with one "gold standard" assay (pMHC-I multimer staining) and the T-Plex assay according to the invention, and the results are shown in FIG.

[0151] pMHC multimer staining is the flow cytometric detection of antigen-specific T cell receptors expressed on the surface of T cells using soluble MHC-peptide oligomers (multimers), such as engineered MHC dimers, pentamers, and / or higher-order oligomers, and biotin-streptavidin-based tetramers covalently linked to fluorescent dyes (Altman et al., Science 274(5284):94-96, 1996). This method allows direct visualization of antigen-specific T cells through the binding of matching / cognate pMHC multimers to T cell receptors expressed on their surface.

[0152] HCMV pp65 495-503 / A2 specific CD8 + T cell line #416 (CMV / A2 T cells) at 500,000 Survivin 96-106 A pool of CD8 / A2-specific T cells was individually spiked with a defined amount (approximately 40-100,000). The spiked test samples were split in half and analyzed with a commercially available pMHC-I multimer stain (a) or a T-Plex assay (b). Figure 11(a) pMHC-I multimer staining. The spiked samples were stained with a commercially available CMV / A2 pMHC-I pentamer (ProImmune) in the presence of 50 nM dasatinib. + / CD3 + pMHC-multimers within T cell populations + The frequencies of pMHC-I multimers are shown. The total number of pMHC-I multimer cells is also extrapolated. Figure 11(b) Corresponding T-Plex assay. Four T-Plex bead pools (10,000 beads each) spiked with cognate CMV / A2-Fc or control pMHC-I-Fc were combined with spiked T cell samples and analyzed in the T-Plex assay. T-Plex assays were performed in 500 μl tubes at 37°C, rotating at 40 rpm for 4 hours. IFN-γ signals from cognate CMV / A2 T-Plex beads (blue / light gray) and representative signals from the corresponding control beads (dark gray) are shown. IFN-γ +The extrapolated total amount of T-Plex beads is shown in red. The top and bottom FACS-plots represent data analysis of the same reaction / bead mix (multiplex detection). Figure 11(c) Linear range assessment. The amount of spiked CMV / A2 T cells calculated based on the counting chamber was determined by the pMHC-I multimer content. + Extrapolated total amount of cells (left panel) or IFN-γ + The amount of T-Plex beads is plotted against the extrapolated total amount (right panel), and a linear regression curve is shown (red dotted line).

[0153] As shown in Figure 11(a), commercially available HCMV pp65 495-503 / A2-multimer (pentamer, ProImmune) staining was performed at 0.005% (2.5x10 5It can be reliably detected at a very low frequency of approximately 20 antigen-specific T cells, which is the published detection limit for pMHC-I multimer staining (Bentzen and Hadrup, Cancer Immunol Immunother. 66(5):657-666, 2017). In contrast, the T-Plex assay has a detection limit of 100–200 antigen-specific T cells and has been shown to be largely independent of surrounding bystander T cells (see Figure 11(b)). Thus, pMHC-I multimer staining is approximately 10 times more sensitive than the T-Plex assay. However, in contrast to the T-Plex assay, it does not allow for functional evaluation of T cell responses or subject recovery. From the perspective of dynamic range, the presence of more than 10,000 cognate T cells severely limits IFN-γ bystander capture on control T-Plex beads, resulting in the loss of clear separation between cognate (APM-matched) and control T-Plex beads (APM-mismatched). Under these circumstances, the performance of the T-Plex assay has been shown to be roughly equivalent to that of the ELISpot assay. However, in standard 96-well plate-based ELISpot, the signal already reaches saturation at 900-1000 spot-forming cells per well, making it impossible to distinguish single spot-forming cells (Karlsson et al., J. Immunol. Methods. 283(1-2):141-153, 2003). In contrast, the T-Plex assay shows a linear relationship between approximately 100 and 10,000 cognate T cells (Figure 11(c)). Advantageously, within this range, the detected IFN-γ-loaded T-Plex beads (IFN-γ + ) reliably reflects the amount of antigen-specific T cells present in the sample. This finding is supported not only by the model T cell lines used in Figure 11, but also by the CD8 + This also applies to antigen-specific T cells within the entire T cell pool. Furthermore, unlike the ELISpot assay, the T-Plex assay can detect multiple antigen-specific T cell responses in parallel in a single reaction.

[0154] Example 4: Optimization of the T-Plex assay and identification of critical assay parameters To improve the sensitivity of the T-Plex assay, we analyzed the effects of parameters such as (1) the composition of the T-Plex beads themselves, (2) the T-Plex reaction time, (3) the tube rolling speed, and (4) the initial proximity of the test sample to the T-Plex beads before rolling (Figure 12). Furthermore, Figure 13 analyzes parameters such as (5) the shape and size of the assay tube used, (6) the presence of costimulatory antibodies on the T-Plex beads, (7) the amount of T-Plex beads used per test, and (8) supplementation of the assay reaction with bystander beads (IFN-γ scavenger beads) that only have IFN-γ capture capacity.

[0155] In all experiments shown in Fig. 12 , HCMV pp65 495-503 / HLA-A2 (CMV / A2)-specific CD8 +Approximately 1,000 cells of T cell lines were used as test samples for the T-Plex assay reaction. T cell lines (Tc) were generated from healthy donor #8667 in Figures 12(a) and (b) and #416 in Figures 12(c) and (d). Unless otherwise noted, T-Plex beads were assembled using covalently conjugated anti-IFN-γ capture monoclonal antibody (αIFN-γ mAb) clone MD-1 (MD-1) and rat α-mouse IgG2a isotype (α-mIgG2a mAb) clone RMG2a in a 3:2 ratio (60% MD-1 / 40% Clone RMG2a). Subsequently, the defined color-coded beads (ID) were loaded with the SCT-based pMHC-I-mIgG2a-Fc construct. Assembled T-Plex beads (4x multiplex / 10,000 beads each) and test samples were rotated at 40 rpm at 37°C for 4 hours. The T-Plex beads were then stained with an αIFN-γ detection mAb (clone 4S.B3) and analyzed. Figure 12(a) shows titration of the ratio of α-mIgG2a / pMHC-I-Fc to α-IFN-γ mAb on T-Plex beads and the amount of two αIFN-γ mAbs used. Representative results of the T-Plex assay are shown as FACS blots of 60:40 αIFN-γ / pMHC-I-Fc T-Plex beads based on αIFN-γ clone NIB42 or clone MD-1 (BioLegend). The IFN-γ signal of the cognate CMV / A2 T-Plex beads (blue / light gray) and a representative signal of the corresponding control beads (dark gray) are shown. The bar graph shows the performance of T-Plex beads depending on the ratio of anti-(α)-IFN-γ and α-mIgG2a / pMHC-I-Fc on the T-Plex beads. Figure 12(b) Kinetics of the T-Plex assay. The T-Plex assay was performed as described above, and the reaction was stopped and analyzed after the indicated incubation time. Figure 12(c) Performance of the T-Plex assay based on rolling velocity. The T-Plex assay was performed in 500 μl tubes at various rotation speeds or in U-bottom 96-well plates under static conditions. CD8 +T cell degranulation was analyzed in parallel reactions using α-CD107a staining as methodologically described by (Betts et al., J. Immunol. Methods. 281(1-2):65-78, 2003). Figure 12(d) Effect of centrifugation of test samples and T-Plex beads before rolling. Test substances and T-Plex beads were loaded into a 500 μl conical tube combined with CO2-saturated assay medium and vortexed. The following steps were performed before rolling as indicated:

[0156] In the experiments shown in Figures 13(a), (c), and (d), T-Plex beads were assembled using covalently coupled αIFN-γ capture mAb (clone MD-1) and rat α-mIgG2a (clone RMG2a) in a 3:2 ratio (60% MD-1 / 40% RMG2a). The pMHC-mIgG2a-Fc construct was then loaded onto defined bead regions (IDs). Unless otherwise specified, the assembled T-Plex beads (4x multiplex / 10,000 beads each) and test samples were rotated at 60 rpm at 37°C for 4 hours and 30 minutes. The T-Plex beads were then stained with αIFN-γ detection mAb and analyzed. Approximately 1,000 beads were detected in Figures 13(a)-(c), and approximately 10,000 beads were detected in Figure 13(d). 495-503 / HLA-A2(CMV / A2) specific CD8 + T cell lines (TCs) were used as test samples for the T-Plex assay reactions, either from healthy donors #8667 or #416. Figures 13(a)-(d) show the IFN-γ signals of the cognate CMV / A2 T-Plex beads (blue, light gray) and one of the three corresponding Ctrl bead signals (dark gray).

[0157] The effects of tube shape, size, and loading volume on the performance of the T-Plex assay were evaluated, and the results are shown in Figure 13(a). T-Plex assays were performed as described above, varying the tube size / shape and loading volume (red) as indicated. Furthermore, the performance of T-Plex beads supplemented with costimulatory mAbs was evaluated, and the results are shown in Figure 13(b). Here, rat α-mIgG2a (Clone RMG2a), αIFN-γ capture mAb (Clone MD-1), αCD28 mAb (Clone 15E8), and αCD2 mAb (RPA-2.10) were covalently coupled to the beads in the indicated ratios. In a second step, pMHC-I-Fc was loaded to generate fully assembled T-Plex beads. The effect of the amount of T-Plex beads was evaluated by titration experiments, and the results are shown in Figure 13(c). T-Plex assays were performed with 60:40 αIFN-γ / pMHC-I-Fc T-Plex beads and 1,000 CMV / A2 T cell test samples. The indicated amounts of beads were used. + The extrapolated total amount of T-Plex beads is shown in red. Furthermore, the effect of IFN-γ scavenger beads on the performance of the T-Plex assay was evaluated, and the results are shown in Figure 13(d). The T-Plex assay was performed using 2 x 10 goat-α-mIgG Dynal beads loaded with only αIFN-γ mAb (MD-1). 5 T-Plex assays were performed in the absence or presence of IFN-γ scavenger beads. T-Plex assays were performed using 10,000 CMV / A2 T cells, demonstrating the outer dynamic range of the T-Plex assay. Median fluorescence intensity (MFI) of the total bead population is shown.

[0158] The capture of effector molecules secreted by activated T cells onto proximity-bound T-Plex beads is a key component of the T-Plex assay. The capture of IFN-γ and other relevant effector cytokines is influenced by multiple parameters. One of the key parameters is the choice and intrinsic properties of the capture antibody. Regarding IFN-γ, the MD-1 clone significantly outperformed the overall brightness and clustering of T-Plex beads loaded with cognate IFN-γ, as well as the actual IFN-γ capture. + Furthermore, a 50:50 ratio of pMHC to IFN-γ capture antibody on the bead surface, a 4-6 hour assay incubation time, and a 500 μL tube filling volume were found to be optimal.

[0159] Additionally, centrifugation of the test sample and T-Plex beads at 60–80 rpm before rolling improves assay sensitivity. In contrast, static incubation of the test sample and T-Plex beads in a 96-well plate without moving them results in APM-independent and indistinguishable bystander IFN-γ loading across the entire T-Plex bead pool. We found that binding of costimulatory antibodies (anti-CD28 antibodies) or reducing the amount of pMHC bound to T-Plex beads reduced assay performance. Using more than 10,000 T-Plex beads per T-cell epitope may slightly increase assay sensitivity. However, a 10,000 T-Plex bead / T-cell epitope assay proved robust for enumerating antigen-specific T-cell populations, with substantial linearity observed between 200 and 10,000 beads. This dynamic range can be further improved by the additional use of beads that exhibit high IFN-γ capture capacity but lack T cell stimulatory capacity (i.e., lacking APMs, called IFN-γ scavenger beads), potentially reducing the overall bystander (antigen-independent) IFN-γ load on all T-Plex beads present in the coculture.

[0160] Example 5: Variation in aAPC assembly and its impact on T-Plex assay performance Figure 14(a) shows a schematic diagram of a variation of aAPC (T-Plex bead) assembly. To obtain the data shown in the upper panel, we used the previously described T-Plex beads, with the modification that color-coded Luminex beads (Beads) were covalently coupled to αIFN-γ capture mAb (Clone MD-1) and rat α-mIgG2a (Clone RMG2a) in a 1:1 (50:50) ratio. Subsequently, defined bead IDs were loaded with soluble pMHC-I-mIgG2a-Fc aAPMs [CMV / A2-Fc and Survivin / A2-Fc] (also shown in Figure 1) derived from the supernatant of CHO-S cell-expressed constructs. To obtain the data shown in the center panel, Luminex beads were covalently coupled with streptavidin and an αIFN-γ capture mAb (Clone MD1) in a 1:1 ratio, followed by loading with purified biotinylated pMHC-I-pCC-mIgG2a-FC-Bio (also shown in Figure 3). To obtain the data shown in the bottom panel, Luminex beads were directly covalently coupled with purified pMHC-I-Fc-STag constructs and an αIFN-γ capture mAb in a 50%:50% (1:1) or 25%:75% (1:3) ratio. Figure 14(b) shows the corresponding conjugation quality control. pHLA-A2 conjugation was analyzed by staining the final pHLA-A2-loaded / conjugated T-Plex beads with an αHLA-A2 mAb of the IgG2b isotype (Clone BB7.2 / BioLegend). The maximum αIFN-γ capture capacity was analyzed by αIFN-γ-PE detection mAb (Clone 4S.B3 / BioLegend) after incubating fully assembled T-Plex beads with 4 ng / ml recombinant IFN-γ (BioLegend) for 2 h at 37°C. HCMV pp65 495-503 / HLA-A2-Fc (CMV / A2-Fc) (blue, light gray) and Survivin 96-104Fluorescence signals from the IFN-γ / HLA-A2-Fc (Survivin / A2-Fc)-bound T-Plex beads and unloaded Luminex beads (dotted line) are shown. Figure 14(c) shows the performance of the corresponding T-Plex assay. The T-Plex assay was performed using the variation of the T-Plex bead assembly shown in (a). One thousand CMV / A2-specific T cells (TC#5561) were placed in a 500 μl tube and combined with T-Plex beads (10,000 beads per 4x multiplex / T cell epitope) and spun at 60 rpm at 37°C for 4 hours before rolling. The IFN-γ signal from the cognate CMV / A2 T-Plex beads (blue / light gray) and a representative signal from the corresponding control beads (dark gray) are shown. The top and bottom FACS plots represent data analysis of the same reaction / bead mix (multiplex detection).

[0161] α-mIgG2a-Fc was covalently coupled to Luminex beads at a ratio of 50:50. Loading of (α-Fc)mAb[RMG2a] / IFN-γ-capture mAb[MD-1] or streptavidin[SAv] / IFN-γ-capture mAb[MD-1] with pHLA-A2-Fc or biotinylated pHLA-A2-pCC-Fc demonstrated very similar pMHC-Fc binding and maximum IFN-γ-capture capacity. However, staining of HLA-A2 (clone BB7.2, IgG2b isotype) with pMHC-Fc-loaded α-Fc-based T-Plex beads yielded approximately four-fold higher fluorescence intensity (MFI) than with pMHC-pCC-Fc-loaded SAV-based T-Plex beads, indicating slightly superior pMHC binding capacity of α-Fc-based T-Plex beads. However, CMV / A2-specific CD8 +In a T-Plex assay (TC#5561) detecting 1,000 T cells, α-Fc-based and SAv-based T-Plex beads performed nearly identically. Thus, different T-Plex bead structures (i.e., IFN-γ-capture mAb / α-mIgG2a-Fc mAb beads or IFN-γ-capture mAb / streptavidin beads loaded with pMHC-Fc or biotinylated pMHC, respectively) were shown to yield favorable and overall very similar T-Plex assay performance.

[0162] To distinguish between antigen-dependent IFN-γ-loaded T-Plex beads (i.e., aAPCs that induced cognate T cell responses) and bystander IFN-γ-loaded T-Plex beads (i.e., aAPCs with nonmatching APMs present in the coculture of all aAPC and T cell populations), it is crucial that the cognate and control T-Plex beads have the same IFN-γ-binding capacity. This is easily achieved by covalently coupling multiple Luminex beads of different colors but homogenous protein-binding capacities and using a shared master mix containing IFN-γ-capture mAb and streptavidin or α-mIgG2a-Fc mAb as a "production batch."

[0163] Example 6: MTB / DR3 CD4 with pMHC-II-Fc-loaded T-Plex beads + Antigen-specific detection of T cell clone RP15.1.1 Figure 15 shows a single CD4 + We present a pMHC-II-based T-Plex assay for the detection of T cell lines (model systems). In particular, Mycobacterium tuberculosis (MTB) heat shock protein 65 (Hsp65) by the T-Plex assay. 1-13 / HLA-DRB1*03:01 / DRA*01(MTB / DR3)-specific CD4 +Detection of T cell line clone RP15.1.1 is shown in Figure 15(a). T-Plex beads (60% αIFN-γ mAb [clone MD-1] / 40% rat α-mIgG2a [clone RMG2a]) were loaded with MTB Hsp651-13 / HLA-DR3-pCC-Fc (purple (light gray) / cognate) or CLIP 103-117 pMHC-II-Fc / DR3-pCC-Fc (dark grey / control) was loaded as in Figure 2. Then, pMHC-II-Fc-loaded T-Plex beads (2x multiplex / 10,000 beads each) were added to 250,000 Survivin 96-104 / HLA-A2 specific CD8 + In the presence of bystander T cells, MTB Hsp65 1-13 / HLA-DR3 specific CD4 + T-Plex beads were combined with the indicated amounts (numbers in red) of T cell clone RP15.1.1 (MTB / DR3 T cells). T-Plex assays were performed in 500 μl tubes at 37°C, rotating at 40 rpm for 4 hours. T-Plex beads were then stained with an αIFN-γ detection mAb (clone 4S.B3) and analyzed. The presence of antigen-specific T cells was confirmed by the presence of IFN-γ higher than that of T-Plex control beads (dark gray). + Subpopulations were demonstrated by the appearance of cognate T-Plex beads. The top and bottom FACS plots represent data analysis of the same reaction / bead mix (multiplex detection). Figure 15(b) shows the performance of T-Plex beads supplemented with costimulatory mAbs. Here, rat α-mIgG2a (clone RMG2a), αIFN-γ capture mAb (clone MD-1), αCD28 mAb (clone 15E8), and αCD2 mAb (RPA-2.10) were covalently coupled to T-Plex beads at the indicated ratios. In the second step, pMHC-II-Fc was loaded to generate fully assembled T-Plex beads, which were finally coupled with 10,000 MTB / DR3 T cells, and the T-Plex assay was performed as described above.

[0164] Using cognate MTB / DR3-associated T-Plex beads, 50,000 to 200 MTB / DR3-specific CD4 + The presence of CMV / A2-specific CD8 T cells was reliably detected (Fig. 15(a)). + As seen in similar experiments with T cell lines #416 or #5561, the resulting IFN-γ + The fraction of the T-Plex bead population was somewhat lower, indicating that stimulation with bead-based aAPCs resulted in the production of MTB / DR3-specific CD4 + This can be explained by the fact that only 40-60% of T cells actually express IFN-γ. Furthermore, T-Plex beads supplemented with additional costimulatory antibodies inhibited the expression of MTB / DR3-specific CD4 + The T-Plex assay performed poorly in detecting T cell lines (Figure 15(b)), consistent with previous T-Plex assay optimization experiments shown in Figure 13(b). In conclusion, we demonstrate that the T-Plex bead-based T-Plex assay concept is robust against T cell lines secreting IFN-γ. h 1-differentiated CD4 + This indicates that the method can also be applied to antigen-specific detection of T cells.

[0165] Example 7: Proof of principle of T-Plex 2 CD4 + Assays for antigen-specific detection of T cells T-Plex of this example 2 Proof-of-concept data (detecting multiple cytokines on the same bead) demonstrates: a) CD4 + The T cell lines produce multiple cytokines upon stimulation with aAPCs of the invention; b) the assay of the third aspect of the invention (T-Plex assay) also works with the cytokines TNFα, IL-4 and IL-2 as a reference; and c) the assay of the fourth aspect of the invention (T-Plex assay) also works with the cytokines TNFα, IL-4 and IL-2 as a reference. 2 The assay) is suitable for multiple levels of multiplexing, i.e., detection of multiple T cell specificities (2 in this example) and multiple cytokines (3 in this example).

[0166] Figure 16(a) shows the MTB / DR3 CD4 + Cytokine intracellular staining (ICS) after 5 h aAPC-based restimulation of T cell lines: MTB Hsp65 / HLA-DR3-pCC-Fc (MTB / DR3-Fc) (cognate) and CLIP 103-117 / DR3-pCC-Fc (CLIP / DR3-Fc) coated goat-α-mouse-IgG-Fc Dynabeads (Invitrogen) were added to an equal amount of MTB Hsp65 1-13 / HLA-DR3-specific CD4 + CD4 T cells were co-cultured with T cell clone RP15.1.1 (MTB / DR3 T cells) in a 96-well U-bottom well for 5 hours at 37°C in the presence of brefeldin A and monensin to block cytokine secretion. + The frequency of cytokine-expressing cells within the T cell population is shown. Figure 16(b) demonstrates the detection of different cytokines using the T-Plex bead platform. T-Plex beads were assembled using a covalently coupled αIFN-γ capture mAb (Clone MD-1) and monoclonal α-mIgG2a in a ratio of 60% to 40%. Alternatively, cytokine capture mAbs binding to IL-2 (Clone MQ1-17H12), IL-4 (Clone 8D4-8), or TNF-α (Clone Mab 1) were used instead of αIFN-γ. Subsequently, pMHC-II-loaded T-Plex bead pools based on different cytokine mAbs were loaded with MTB / DR3-Fc (purple (light gray) / cognate) or CLIP / DR3-Fc (dark gray / control), and finally, MTB / DR3-specific CD4 +The assay was performed by placing 10,000 beads in a 500 μl tube and rolling at 60 rpm at 37°C for 5 hours (2x multiplex). After the T-Plex reaction, the T-Plex beads were stained with the corresponding cytokine-detecting mAbs (all conjugated to the fluorescent dye phycoerythrin (PE)) and finally analyzed by FACS. The top and bottom T-Plex assay FACS-plot pairs represent data analysis from the same reaction / bead mix. Figure 16(c) shows the T-Plex assay. 2 This is the proof-of-principle data for the assay. 2 The beads were assembled by covalently coupling αIFN-γ, αTNF-α, and IL-4 capture mAbs (each 1 / 5 (20% of the total Luminex bead protein binding capacity) and monoclonal α-mIgG2a (2 / 5 [40%]). The beads were then loaded with pMHC-II and T-Plex. 2 The beads were incubated with MTB / DR3-specific T cells, and the T-Plex assay was performed as described above. 2 The beads were stained with the corresponding cytokine-detecting mAb conjugated with different fluorescent dyes (Brilliant violet 421 nm (BV421), PE, PE / Cy7) as shown in the figure and analyzed by FACS. The four T-Plex assay FACS plots shown are all from the same T-Plex assay. 2 This is obtained from the reaction / bead mix.

[0167] MTB / DR3-specific CD4 + When co-cultured with T cell clones, cognate T-Plex 2 The beads were partially loaded with a combination of effector cytokines (IFN-γ, TNF-α, IL-4) and detected simultaneously in a single multidimensional multiplex reaction. 2 The beads were MTB / DR3-specific CD4 + Suitable for providing functional profiles of T cell clones, which can be achieved by T-Plex 2 This represents proof of principle of the concept.

[0168] Example 8: Antigen-specific T cell detection using the T-Plex assay does not change the phenotype of the original sample.

[0169] The data from this example, also shown in Figure 17, demonstrate the effect of performing the assay of the present invention on a sample of T cells. As further described directly below, samples characterized in parallel by pMHC-multimer analysis were analyzed by the T-Plex assay (beads were analyzed by FACS), and the T cells were returned to culture. After six days, the phenotype of the sample previously analyzed by the T-Plex assay was compared to that of an "untouched" T cell sample cultured in parallel for the same six days. No obvious changes / differences were observed between the phenotypes of these samples. Thus, analysis using the T-Plex assay results in little phenotypic change in the cells of the sample.

[0170] Figure 17(a) shows pMHC-I multimer staining of healthy subject (HD) sample #3637. HLA-A2 + A T cell sample from healthy donor (HD) #3637 was first analyzed by pMHC-I multimer staining (no multiplex). + pMHC-I multimers within T cell populations + The cell frequency is indicated by the black / horizontal numbers: 2.5x10 6 Each antigen-specific CD8 in PBMC + The total amount of T cells is extrapolated and indicated by red vertical numbers. Figure 17(b) shows the corresponding data for the T-Plex assay-based analysis of T cell samples from the same donor. To analyze whether the T-Plex run induces antigen-specific proliferation, PBMCs from HD#3637 were labeled with CellTrace violet (CTV / Invitrogen) and then untouched CD8 +T cells were isolated by dissolving 2.5x10 pMHC-I-Fc-loaded T-Plex bead pools (4x multiplex (CMV / A2; Flu / A2; EBV / A2; Survivin / A2) / 10,000 beads each) in a 500 μl tube. 6 ,10x10 6 CD8 isolated from total PBMCs + were incubated with T cells. The test samples and T-Plex beads were centrifuged at 1500 rpm for 5 minutes before rolling at 60 rpm for 5 hours at 37°C. After 5 hours, the T-Plex beads were magnetically separated from the T cells and analyzed. The T-cell samples were returned to culture together for 6 days. Figure 17(c) shows the phenotype of the samples after the T-Plex assay compared to an intact control T cell culture. CTV-labeled HD#3637 CD8 cells cultured for 6 days. + T cells (with or without T-Plex assay) were additionally stained with pMHC-I multimers before staining for lineage and activation markers. + pMHC-I multimers within T cell populations + The middle and bottom rows of Figure 17(c) show the frequency of cells expressing pMHC-I multimers. + CD8 + T cell proliferation marker (CTV dim ) and activation markers (CD25 + / 4-1BB + ) Expression ratio.

[0171] Example 9: Soluble pMHC-I-Fc molecules were successfully produced using eukaryotic cells and exhibited antigen-specific binding.

[0172] As shown in Figure 18(a), the dimeric disulfide-trapped (dt) peptide-MHC-class I immunoglobulin Fc fusion molecule (pMHC-I aAPM) consists of two single polypeptide chains containing covalently linked T cell epitope peptide ligands (9–10 amino acids), human β2 microglobulin (β2m), the ectodomain of an HLA class I allele, and constant heavy chain (CH) domains 2 and 3 of mouse immunoglobulin isotype IgG2a. The dotted line indicates a flexible glycine-serine linker. An intramolecular disulfide trap between the C-terminal peptide extension and a cysteine ​​(C) residue instead of the MHC-I tyrosine (Y) residue further stabilizes the pMHC complex. The C-terminal Strep-Tag II (STag) sequence allows affinity purification under neutral conditions using Strep-Tactin (IBA Lifesciences). Figure 18(b) shows the results of expression and structural conformation verification of dt-pHLA-A2-Fc. 96-104 CHO-S cells expressing pMHC-Fc-STag were intracellularly stained with α-HLA-A2 mAb (clone BB7.2) (blue, light gray) or α-mIgG2a mAb (clone RMG2a) (dark gray) on day 3 posttransfection. Figure 18(c) shows the results of dt-pHLA-A2-Fc-STag affinity chromatography. CHO-S cells transiently expressing pMHC-Fc-STag for 6 days were purified at pH 7.4 using a Strep-Tactin high-capacity resin-packed column. After Coomassie staining, 10% SDS-PAGE was performed under non-reducing and reducing conditions. M: marker; CR: crude / CHO-S supernatant; FT: flow-through; Wp: pooled buffer used for washing; dial.prod: 2.5 μg / lane of dialyzed product. Figure 18(d) shows the results of verifying the antigen-specific binding of the dt-pHLA-A2-Fc-STag protein. 96-104 / HLA-A2 specific CD8 +T cell lines were stained with cognate (red / dark gray) and control in the presence of dasatinib (50 nM). (Blue, light gray) pMHC-I multimers were stained at 25 μg / ml, followed by lineage marker staining. (Left panel) Commercially available pHLA-A2 pentamer (ProImmune); (middle panel) dt-pHLA-A2-Fc-STag and allophycocyanin-conjugated (APC) Strep-Tactin (IBA Lifesciences) conjugate; (right panel) Sequential staining with dt-pHLA-A2-Fc-STag and biotinylated α-mIgG2a mAb (α-mIgG2a-Biotin and Streptavidin-APC). To exclude dead cells, T cells were labeled with ZombieAqua (BioLegend) before multimer staining. MFI: median fluorescence intensity; FMO: fluorescence minus 1 background control.

[0173] Example 10: Successful production of soluble pMHC-II-Fc molecules and verification of antigen-specific binding As shown in Figure 19(a), peptide-MHC-class II monomeric immunoglobulin Fc fusion constructs (pMHC-II aAPMs) consist of two separate polypeptide chains. The MHC-II β chain is fused to the antigenic peptide at its N-terminus via a glycine-serine linker. The C-terminal β chain ectodomain (β1-β2) is fused to a parallel coiled-coil (pCC) basic zipper, followed by the hinge domain and CH2 and CH3 of mIgG2a (Fc), site-specifically biotinylated with His6-ag and AviTag at the C-terminus. The monomorphic α chain ectodomain (α1-α2) is fused to the complementary acidic pCC-Fc and StrepTag-II at the C-terminus. Figure 19(b) shows the expression and conformational validation results of pHLA-DR3-Fc. MTB Hsp65 1-13CHO-S cells expressing / HLA-DRB1*03:01 / DRA*01 (MTB / DR3)-Fc-His / Avi / STag were intracellularly stained with α-HLA-DR mAb (Clone L243, light gray) [orange] or α-mIgG2a mAb (Clone RMG2a, dark gray) 3 days after transfection. Figure 19(c) shows the results of pHLA-DR3-Fc affinity chromatography. CHO-S cells transiently expressing pDR3-Fc-STag for 6 days were purified at pH 7.4 using a Strep-Tactin high-capacity resin-packed column. After Coomassie staining, 10% SDS-PAGE was performed under non-reducing and reducing conditions. Figure 19(d) shows the results of testing MTB / DR3-Fc specificity and bead-bound stimulatory activity using cognate CD4+ T cell clones. MTB / DR3-specific CD4 + The results are shown for a 5-hour co-culture of T cell clone RP15.1.1 with bead- or cell-based aAPCs. The bead-based aAPCs were either MTB / DR3-Fc or CMV pp65. 510-522 Goat-α-mouse IgG-Fc-(GαM) Dynabeads preloaded with HLA-DR3 (CMV / DR3-Fc, control) were used. T2 cells expressing HLA-DR3 and HLA-DM (T2.DR3.DM) were incubated with 10 μM MTB Hsp65. 1-13 peptide or HCMV pp65 as a control 510-522 The cells were pulsed with β-lactam overnight and used as aAPCs. + Stimulation of T cell clone RP15.1.1 is demonstrated by induction of cytokine expression analyzed by intracellular staining for TNF-α and IFN-γ after lineage marker staining.

[0174] Many modifications and other embodiments of the inventions described herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is to be understood, therefore, that the inventions are not to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. An artificial antigen-presenting cell (aAPC) for detecting effector molecules of T cells that respond to presentation of an antigenic peptide sequence, the aAPC comprising: (a) a surface, the surface being a surface of a particle selected from the group consisting of a rigid spherical particle, a non-spherical particle, and a fluid lipid bilayer-containing system, the particle itself being coded so as to be distinguishable when used in a multiplexed assay to assess antigen-specific T cell responses; (b) one or more artificial antigen-presenting molecules (aAPMs) attached to the surface via attachment sequences or via direct chemical bonding; and (c) one or more surface-attached capture antibodies specific for one or more effector molecules released from T cells in response to presentation of the antigenic peptide sequence. Including, Each of the one or more aAPMs comprises an identical antigen peptide sequence.

2. The aAPC of claim 1 , wherein the one or more capture antibodies are specific for the same effector molecule or comprise one or more groups of capture antibodies, each group being specific for a different effector molecule.

3. the aAPC further comprises one or more costimulatory molecules attached to its surface to enhance the T cell response to presentation of the antigenic peptide sequence; (d) the one or more costimulatory antibodies are specific for the same costimulatory receptor, or (e) the one or more costimulatory antibodies comprise one or more groups of costimulatory antibodies, each group being specific for a different group of the same costimulatory receptor;

4. 3. The aAPC of claim 1 or claim 2, wherein the particles are color-coded, the color coding indicating the antigen peptide sequence of the aAPM bound to the aAPC.

5. aAPM, a monomeric, dimeric or multimeric molecule comprising a soluble antigenic peptide-loadable or peptide-loaded MHC class I or MHC class II portion; or β 2 - a tagged recombinant soluble MHC-I molecule covalently linked to a microglobulin and assembled with a synthetic peptide; or A single polypeptide sequence comprising, in order from amino terminus to carboxyl terminus, an antigen-presenting domain, a dimerization domain, an immunoglobulin (Ig) Fc domain, and an attachment sequence, wherein the sequence of the antigen-presenting domain comprises an N-terminal antigen peptide sequence. The aAPC of claim 1,

6. (a) the dimerization domain comprises an IgG hinge region; (b) the Ig Fc domain is a mouse IgG2a Fc region containing an N297Q mutation or a human IgG1 Fc region containing an N297Q mutation; and (c) the attachment sequence is a peptide tag for attaching aAPM to a surface; The aAPC of claim 5.

7. (a) the IgG hinge region comprises SEQ ID NO: 1 or SEQ ID NO: 2; (b) the IgG Fc region comprises SEQ ID NO:3 or SEQ ID NO:4; (c) The aAPC of claim 6, wherein the peptide tag enables attachment of the aAPM to a surface via affinity-based binding or conjugation to the surface.

8. 6. The aAPC of claim 5, wherein the antigenic peptide sequence is a peptide sequence of an antigen selected from the group consisting of a viral antigen; a bacterial antigen; a fungal antigen; a parasitic antigen; an autoimmune antigen; an allergy-related antigen, and a tumor antigen, and the antigenic peptide sequence is selected from the group consisting of HCMV pp65 495-503 (SEQ ID NO: 10); EBV BMLF-1 259-267 (SEQ ID NO: 13); influenza virus MP 58-66 (SEQ ID NO: 14), NY-ESO-1 157-165 / 165V (SEQ ID NO: 11); and Survivin 96-104 / 97M (SEQ ID NO: 12).

9. The aAPC of claim 5 or 6, wherein the antigen-presenting domain of a single polypeptide sequence comprises, in order from the amino terminus to the carboxyl terminus, an antigenic peptide sequence, a first linker sequence, and an MHC class I portion.

10. From the amino terminus to the carboxyl terminus, the MHC class I moieties are: 2 The aAPC of claim 5, comprising a -microglobulin sequence, a second linker sequence, an MHC class I HLA-A*02:01 α1 sequence, an MHC class I HLA-A*02:01 α2 sequence, and an MHC class I HLA-A*02:01 α3 sequence.

11. the first linker sequence comprises a first cysteine ​​residue and the MHC class I HLA-A*02:01 α1 sequence comprises a second cysteine ​​residue, the first and second cysteine ​​residues forming a disulfide trap that enhances binding between the antigenic peptide sequence and the MHC class I portion of the antigen-presenting domain via a covalent bond; or The aAPC of claim 10, wherein the first linker sequence comprises SEQ ID NO:

30.

12. the MHC class I portion comprises SEQ ID NO: 31 or SEQ ID NO: 32; or The aAPC of claim 11, wherein the aAPM comprises, in order from the amino terminus to the carboxyl terminus, an antigenic peptide sequence and SEQ ID NO:

33.

13. The aAPC of claim 5 , wherein the antigen-presenting domain of the single polypeptide sequence comprises, in order from the amino terminus to the carboxyl terminus: an antigenic peptide sequence and an MHC class II portion.

14. The MHC class II portion is, in order from amino terminus to carboxyl terminus: MHC class II HLA-DRβ1 and MHC class II HLA-DRβ2 sequences, MHC class II HLA-DRα1 and MHC class II HLA-DRα2 sequences, which are not linked to an antigenic peptide sequence; or the MHC class II portion comprises the DRB1*03:01 sequence of SEQ ID NO: 50 and the DRA*01:01 sequence of SEQ ID NO: 51; The aAPC of claim 13.

15. 14. The aAPC of claim 13, wherein the dimerization domain further comprises a parallel coiled-coil acidic or basic zipper motif, or wherein the dimerization domain further comprises a parallel coiled-coil basic zipper motif of SEQ ID NO: 53 or an acidic zipper motif of SEQ ID NO:

54.

16. aAPM comprises, in order from amino terminus to carboxyl terminus, the antigenic peptide sequence and SEQ ID NO:55; and SEQ ID NO:

56. The aAPC of claim 13.

17. aAPC comprises a dimer comprising aAPM, (a) the dimer is a homodimer or heterodimer of two aAPMs as defined in claim 5, or (b) the dimer is a heterodimer of aAPM as defined in claim 13 and a second molecule; The aAPC of claim 5.

18. A composition comprising a plurality of aAPCs according to claim 5, 7, 12 or 14, wherein the composition comprises a plurality of identical aAPCs, and the identical aAPCs comprise a single capture molecule specific for each single effector molecule, or a plurality of capture molecules specific for each of a plurality of effector molecules.

19. A composition comprising multiple groups of aAPCs according to claim 1 or 2, wherein all aAPCs in each group are identically coded, the antigen peptide sequences presented by the aAPCs in each group are identical within the group but different between groups, and the aAPCs in each group comprise a single capture molecule specific for a single respective effector molecule, or multiple capture molecules specific for multiple respective effector molecules.

20. 1. An assay for determining an antigen-specific T cell response, comprising the steps of: (a) contacting a T cell with the composition of claim 18 under conditions and for a time suitable to elicit a response from the T cell upon presentation of the antigenic peptide sequence by the same aAPC; (b) separating aAPCs from T cells; (c) contacting the aAPC with a detection antibody against one or more effector molecules for which the one or more capture molecules of the aAPC are specific; (d) analyzing the release of effector molecules of T cells by detecting effector molecules captured by one or more capture molecules of the aAPC; This determines the T cell response specific to the antigen peptide sequence presented by the same aAPC.

21. 1. An assay for determining multiple antigen-specific T cell responses, comprising the steps of: (a) contacting T cells with the composition of claim 19 under conditions and for a time suitable to elicit a response from the T cells upon presentation of each of the different antigenic peptide sequences presented by the aAPCs of each group; (b) separating aAPCs from T cells; (c) contacting the aAPCs with a detection antibody against one or more effector molecules for which one or more capture molecules of the aAPCs in each population of aAPCs are specific; (d) identifying and isolating aAPCs from each of said populations of aAPCs; (e) analyzing the release of effector molecules of T cells by detecting effector molecules captured by one or more capture molecules of aAPCs within each population of aAPCs; This determines multiple antigen-specific T cell responses specific for each antigenic peptide sequence presented by each population of aAPCs.

22. - the T cells are contained in a fraction of peripheral blood mononuclear cells (PBMC) or are purified T cells, and - the step of separating aAPCs from T cells comprises washing the aAPCs under conditions suitable for maintaining T cell viability, and then recovering the separated T cells for further in vitro cell culture; 22. The assay of claim 20 or claim 21.

23. 23. The assay of claim 22, wherein the contacting in step (a) comprises continuously moving the mixture of T cells and the composition so as to produce a uniformly mixed suspension of T cells and aAPCs.

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