Multiplex MHC peptide binding and t cell detection method / quantifiable multiplex MHC binding assay

Abstract

The invention is directed to a method for detecting at least one target peptide by binding to a MHC protein by providing a plurality of different MHC proteins with placeholder peptides labelled with at least one fluorescent marker thereby obtaining a plurality of labelled MHC proteins.

Description

MULTIPLEX MHC PEPTIDE BINDING AND T CELL DETECTION METHOD / QUANTIFIABLE MULTIPLEX MHC BINDING ASSAYBACKGROUND

[0001] The present invention is directed to a method for detecting the binding restriction of at least one peptide to an MHC protein allotype (e.g. HLA protein) present in a mixture of different MHC protein allotypes immobilized on beads and the use of the method for identifying peptide / MHC / bead complexes capable of activating T cells, wherein the peptide represents a potential new immunogenic or immunodominant MHC-presented T cell epitope.

[0002] The prediction of antigen-specific T cell responses is of utmost importance in the fields of adaptive immunity, cellular therapy, and drug development. Antigens, specific for cancer or pathogens, contain so called T cell epitopes. Those epitopes are loaded onto major histocompatibility complexes (MHCs) as proteolyzed peptides, forming a peptide / MHC (pMHC) structure on the surface of an antigen presenting cell (APC).

[0003] To achieve antigen-specific activation of a T cell, its T cell receptor (TCR) must bind with sufficient affinity and duration to the pMHC of an APC to form the immunological synapse, which induces a T cell activating signaling cascade. Since each T cell expresses a unique TCR sequence encoding the TCR, whereby every TCR can show an optimal affinity to a certain pMHC, a match of the MHC-presented peptide (epitope) and the corresponding / cognate TCR sequence is required for T cell activation. As a result, T cells are activated upon a specific, usually clonotypic interaction, requiring binding with sufficient affinity to the MHC-presented epitope in an antigen specific fashion. Mathematically, the human immune system can give rise to 1014to 1016different TCR sequences, although the real number of unique T cells expressing individual TCRs is several orders of magnitude lower.

[0004] MHC proteins are divided into two classes, MHC class I and MHC class II proteins, capable of activating CD8+ and CD4+ T cells respectively when presenting T cell epitopes on the surface.

[0005] In humans, MHC proteins are encoded by genes mapping to the most polymorphic region in the human genome: the human leukocyte antigen (HLA) locus encoding for the human MHC I proteins HLA-A, -B and -C and MHC II proteins HLA-DR, -DQ and -DP, with several tens of thousands of variants known on the protein level. Each of these human MHC protein allotype variants is capable of binding and presenting its own different peptide repertoire, mostly differing when comparing one MHC variant to another. Hence, one antigen usually contains multiple potential MHC-presented T cell epitopes, which are restricted to a certainMHC variant. Thus, which of the peptide epitopes of a given antigen drives T cell activation and is therefore an immunogenic peptide epitope, depends on the individual HLA allelic background of a person.

[0006] The identification of potential immunogenic / immunodominant T cell epitopes (peptide presented by MHC proteins) from cancer-, pathogen- and auto-antigens is essential for multiple health related applications:1) Personalized cancer therapies targeting predicted T cell epitopes, derived from mutated cancer-specific proteins.2) Novel vaccines selected and designed to contain a broad spectrum of T cell epitopes for the most common MHCs.3) New protein drugs which undergo de-immunization during development via elimination of T cell epitopes from the sequence to avoid anti-drug immune responses.4) Risk of autoimmune disorders can be estimated by the identification of T cell epitopes in the sequence of wildtype proteins.

[0007] Based on these applications there is the need for two different approaches for the identification of epitopes and their corresponding MHC allotype variant within a potential antigen:1. A (semi-)personalized approach, whereby a potential antigen (from cancer, autoimmune diseases or infection) is analyzed for the presence of T cell epitopes considering a person's individual MHC background. E.g. to design a tailored therapy to target that antigen.2. A population representative approach. Here an antigen is screened for the presence of T cell epitopes considering the most abundant alleles in the population, to evaluate the chance, if this antigen can mount a T cell response in the majority of the population. This is essential in vaccine development and risk mitigation during drug development.

[0008] EP2800974 relates to a method for producing an examination reagent, wherein a helper ligand which enables the folding of the MHC class I protein is added to an initial solution containing a receptor protein or a receptor protein complex, more particularly an MHC class I protein or a multimeric MHC class I protein complex. An examination peptide and preferably a ligand exchanger / ligand releaser is added to the folded MHC class I protein or the multimeric MHC class I protein complex, so that an analysis solution of the examination reagent can be obtained which has the MHC class I protein with an examination peptide. Although this method can be used in order to identify T cell stimulating peptide presenting by MHC molecules, itrequires T cells expressing the reactive TCR recognizing the pMHC molecule and is not useful in order to analyze a cohort of MHC allotype variants in parallel. In addition, the readout (antigen-specific T cell staining) is only indirectly correlating the binding of the peptide to a certain MHC variants, as this is not directly measured.

[0009] EP 1648919 is based on the discovery that MHC monomers immobilized to a solid surface are capable of activating T-cells that recognize specific peptides in the context of MHC Class I or Class II molecules. Methods for detecting T-cells responding to MHC monomers, and methods for measuring the frequency of specific and activated T-cells in a heterogeneous population are provided in this patent application. This patent application also provides systems and kits useful for conducting the methods of the present invention.

[0010] However, the method disclosed in this patent application requires the use of Ag- specific cells which need to be brought in contact with pMHC immobilized on a solid phase and does not offer a multiplex-based solution.

[0011] EP1692504 focusses on a solution-based methods for identifying an MHC -binding peptide or measuring affinity of MHC -binding peptides for an MHC monomer, or modified MHC monomer by incubating at least one MHC monomer or modified MHC monomer having a bound template MHC -binding peptide, an excess amount of a competitor peptide, and a tracer MHC -binding peptide tagged with a detectable label so as to allow competition binding between the three peptides. At least a portion of the competitor peptide exchanges with the template peptide and a difference in signal produced by the detectable label in the total sample as compared with signal produced solely by monomers after the competition assay is obtained and used to calculate affinity of the competitor peptide for the monomer. These methods are useful in peptide discovery programs and exchanged monomers can be further tested for activity in tetramer cell staining assays. However, EP 1692504 is restricted in the detection of only one or maximally a few peptides simultaneously.

[0012] EP3455630 describes a peptide exchange system and method. In this patent application, a method for quantified peptide exchange comprising a MHC molecule bound to a first peptide, wherein the first peptide is labeled with a first label, comprising an optional peptide exchange factor and a second peptide (examination peptide), is described. The method further comprises a capture system: Anti MHC antibodies and / or magnetic capture beads. This invention can only be applied to one or only very few pMHC complexes in parallel.

[0013] US9399795B2 describes a bead-based system in which beads, also termed microspheres, carrying a plurality of different chemical functionalities are randomly distributed on a substrate comprising a patterned surface of discrete sites that can bind the individualmicrospheres. The invention is focused on the bioactive agent that comprises an amplified nucleic acid. This method was not claimed for the prediction of peptide binding to MHC molecules present in a mixture of MHC molecules.

[0014] US20070259449A1 relates to the formulation of a fully integrated system for the implementation of biochemical multiplex analysis on a surface in a miniaturized format. The method describes making a bead array comprising a substrate with a solution of different beads without an optical signature and randomly associating onto sites. This method is claimed or related to the prediction of peptide binding to MHC molecules present in a mixture of MHC molecules.OBJECT OF THE INVENTION

[0015] The objective of the invention is to provide a multiplex method to detect the binding of at least one antigenic peptide present in a mixture of peptides to one or more MHC allotype protein variants present in a mixture of different immobilized MHC allotype protein ns via flow cytometric analysis and sorting. Current in vitro assays are not capable of multiplexing the determination of the binding of a peptide(s) to MHC(s) of a T cell epitope. Whether a single peptide is, or a pool of peptides contains a T cell epitope, respectively, is determined only for one MHC at a time in a single reaction. Hence, mid- or high-throughput assays are not possible with present approaches.SUMMARY

[0016] It was found that Multiplex beads (color-coded micron-sized beads which are optionally magnetic) decorated with specific MHCs, wherein one specific MHC allotype variant is immobilized onto one color- and / or size-coded bead, and, a preloaded peptide (so- called placeholder peptide) bound to the MHC is labeled (e.g., fluorescence labeled), would allow for the novel, simultaneous detection of peptide binding to a specific MHC out of a pool of peptides as well as the multiplicity of MHCs binding to one specific peptide.

[0017] Accordingly, the invention is directed to a method for detecting at least one target peptide by binding to a MHC protein characterized in a) providing a plurality of different MHC proteins with placeholder peptides labelled with at least one fluorescent marker thereby obtaining a plurality of labelled MHC proteinsb) binding of the labelled MHC proteins to color-coded particles comprising at least one fluorescent dye, wherein the emission spectrum of the fluorescent dyes code for different MHC proteins, thereby obtaining a plurality of labelled color-coded particles c) providing the plurality of labelled color-coded particles with at least one target peptide, wherein at least one target peptide is bound by at least one MHC protein of at least one labelled color-coded particle by replacing the respective placeholder peptide from the MHC protein, thereby obtaining a mixture of color- coded particles bound to a target peptide and color-coded particles bound to placeholder peptides d) detecting the emission spectrum of the color-coded particles bound to a target peptide and the color-coded particles bound to placeholder peptides, thereby detecting the target proteins bound to the MHC protein for which the emission spectrum codes.

[0018] Preferable, the emission spectrum of the color-coded particles bound to a target peptide and the color-coded particles bound to placeholder peptides is detected after isolating the particles, for example by flow-cytometry.

[0019] Thus, due to the unique features of these beads, the different specific alleles can be distinguished based on their bead-specific feature (e.g., specific size or color). Such an approach can be used to identify antigen-specific new epitopes relevant for T-cell mediated immunity in the context of immunogenicity risk assessment or vaccine development.

[0020] The Multiplex-based analysis of these beads can then even be combined with several down-stream application of isolated beads (e.g. via Flow sorting) and the direct analysis of the bound peptide. This would then also allow for the analysis of multiple peptides to multiple alleles.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 shows the general concept of the method of the invention

[0022] Fig. 2 and 4 show experimental details

[0023] Fig.3 shows the loading of color-coded particles with MHC complexes containing a fluorescent placeholder peptide shown for one MHC protein (MHC allotype).DETAILED DESCRIPTION

[0024] Human MHC (HLA) alleles are encoded by oligogenic and highly polymorphic genes. Within the human population, there are > 10.000 different HLA class I and HLA class II allele variants known. The polymorphisms on protein level usually cluster to the site where the peptide binds, favoring the presentation of certain peptides with certain consensus sequences. Thus, in the context of defined infectious or cancer diseases, the peptides presented to T cells triggering antigen-specific T cell responses depend on the HLA type of an individual.

[0025] Bringing recombinant and bead immobilized MHC complexes preloaded with a labeled placeholder peptide in contact with one or more examination peptide(s) and optionally with a MHC loading enhancer (MLE) / peptide exchange catalyst (PEC) lead to a loss of the peptide-conjugated label signal on the bead if the examination peptide is an antigenic peptide and is, thus, binding to the analyzed MHC protein variant.

[0026] In such a method, the binding of a particular peptide, for example a SARS-CoV-2 Spike protein-derived antigenic peptide, can be analyzed with respect to the interaction with a certain MHC allotype variant. However, it is often not clear, to which MHC allotype variant a peptide will bind. Thus, it is often not even clear whether an examination peptide will bind to one or more MHC allotype variants.

[0027] The invention offers a solution allowing the simultaneous analysis of multiple MHC allotype variants with respect to the interaction / binding with an examination peptide (or even more examination peptides).

[0028] This can be achieved by binding a multiplicity of MHC allotype variants to a multiplicity of multiplex beads which are unique with respect to their size and / color (fluorescence). The immobilization of the MHC molecules on the bead surface (e.g. amino dextran) can be either covalent / direct or non-covalent indirect (e.g. binding of biotinylated pFITC / MHC molecules to streptavidin molecules covalently conjugated to the bead surface).

[0029] Furthermore, the pFITC / MHC molecule can be bound to the beads via a linker (e.g. peptide linker).

[0030] It is important that one MHC allotype variant is bound to one bead type which can be identified by e.g. flow cytometry. In this way, the color / size coding is directly related to a certain MHC allotype variant.

[0031] For the method to work, the pre-bound placeholder peptide must be labelled (for instance fluorescently) with at least one type dye molecule, e.g. with FITC. During the examination a different non-fluore scent peptide will likely replace the pre-bound labelled peptide, producing the loss in fluorescence intensity e.g. FITC signal on one specific bead (e.g. analysed by Flow cytometry). Such loss in fluorescence is the indication of a successfulbinding / replacing effect of the examination peptide to a particular MHC allotype which can be uniquely identified by the coded beadColor coded particle

[0032] The term “color coded particle” refers to any material composed of beads, particles (micro or nano sized) labeled with at least 1 color which is not readily solvable in aqueous systems usually used for cell handling. The term does not necessarily refer to a certain hardness or a composition / material.

[0033] Color coded particles as used in the present invention may be manufactured from any material as long as the solvability in aqueous systems is so low that the particle remains observable or detectable during the method of the invention. For example, color coded particles may comprise polystyrene, polydextran, polymethylmetaacrylate, alloptionally chemically modified with reactive groups to bind dyes or oligonucleotides. Suitable reactive groups are for example amino or carboxylic groups.

[0034] Color coded particles useful for the present invention may be prepared with methods known to the skilled person or as described in the literature. For example, they can be prepared by incorporating dyes into pre-formed polymer beads either by swelling of particles in organic solvent mixtures containing dyes either at room temperature (US 6514295 Bl) or at elevated temperatures (US 7507588 B2). A further method involves shifting of phase equilibria due to water addition to force hydrophobic dyes into the polymer phase (US 6964747 B2). Solid micro-sized beads can also be prepared by polymerization of monomer mixtures including dye labeled monomers (J. Am. Chem. Soc. 2004, 126, 21, 6562-6563) or physical entrapment of hydrophobic dyes during particle formation by polymerization (US 5073498).

[0035] Color coded particles “as such” are commercially available from Miltenyi Biotec B.V. & Co. KG.

[0036] The color-coded particles may have a mean diameter between 1 and 30pm.

[0037] Preferable, the color-coded particles are provided in 2 to 50 different ranges of mean diameters wherein the different ranges of diameters can be detected by light scattering.

[0038] In a embodiment of the method according to the invention, a plurality of color- coded particles is provided wherein each color-coded particles comprises a different MHC protein.

[0039] For example, the color-coded particles may comprise at least two dyes having different emission spectra with a difference in emission maxima at least 10 nm, more preferable at least two dyes having different emission spectra with a difference in emission maxima atleast 20 nm. While an increasing number of different dyes improves the quality and amount of information, in practical use, 2 to 10 different dyes are sufficient. 1 dye is sufficient in case of only using the concentration as selection criterium.

[0040] The concentration and the difference in emission maxima of the dyes are preferably selected in a way that discrimination of at least 30, preferably at least 50 different color-coded particles is possible.

[0041] The fluorescence color of the particles is preferable detected in the electromagnetic spectrum between 300 and 900 nm and / or detected via forward scatter and side scatter light.

[0042] Useful dyes are for example protein-based, such as phycobiliproteins, polymeric, such as polyfluorenes, small organic molecule dyes, such as xanthenes, like fluorescein, or rhodamines, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethenes, or metallo-organic complexes, such as Ru, Eu, Pt complexes. Besides single molecule entities, clusters of fluorescent proteins or small organic molecule dyes, as well as nanoparticles, such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymer nanoparticles can also be used as fluorescent moieties.

[0043] In variants of the invention, the MHC proteins are bound to the color-coded particles via an affinity unit, such as streptavidin, avidin, biotin, thymine or neutravidin. barcoding oligonucleotides

[0044] The color-coded particles may comprise further a barcoding oligonucleotide having 2 to 10 nucleotides coding for the mixture of fluorescent dyes in each particle and wherein the peptide-loaded color-coded particles are sequenced after step e).

[0045] Such oligonucleotides are referred to as “barcode” since they allow identifying a single target by their unique sequence.

[0046] The oligonucleotide sequences may contain the naturally occurring cytosine (C), adenine (A), guanine (G)and thymine (T) are preferred. By randomly polymerizing these units, a library of oligonucleotides with different sequences can be obtained. For example, a library randomly producing oligonucleotides comprising 10 nucleotide residues will have 410 = 1048576 members.

[0047] The color-coded particles may further comprise further an oligonucleotide having 2 to 10 nucleotides as PCR primer and wherein the peptide-loaded color-coded particles are multiplied by PCR before sequencing.

[0048] The oligonucleotide sequences may code for a PCR handle or a color specific barcode or a bead specific barcode and are bound to each other either directly or via further oligonucleotide units as spacer unit. The spacer units may be the same or different oligonucleotides comprising each 0 to 30 nucleotide residues. Preferable, the spacer units are non-specific oligonucleotides.

[0049] The technique to produce oligonucleotides and libraries thereof is well known to a person skilled in the art, as well as the technologies to amplify isolated oligonucleotides to obtain larger amounts thereof. US 9388465 summarizes these technologies.Method

[0050] The method is explained in detail according to Figs. 1 and 3.

[0051] Fig.1 shows the principle of the multiplex MHC (HL A) peptide binding assay of the invention. Upper part: Differently color-coded particles (Particle No.l, -No.2 and -No.3) are bound to different specific human MHC proteins (HLA No.l, -No.2, No.3). The color of the particle, herein as yellow, blue and red is linked to the specific human MHC protein. In addition, each specific MHC protein is bound to a placeholder peptide labeled with a fluorescence dye indicated in green (e.g. FITC). Middle and lower part: If a peptide (= a potential T cell epitope) is added to the mixture of color-coded particles bound to different MHC molecules loaded with a fluorescence-labeled placeholder peptides, it may bind to one or more of the specific human MHC proteins (HLAs) bound to the different color-coded particles by replacing the pre-bound fluorescence-labeled peptide from the MC binding groove. This will lead to the reduction of the respective fluorescence for the cognate MHC molecule when detected in a flow cytometry-based assay. Lower plots: This in turn will lead to a loss of placeholder fluorescence, what can be detected e.g. via flow cytometry when plotting the fluorescence of the different particle against the fluorescence of the placeholder peptide label. If a potential immunogenic T cell epitope binds with moderate to high affinity to one of the specific human MHC proteins (HLAs), it will bind to it and replace the placeholder peptide. As the presentation capability of the immune system to present peptides to T cells is one - if not the most relevant event during an Ag-specific immune response, the prove that the peptide binds to a specific MHC protein immobilized on a color-coded particle, strongly indicates, that this is a real T cell epitope.

[0052] Fig. 3 shows the method of the invention more detailed. Here, biotinylated pFITC / MHC complexes, bound to a FITC-labeled placeholder peptide are immobilized on Streptavidin PMMA beads. After incubation, the pFITC / MHC decorated particles aresupplemented with a peptide exchange catalyst and the examination peptides at defined concentration and incubated for a defined time. The particles are then analyzed by Flow cytometry. ). Note: The peptide loading reaction takes finally place in a mixture of color- coded particle bound to different MHC proteins presenting labeled peptide (“multiplex”), but for simplicity the reaction for only one MHC allotype is represented (“monoplex”). Critical parameters for a multiplex experiment are the concentration of the pFITC / MHC, the number of beads, and the concentration of the examination peptide, the peptide exchange catalyst (can strongly vary depending on the respective MHC protein / MHC allotype) and the incubation time.

[0053] Preferable, replacing the placeholder peptide is performed in presence of at least one exchange catalyst, for example with a short peptide consisting of 2-5 amino acids, short peptides consisting of 2-5 amino acids with modified C- and / or N-termini, adamantly-based small-molecule compounds, HLA-DM protein, Tapasin protein or TAPBPR protein.

[0054] Replacing the placeholder peptide is preferable performed at a pH between 4.5-9.5

[0055] The MHC proteins may be selected from human classical MHC class I proteins, human classical MHC II proteins or human non-classical MHC proteins.

[0056] To further increase the resolution of the detection process, the method according to the invention may further comprise detecting the emission spectrum of the peptide-loaded color-coded particles, the emission spectrum of the labelled color-coded particles is detected.

[0057] The method can be especially performed with target peptides originating from viral-, cancer- or autoimmune T cell immune responses.Use of the method

[0058] The method of the invention can be used for various applications in research, diagnostics, and cell therapy.

[0059] Another object of the invention is therefore the use of the method as disclosed herein for identifying target peptides capable of activating and / or expanding T cells, regulatory T cell, NKT cells, g / d T Cells and / or TCR engineered cells.

[0060] In a first use of the invention, examination peptides are detected for their capability to bind to certain MHC proteins present in a mixture of 2 or more (up to 100) different MHC proteins. The sample may comprise one peptide or even a mixture of peptides. At least one examination peptide is added to a mixture of color-coded particle bound to different MHCs, whereby the MHC are “prebound” to peptides which are labelled with e.g., FITC (pFITC / MHC). The label of the peptide can be identical for all respective MHC allotypevariants. The reaction can take place between 0,05 to 24 h. The examination peptide may eventually compete the fluorescently labelled peptide from the binding groove of a certain MHC protein, what leads to the loss of the signal of the peptide label when subsequently analysed for the particle colour, encoding the information for the MHC protein and the label of the peptide. The examination peptides relate to viral-, cancer- or autoimmune T cell immune responses.

[0061] In a second use of the invention, the exchange of the labelled prebound placeholder peptide present in the MHC binding groove requires catalysis by the addition of one or more peptide exchange catalyst (e.g., TAPBPR, Tapasin, 2-5mer peptides, small molecules, or HLA-DM) after the addition of the examination peptide.

[0062] Another use of the invention refers to the feature that the color-coded / labelled particles are not directly / covalently bound to the particle but indirectly via a carrier such as e.g., Streptavidin, avidin or neutravidin, which itself is covalently bound to the particle, allowing the binding of the particle-bound Streptavidin, avidin or neutravidin, to biotinylated MHC proteins loaded with labelled placeholder peptides.

[0063] In one use of the invention the particles bound to the examination peptide are isolated from the mixture / plurality of particles not bound to the examination peptide by flow sorting or magnetic-activated cell sorting. The particles bound to the examination peptide are then used to activate an antigen-specific T cell analytics and / or T cell expansion, comprising contacting the T cell with the isolated particle thereby activating the antigen-specific T cell. The T cells are either present in a biological specimens such as human or murine blood, human or murine peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood or in a more purified form (e.g., CD3-, CD4- CD8-enriched T cell fractions). Alternatively, T cell lines or T cell clone can be used. T cell response is detected by Cytokine detection via e.g., intracellular Cytokine staining of activated T cells and compared to non-activated cells or by detecting T cell proliferation.

[0064] In one selection of the invention the activation and / or proliferation of such T cells responding to particles bound to the examination peptide would require contacting the T cell with the isolated particle and with co-stimulatory molecules or reagents (e.g., anti-Cd28, anti- Cd3, either soluble or immobilized on a carbohydrate backbone) and / or the addition of interleukins (IL-2, IL7, IL- 15).

[0065] In another use of the invention the color-coded particles comprise further a barcoding oligonucleotide having 2 to 10 nucleotide residues coding for the mixture of fluorescent dyesand wherein the peptide-loaded color-coded particles are sequenced. The oligonucleotide may also function as a label for the placeholder peptide in the MHC binding groove.EXAMPLESExample 1 : Cloning, expression and purification of rec. MHC proteins from E, coli and subsequent biotinylation

[0066] For the expression of recombinant MHC molecules in E. coli, synthetic genes encoding for the extracellular domains of a chain (also termed heavy chain) and P2-microglobulin (P2m) were each cloned into the bacterial expression plasmid pET9a separately. An AviTag, allowing enzymatic site-specific biotinylation of the pMHC complexes, was genetically fused to the C- terminus of the extracellular domain of the A*0201 heavy chain, resulting in the expression plasmids pET9a-P2m, pET9a-A* 0201 -His, and pET9a-A*0201-His-Cys. After transformation into E. coli BL21(DE3), selected clones were grown in LB medium, and expression in shaker flasks was induced by the addition of IPTG. After 3 hours, the cells were harvested. After cell disruption, inclusion bodies were isolated, washed, and then solubilized in 6 M GdnHCl, 20 mM Tris, pH 8. Solubilized inclusions bodies were centrifuged for 5 min, and the supernatant was used for refolding of the pMHC complex by rapid dilution.

[0067] Therefore, P2m and chemically synthesized FITC-labeled peptides were added to the refolding buffer (100 mM Tris-HCl (pH 7.7), 400 mM L-arginine, 5 mM red. Glutathione (GSH), 0.5 mM ox. Glutathione (GSSG), 2 mM EDTA) at concentrations of 25 mg / L and 10 mg / L, respectively, and incubated for 2 hours at 4 ° C. Then A*0201 heavy chain at a concentration of 30 mg / L was added and the refolding mix was further incubated for 96 h at 4° C. The pMHC complex was purified by anion exchange chromatography using an Akta pure system, buffer A (20 mM Tris, 15 mM NaCl, pH 8) and buffer B (20 mM Tris pH 8.5, 1 M NaCl). The refolding mixture was diluted with buffer A until the conductivity was between 4- 5 mS / cm. An anion exchange Q adsorber was assembled using two prefilters (0.45 pm and 0.22 pm MillexGV filter).

[0068] After equilibration with running buffer (20 mM Tris, pH 8.5, 20 mMNaCl), the sample was loaded using a peristaltic pump with a flow rate of 40 ml / min. The next steps were performed via Akta.

[0069] The adsorber was connected and washed (without the prefilter systems) using 200 ml of running buffer with a flow rate of 4 ml / min. The elution was performed with a step-elution starting with 200 and then 400 up to 1000 mM NaCl in running buffer with 4 ml / min. Protein-containing fractions were monitored at 280 nm and collected. Table 1 shows an overview of purified pMHC complexes.Table 1 : Yields and purities of generated pMHC complexes loaded with a FITC-labeled placeholder peptideSequence of placeholder peptide:Biotinylation of pMHC complexes

[0070] pMHC complexes were biotinylated using BirA biotin-protein ligase. Before buffer exchange against 20 mM Tris, pH 7,8 was carried out using Ultra filtration (Amicon Ultra-15 Centrifugal Filter 10 kDa MWCO Millipore). pMHC complexes were then concentrated to a mass concentration of >= 3 mg / mL. The biotinylation reaction was carried out according to manufactures instruction (Bulk BirA: BirA biotin-protein ligase bulk reaction kit, Avidity ILLC).

[0071] Afterwards, the reaction mix was resuspended and incubated over night at 2 - 8°C. Next day, lodoacetamide (IAA) was added to the reaction at a final concentration of 10 mM and incubated for 30 min at RT in the dark. The reaction mix was purified by SEC in PE buffer using a HiLoad 16 / 600 Superdex 200 pg column (Cytiva) and fractions containing thebiotinylated pMHC complexes were pooled and concentrated to -1.15 mg / mL. The success of biotinylation was analysed by Native PAGE By loading mixtures of 1 pg Streptavidin and increasing molar ratios of Streptavidin / biotinylated pMHC (1 : 1, 1:2, 1 :4 and 1 :6).Example 2: Conduction and analysis of multiplex MHC peptide binding assay

[0072] For the coupling of biotinylated pFITC / MHC monomers (A*0101-His-Cys / pp50 (245-253)_V3 FITC-bio, “pFITC / MHC I” and A*0201-His-Cys / pp65 (495-503)_V4 FITC- bio, “pFITC / MHC II”), which are linked to MHC-specific placeholder peptides, to Streptavidin-coated beads, populations of color-coded Streptavidin Poly(methyl methacrylate) (PMMA) Red4 Beads were purchased from Poly An (Red 4 0.2 / 5.5 PMMA Beads; Cat. No. 10652005; LOT MG220113SAV1 and MG220113SAV5). These bead populations (color- coded particles) can be discriminated from each other by varying APC staining intensities (figure 2).

[0073] Coupling of Red4 0.2 and Red4 5.5 beads to biotinylated and FITC-peptide- loaded A*0101-monomers and A*0201 -monomers, respectively, was accomplished through incubation of 4,2 nM pFITC / MHC monomer / sample with 2,4E4 beads / sample upon constant rotation at 500 rpm (Eppendorf ThermoMixer® C, Cat. No. 5382000015) for 15 min at room temperature. Afterwards 2E4 pFITC / MHC coupled beads per exchange reaction were plated out on a 96-well V-bottom plate and mixed with peptide-exchange reaction mix at a final volume of 100 pL. The peptide exchange reaction mix included 400 pM MHC allotype-specific examination peptide / sample as well as 5 mM MHC allotype-specific peptide exchange catalyst / sample. In duplex settings, in which peptides on both pFITC / MHC monomers should be exchanged, the peptide-exchange reaction mix contained the specific examination peptide and peptide exchange catalyst for both reactions. Samples were incubated for 16 h, at room temperature, protected from light and under constant rotation at 450 rpm. Afterwards, samples were transferred to a 96-well filter plate (Coming FilterEX® 96-well Filter Plates (0,2 pm PVDF membrane), Cat. No. 110-021-067). Twice washing of the samples using 250 pL PBSZEDTA buffer (PEB) and centrifugation at 500 g for 2 min. Finally, samples were resuspended in 120 pL PEB. Data acquisition was performed on a MACSQuantl6.

[0074] As the placeholder peptide, which is coupled to the MHC-monomer, is fluorescently labeled with FETC-fluorochrome, the release of the placeholder peptide caused by the exchange with the examination peptide is directly linked to a decrease in FITC mid fluorescent intensity (MFI, see figure 3 for overview of a general concept shown for one MHC protein immobilized on a color-coded particle).

[0075] Figure 4 shows the results of an exemplary MHC peptide exchange experiment using human MHC allotype variants (HLAs). Therein, the absent FITC signal of two pure populations of Streptavidin PMMA Beads (Figure 4A) can be compared to the increased FITC MFI values of beads successfully coupled to the pFITC / MHC monomers (figure 4B, black histograms).

[0076] In more detail, Fig.4 shows the detection of binding of an examination peptide to MHC / bead complexes via detection of loss of placeholder fluorescence. (A) Evaluation of FITC signal from pure Streptavidin Red4 0.2 and 5.5 PMMA beads. Gating was performed on all beads via the forward- / side-scatter (left plot), followed by the discrimination of the two bead populations by APC staining intensities (middle plots). Lastly, the absent FITC signal is shown for both bead populations (righter plots).

[0077] (B) Characterization of pFITC / MHC-decorated Red4 0.2 and 5.5 beads after single peptide exchanges (left plots) and duplex exchange (right plots). Reduced FITC-MFIs (color) are observable among single-peptide MHC exchange for the human MHC complex HLA A*0101-allotype (upper-left histogram, orange graph) and for HLA A*0201 -allotype (lower- left histogram, blue graph), compared to unexchanged pFITC / MHC complexes (black) to corresponding beads, pre-peptide exchange FITC-MFI values (corresponding black curves).

[0078] Lastly, the results of the single-MHC (figure 2B, upper and lower left histograms) and duplex-MHC experiments are provided (See Figure 2B; upper and lower right histograms).

[0079] Fig.2 shows Streptavidin Red4 0.2 / 5.5 Bead Populations with different color codes in the APC detection wavelength. Characterization of pure, non-decorated PMMA beads. Shown is the gating strategy to discriminate between different color-codes of Streptavidin 0.2 and 5.5 Red4 PMMA Beads: After gating on all beads using the forward- and side scatter (upper left plot), doublets were excluded trough forward-scatter gating (upper, right plot). Lastly, Red2 0.2 and 5.5 PMMA beads can be discriminated by the gating on the two distinct color-coded populations (lower plots). Created with Biorender.com.

Claims

Claims1. Method for detecting at least one target peptide by binding to a MHC protein characterized in a) providing a plurality of different MHC proteins with placeholder peptides labelled with at least one fluorescent marker thereby obtaining a plurality of labelled MHC proteins b) binding of the labelled MHC proteins to color-coded particles comprising at least one fluorescent dye, wherein the emission spectrum of the fluorescent dyes code for different MHC proteins, thereby obtaining a plurality of labelled color-coded particles c) providing the plurality of labelled color-coded particles with at least one target peptide, wherein at least one target peptide is bound by at least one MHC protein of at least one labelled color-coded particle by replacing the respective placeholder peptide from the MHC protein, thereby obtaining a mixture of color- coded particles bound to a target peptide and color-coded particles bound to placeholder peptides d) detecting the emission spectrum of the color-coded particles bound to a target peptide and the color-coded particles bound to placeholder peptides, thereby detecting the target proteins bound to the MHC protein for which the emission spectrum codes.

2. Method according to claim 1 characterized in that the color-coded particles comprise a mixture of 2 to 50 different fluorescent dyes wherein the fluorescent dyes have different emission spectra thereby creating a combined emission spectrum coding for the at least one MHC protein.

3. Method according to claim 1 or 2 characterized in that replacing the placeholder peptide is performed in presence of at least one exchange catalyst.

4. Method according to any of the claims 1 to 3 characterized in that the MHC proteins are selected from human classical MHC class I proteins, human classical MHC II proteins or human non-classical MHC proteins.

5. Method according to any of the claims 1 to 4 characterized in that the color-coded particles comprise further a barcoding oligonucleotide having 2 to 10 nucleotides coding for the mixture of fluorescent dyes in each particle and wherein the peptide-loaded color-coded particles are sequenced after detection of the target peptides.

6. Method according to claim 5 characterized in that the color-coded particles comprise further an oligonucleotide having 2 to 10 nucleotides as PCR primer and wherein the peptide-loaded color-coded particles are multiplied by PCR before sequencing.

7. Method according to any of the claims 1 to 6 characterized in that the color-coded particles have a mean diameter between 1 and 30pm.

8. Method according to claim 7 characterized in that the color-coded particles are provided in 2 to 50 different ranges of mean diameters wherein the different ranges of diameters can be detected by light scattering.

9. Method according to any of the claims 1 to 8 characterized in that further to detecting the emission spectrum of the peptide-loaded color-coded particles, the emission spectrum of the labelled color-coded particles is detected.

10. Method according to any of the claims 1 to 9 characterized in that the MHC proteins are bound to the color-coded particles via an affinity unit.

11. Method according to claim 10 characterized in that the affinity unit comprises Streptavidin, avidin, biotin, thymine or neutravidin.

12. Method according to any of the claims 1 to 11 characterized in that the target peptides origin from to viral-, cancer- or autoimmune T cell immune responses.

13. Method according to any of the claims 1 to 12 characterized in that the emission spectrum of the color-coded particles bound to a target peptide and the color-coded particles bound to placeholder peptides is detected after isolating the particles.

14. Method according to claim 13 characterized in that detecting the emission spectrum and isolating the particles. is performed by flow-cytometry.

15. Use of the method according to any of the claims 1 to 14 for identifying target peptides capable of activating and / or expanding T cells, regulatory T cell, NKT cells, g / d T Cells and / or TCR engineered cells.

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