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WO2025125528A1 - Procédé de liaison multiplexe cmh-peptide et de détection des lymphocytes t / essai quantifiable de liaison multiplexe cmh-peptide - Google Patents

Procédé de liaison multiplexe cmh-peptide et de détection des lymphocytes t / essai quantifiable de liaison multiplexe cmh-peptide Download PDF

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Publication number
WO2025125528A1
WO2025125528A1 PCT/EP2024/086127 EP2024086127W WO2025125528A1 WO 2025125528 A1 WO2025125528 A1 WO 2025125528A1 EP 2024086127 W EP2024086127 W EP 2024086127W WO 2025125528 A1 WO2025125528 A1 WO 2025125528A1
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WIPO (PCT)
Prior art keywords
mhc
color
peptide
coded particles
coded
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Pending
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PCT/EP2024/086127
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English (en)
Inventor
Charlyn DÖRNTE
Marek Wieczorek
Jonathan Fauerbach
Volker Nölle
Marc Schuster
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Miltenyi Biotec GmbH
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Miltenyi Biotec GmbH
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Publication of WO2025125528A1 publication Critical patent/WO2025125528A1/fr
Pending legal-status Critical Current
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing

Definitions

  • 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.
  • an MHC protein allotype e.g. HLA protein
  • T cell epitopes 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).
  • MHCs major histocompatibility complexes
  • pMHC peptide/MHC
  • T cell receptor 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.
  • 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.
  • the human immune system can give rise to 10 14 to 10 16 different TCR sequences, although the real number of unique T cells expressing individual TCRs is several orders of magnitude lower.
  • 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.
  • 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.
  • HLA human leukocyte antigen
  • 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.
  • one antigen usually contains multiple potential MHC-presented T cell epitopes, which are restricted to a certain MHC variant.
  • 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.
  • Novel vaccines selected and designed to contain a broad spectrum of T cell epitopes for the most common MHCs.
  • 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.
  • 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.
  • this method can be used in order to identify T cell stimulating peptide presenting by MHC molecules, it requires 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.
  • the readout is only indirectly correlating the binding of the peptide to a certain MHC variants, as this is not directly measured.
  • 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.
  • 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.
  • EP 1692504 is restricted in the detection of only one or maximally a few peptides simultaneously.
  • EP3455630 describes a peptide exchange system and method.
  • 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.
  • 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 individual microspheres.
  • 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.
  • 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.
  • 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.
  • the different specific alleles can be distinguished based on their bead-specific feature (e.g., specific size or color).
  • bead-specific feature e.g., specific size or color.
  • HLA Human MHC
  • HLA class I and HLA class II allele variants 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.
  • the binding of a particular peptide for example a SARS-CoV-2 Spike protein-derived antigenic peptide
  • a particular peptide for example a SARS-CoV-2 Spike protein-derived antigenic peptide
  • MHC allotype variant a peptide will bind.
  • an examination peptide will bind to one or more MHC allotype variants.
  • 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).
  • 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).
  • the pFITC/MHC molecule can be bound to the beads via a linker (e.g. peptide linker).
  • a linker e.g. peptide linker
  • 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.
  • the pre-bound placeholder peptide must be labelled (for instance fluorescently) with at least one type dye molecule, e.g. with FITC.
  • FITC a type dye molecule
  • FITC signal e.g. FITC signal on one specific bead (e.g. analysed by Flow cytometry).
  • loss in fluorescence is the indication of a successful binding/replacing effect of the examination peptide to a particular MHC allotype which can be uniquely identified by the coded bead
  • each color-coded particles comprises a different MHC protein.
  • 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.
  • 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.
  • 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.
  • each specific MHC protein is bound to a placeholder peptide labeled with a fluorescence dye indicated in green (e.g. FITC).
  • HLAs human MHC proteins
  • 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.
  • HLAs human MHC proteins
  • 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.
  • Fig. 3 shows the method of the invention more detailed.
  • biotinylated pFITC/MHC complexes, bound to a FITC-labeled placeholder peptide are immobilized on Streptavidin PMMA beads.
  • the pFITC/MHC decorated particles are supplemented 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. ).
  • 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.
  • the method of the invention can be used for various applications in research, diagnostics, and cell therapy.
  • 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.
  • one or more peptide exchange catalyst e.g., TAPBPR, Tapasin, 2-5mer peptides, small molecules, or HLA-DM
  • 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.
  • a carrier such as e.g., Streptavidin, avidin or neutravidin
  • 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).
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • Table 1 Yields and purities of generated pMHC complexes loaded with a FITC-labeled placeholder peptide
  • reaction mix was resuspended and incubated over night at 2 - 8°C.
  • 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 the biotinylated pMHC complexes were pooled and concentrated to -1.15 mg/mL.
  • 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.
  • 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).
  • 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.

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Abstract

L'invention concerne un procédé de détection d'au moins un peptide cible par liaison à une protéine du CMH par fourniture d'une pluralité de différentes protéines du CMH avec des peptides de remplacement marqués avec au moins un marqueur fluorescent, ce qui permet d'obtenir une pluralité de protéines du CMH marquées.
PCT/EP2024/086127 2023-12-14 2024-12-13 Procédé de liaison multiplexe cmh-peptide et de détection des lymphocytes t / essai quantifiable de liaison multiplexe cmh-peptide Pending WO2025125528A1 (fr)

Applications Claiming Priority (2)

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EP23216590 2023-12-14
EP23216590.2 2023-12-14

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WO2025125528A1 true WO2025125528A1 (fr) 2025-06-19

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WO2021122185A1 (fr) * 2019-12-20 2021-06-24 Miltenyi Biotec B.V. & Co. KG Détection de cellule réversible par le biais du cmh avec des conjugués ayant une fraction de détection clivable par voie enzymatique
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CA3215077A1 (fr) * 2021-04-07 2022-10-13 Thomas J. Malia Multimeres de peptide-mhc-immunoglobuline et leurs procedes d'utilisation
WO2023025851A1 (fr) * 2021-08-24 2023-03-02 Immatics US, Inc. Sélection de cellules immunitaires à l'aide de complexes peptide-cmh générés par échange de ligands conditionnel
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US20070259449A1 (en) 1996-04-25 2007-11-08 Michael Seul Method of making a microbead array with attached biomolecules
US6514295B1 (en) 1997-10-14 2003-02-04 Luminex Corporation Precision fluorescently dyed particles and methods of making and using same
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WO2022087154A1 (fr) * 2020-10-20 2022-04-28 Repertoire Immune Medicines, Inc. Multimères peptidiques de classe ii du cmh et leurs utilisations
CA3215077A1 (fr) * 2021-04-07 2022-10-13 Thomas J. Malia Multimeres de peptide-mhc-immunoglobuline et leurs procedes d'utilisation
WO2023025851A1 (fr) * 2021-08-24 2023-03-02 Immatics US, Inc. Sélection de cellules immunitaires à l'aide de complexes peptide-cmh générés par échange de ligands conditionnel
WO2023225207A2 (fr) * 2022-05-19 2023-11-23 Biontech Us Inc. Procédé et systèmes de prédiction d'épitopes spécifiques des hla de classe ii et caractérisation de lymphocytes t cd4+

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