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EP-4740019-A1 - A METHOD OF IDENTIFYING MHC-BINDING PROTEINS AND INTERACTING PEPTIDES IN A SAMPLE

EP4740019A1EP 4740019 A1EP4740019 A1EP 4740019A1EP-4740019-A1

Abstract

The present invention relates to a method of identifying one or more targets of interest in a biological sample. The method comprises the steps of obtaining a biological sample that comprises a peptide-MHC binding molecule, adding to the sample a capturing agent (CA) that binds the peptide-MHC binding molecule, wherein the peptide-MHC binding molecule binds a target of interest, and/or is a target of interest. Enriching the peptide-MHC-binding molecules from said sample to obtain a test sample, treating the test sample with a proteolytic enzyme to obtain proteolytic fragments of inter alia the target(s) of interest, identifying one or more of said proteolytic fragments by means of Mass Spectrometry, thereby identifying the target(s) of interest (Fig. 1).

Inventors

  • Schräder, Christoph
  • SCHUSTER, HEIKO
  • Kowalewski, Daniel Johannes
  • ROSTOCK, Lida
  • HASENEDER, Sarah

Assignees

  • Immatics Biotechnologies GmbH

Dates

Publication Date
20260513
Application Date
20240705

Claims (14)

  1. 1. A method of identifying one or more target(s) of interest in a biological sample, wherein the method comprises the steps of: a) obtaining a biological sample comprising a peptide-MHC binding molecule, which comprises a binding domain of a T-cell receptor (TCR); b) adding to the biological sample a capturing agent (CA) that binds the peptide- MHC binding molecule, wherein the peptide-MHC binding molecule: (i) binds a target of interest, and/or (ii) is a target of interest c) enriching the peptide-MHC -binding molecule from said sample to obtain a test sample; d) treating test sample with a proteolytic enzyme to obtain proteolytic fragments of the one or more target(s) of interest; e) identifying one or more of said proteolytic fragments by means of mass spectrometry (MS), thereby identifying the one or more target(s) of interest.
  2. 2. The method according to claim 1, wherein the peptide-MHC binding molecule is a bispecific binding molecule, preferably additionally comprising a binding domain of an antibody.
  3. 3. The method according to any one of the preceding claims, wherein the one or more target(s) of interest individually a) is, b) comprises or c) is comprised in, at least one selected from the group consisting of • a peptide-MHC binding molecule, or a target binding fragment thereof, • a peptide-MHC complex (pMHC) and/or • a target peptide (TP).
  4. 4. The method according to any one of the preceding claims, further comprising the step of quantifying the one or more target(s) of interest.
  5. 5. The method according to any one of the preceding claims, further comprising adding to the biological sample at least one of: a) a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule, and/or b) a peptide-MHC complex comprising a stable isotope label, the complex being specifically bound by the peptide-MHC binding molecule.
  6. 6. The method according to any one of the preceding claims, wherein identifying one or more proteolytic fragments by means of MS encompasses at least one method selected from the group consisting of: mass spectrometry (MS), tandem mass spectrometry (MS/MS) and liquid chromatography coupled with mass spectrometry (LC-MS, LC-MS/MS).
  7. 7. The method according to any one of the preceding claims, wherein the biological sample is a human or animal sample.
  8. 8. The method according to claim 7, wherein the human or animal of which the biological sample was obtained has been treated with the peptide-MHC binding molecule or a molecule encoding the peptide-MHC binding molecule prior to obtaining the sample.
  9. 9. The method according to any one of claims 3 to 8, wherein the MHC molecule within the peptide-MHC complex is MHC class I, optionally is HLA.
  10. 10. The method according to any one of the preceding claims, wherein the peptide-MHC binding molecule comprises at least one further functional entity selected from the group consisting of • an entity that extends serum half-life, preferably a FC domain, and/or • a toxic entity.
  11. 11. The method according to any one of the preceding claims, wherein the capturing agent (CA) binds the peptide-MHC binding molecule, a) within a target binding domain thereof, or b) outside of a target binding domain thereof, preferably to the FC domain if present.
  12. 12. The method according to any one of the preceding claims, wherein the capturing agent is selected from the group consisting of: protein A, peptide-MHC complex, an anti-Fc domain antibody, CD3, an anti-CD3 binder, the constant or variable domain of a TCR alpha or beta chain, a binder that binds to a binder to the constant or variable domain of a TCR alpha or beta chain, an antiidiotypic binder, and albumin or a fragment thereof.
  13. 13. The method according to any one of the preceding claims, wherein the step of enriching the peptide-MHC binding molecule involves releasing the same from the capturing agent.
  14. 14. The method according to any one of the preceding claims, wherein the biological sample is, or comprises, at least one element of the list consisting of: serum, plasma, tissue, tumor sample; or combinations thereof.

Description

A method of identifying MHC-binding proteins and interacting peptides in a sample Field of the invention The present application relates to a method of identifying one or more target(s) of interest (e.g. MHC -binding proteins) in a sample. Incorporation by Reference All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there are any inconsistencies between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended. Background The major histocompatibility complex (“MHC”, also called “HLA” in the context of the present specification) is a gene cluster on chromosome 6 which is common to most vertebrates encoding for different genes, which play a fundamental role in histocompatibility and the adaptive immune system. In humans this cluster is often also commonly referred to as human leukocyte antigen (HLA). MHC molecules are expressed on all cells of a mammal with the exception of erythrocytes. Their main function is to present short peptides derived from intracellular proteins to cytotoxic T lymphocytes (CTLs) (Boniface and Davis, 1995; Goldberg and Rizzo, 2015b; Gruen and Weissman, 1997; Rock and Shen, 2005). Such short peptides are sometimes also called “T cell epitopes”. T cell epitopes are essentially peptide fragments of intra- or extracellular proteins (so called “parental proteins”) which are presented by MHC class I or II molecules on the surface of antigen presenting cells (APCs). T cell epitopes have a length of typically 8 - 11 amino acid (AA) residues (when being presented by MHC class I molecules) or typically 15 - 24 AA residues (when being presented by MHC class II molecules). Such parental proteins are intracellular proteins expressed by the respective cells, and part thereof is then fragmented by the proteasome complex, so that the resulting fragments are loaded on MHC class I proteins which are then displayed on the surface of the cells. The resulting complexes are called “peptide-MHC complexes” (or ”pMHC”, or “pHLA”, in the context of the present specification). Alternatively, such parental proteins are extracellular proteins that are endocytosed by cells, fragmented and then loaded on MHC class II proteins which are then displayed on the surface of the cells. CTLs express CD8 co-receptors, in addition to T cell receptors (TCRs). When a CTL's CD8 receptor docks to an MHC class I molecule on a target cell, if the CTL's TCR fits the epitope represented by the complex of MHC class I molecule and presented peptide, the CTL triggers the target cell lysis by either releasing a cargo of cytolytic enzymes or rendering the cell to undergo programmed cell death by apoptosis (Delves and Roitt, N Engl J Med. 2000 Jul 6;343(l):37-49; Lustgarten et al., Eur J Immunol. 1991 Oct;21(10):2507-15). Thus, MHC class I helps mediate cellular immunity, a primary means to address intracellular pathogens, such as viruses and some bacteria, including bacterial L forms or bacterial genera Shigella and Rickettsia (Madden et al., Cell. 1993 Nov 19;75(4):693-708). Furthermore, this process is also of utmost importance for the immunological response and defense against neoplastic diseases such as cancer (Coulie et al., Nat Rev Cancer. 2014 Feb;14(2): 135-46.; Urban and Schreiber, Annual Review of Immunology 1992 10: 1, 617-644, 1992). Peptide-MHC (pMHC) complexes have been discussed as a target for therapeutic intervention, by means of entities binding to the combination of MHC and a given peptide presented by the MHC. Such entities are, for example, • adoptive T cells comprising a native or engineered T cell receptor (“TCR-T)” (Liu Y, et al., Front Oncol. 2022 Jan 25; 11) • molecules consisting of, or comprising a T cell receptor (TCR) or a target binding fragment thereof (W02019012141A1), and/or • molecules consisting of, or comprising a TCR mimic antibody (TCRmAb), or a target binding fragment thereof (Dubrovsky et al, Oncoimmunology. 2015 Jun 1;5(1)) T cell receptors (TCRs), in their original form, are molecules of the immune system that are capable to bind to fragments of intra- or extracellular proteins (T cell epitopes) presented by molecules of the Major Histocompatibility Complex (MHC) class I or II on the surface of antigen presenting cells (APCs). TCRs bind to the area of the MHC which presents the T cell epitopes and are hence specific for the complex of the two. They do not bind to the naked MHC or a free-floating T cell epitope. Antibodies can be developed to specifically bind these peptide-MHC complexes, similar to the recognition of such complexes by TCRs. These antibodies are referred to as TCR mimic antibodi