A method of identifying mhc-binding proteins and interacting peptides in a sample

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).

Description

[0001] A method of identifying MHC-binding proteins and interacting peptides in a sample

[0002] Field of the invention

[0003] The present application relates to a method of identifying one or more target(s) of interest (e.g. MHC -binding proteins) in a sample.

[0004] Incorporation by Reference

[0005] 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.

[0006] Background

[0007] 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).

[0008] 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).

[0009] 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).

[0010] 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.

[0011] 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).

[0012] 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)

[0013] • molecules consisting of, or comprising a T cell receptor (TCR) or a target binding fragment thereof (W02019012141A1), and / or

[0014] • 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))

[0015] 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.

[0016] 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 antibodies (TCRmAb).

[0017] Molecules consisting of, or comprising, a TCR mimic antibody or a T cell receptor or a fragment thereof can be administered systematically, and then bind to cells displaying on their surface peptide-MHC complexes that present a target peptide, i.e., a peptide to which, when presented by MHC, the target binding part of a TCR mimic antibody or a T cell receptor can bind.

[0018] Based on state of the art, immunoproteomic methods as e.g. developed and patented by the applicant of the present invention (AbsQuant® method, W02016107740A1, Xpresident® method W020031043), T cell epitopes can be identified which are presented by MHC molecules primarily on tumor tissue, and not on healthy tissue. Such T cell epitopes are often called “tumor associated peptides” (“TUMAPs”). Such TUMAPs stem from parental proteins that are expressed in the respective cell, and part of which are fragmented in the proteasome process and then delivered to MHC. If the respective cell is cancerous, this can either result in a) expression of canonical wildtype proteins, yet in a wrong ontogenetic phase (e.g., embryonal protein expressed by a tumor in an adult patient) b) expression of canonical wildtype proteins, yet in a wrong tissue c) expression of canonical wildtype proteins, yet in an exaggerated quantity d) expression of a mutated protein, resulting in non-naturally occurring T cell epitopes (cancer-neoepitopes)

[0019] After binding to a pMHC presenting such peptide, the molecule consisting of, or comprising, a TCR mimic antibody or a T cell receptor or fragment thereof, can evoke an anti-tumor response, e.g., by means of a conjugated effector domain. Such effector domain can, for example, be a T cell engager, like a CD3 binder (e.g., an anti CD3 antibody) or binder to a constant or variable domain of a TCR alpha or beta chain (e.g., a respective antibody).

[0020] Pharmacokinetics and pharmacodynamics, as well as tissue distribution, of such molecules consisting of, or comprising, a TCR mimic antibody or a T cell receptor or fragment thereof are not well understood so far. Contrary to typical antibodies, which bind to cell surface proteins or soluble proteins, the binding reaction the target binding domains of TCR mimic antibodies or T cell receptors are involved in is more complex, as it comprises two further elements instead of only one, namely the MHC and the target peptide presented thereby. The complexity of the binding reaction is shown in an illustrative fashion in Fig. 3.

[0021] This degree of complexity makes investigations of pharmacokinetics and pharmacodynamics of therapeutic entities comprising a peptide-MHC binding molecule very difficult. The present invention addresses these issues.

[0022] Summary of the Invention

[0023] The present invention essentially relates to methods for identifying and optionally quantifying one or more target(s) of interest in a biological sample utilizing the interactions between a target peptide, an MHC and a peptide-MHC binding molecule. Since three entities are involved in the binding, it is different from and more complex than e.g. antibody-antigen interactions. The present invention provides several approaches that are uniformly suitable to identify and optionally quantify the interaction between the three variables, i.e. target peptide, MHC and MHC binding molecule. The inventive method utilizes either “stationary” peptide-MHC complexes to capture binding molecules, or “stationary” binding molecules to capture peptide- MHC complexes, or “stationary” capturing agents which capture binding molecules that may or may not be engaging with (i.e., bound to) peptide-MHC complexes, which are just several aspects of the same idea. In other words, binding molecules may be captured as free entities, or with their binding sites being occupied with a peptide-MHC complex. Labelled target peptides, peptide-MHC complexes or binding molecules may be added to the sample as needed to facilitate identification and optionally quantification of the binding interaction.

[0024] The present invention thus relates to a method of identifying one or more targets(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:

[0025] (i) binds a target of interest, and / or

[0026] (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 peptide(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.

[0027] In other words the present invention regards a method for the identification of peptide-MHC binding molecules or other targets (e.g. peptides) of interest that interact with the peptide-MHC binding molecules in a biological sample. A capturing agent is then used to enrich the peptide- MHC binding molecules and optionally further target(s) of interest attached thereto. Identifying the target of interest (TOI) may encompass quantifying the one or more target(s) of interest.

[0028] In a preferred embodiment of the method the peptide-MHC binding molecule is a bispecific binding molecule. More preferably the peptide-MHC binding molecule is additionally comprising a binding domain of an antibody.

[0029] In a preferred embodiment the method may further comprise adding to the biological sample and / or the test sample at least one of a) a stable isotope labelled (SIL) variant of the peptide-MHC binding, and / or b) a peptide-MHC complex bearing a stable isotope labelled target peptide (TP), the complex being capable of specifically binding to the peptide-MHC binding molecule.

[0030] In a preferred embodiment step e) 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).

[0031] In a preferred embodiment, the biological sample is a human or animal sample. In a preferred embodiment the biological sample is a human sample. In a preferred embodiment the biological sample is obtained from a mammal.

[0032] In a preferred embodiment the human or animal of which the biological sample was obtained has been treated with the peptide-MHC binding molecule prior to obtaining the sample. In a preferred embodiment the human or animal of which the biological sample was obtained has been treated with one or more molecule(s) encoding the peptide-MHC binding molecule.

[0033] In a preferred embodiment the one or more target(s) of interest a) is, b) comprises or c) is comprised in, at least one selected from the group consisting of an unbound (“free”) or bound peptide-MHC binding molecule, or a target binding fragment thereof; a peptide-MHC complex (pMHC) and / or a target peptide (TP). In a preferred embodiment the target of interest is a peptide-MHC binding molecule or a target binding fragment thereof. In a preferred embodiment the target of interest is a peptide-MHC complex. In a preferred embodiment the target of interest is a target peptide. In a preferred embodiment the targets of interest are a peptide-MHC binding molecule (or a target binding fragment thereof) and a peptide-MHC complex. In a preferred embodiment the targets of interest are a peptide-MHC binding molecule (or a target binding fragment thereof) and a target peptide. In a preferred embodiment the targets of interest are a peptide-MHC binding molecule (or a target binding fragment thereof) and a cell (preferably a T-cell) bound by the bispecific peptide-MHC binding molecule.

[0034] In the methods of the invention, the unbound or bound peptide-MHC binding molecule or target binding fragment thereof may be at least one of a T cell receptor (TCR) or a target binding fragment thereof, and / or TCR mimic antibody (TCRmAb), or a target binding fragment thereof.

[0035] In a preferred embodiment the MHC molecule within the peptide-MHC complex may be MHC class I, optionally HLA.

[0036] In a preferred embodiment the capturing agent (CA) may be a) a capturing agent that binds to a domain of the peptide-MHC binding molecule within the target binding domain thereof, or (b) may be a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof.

[0037] In a preferred embodiment 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, a antiidiotypic binder, and albumin or a fragment thereof.

[0038] In a preferred embodiment the capturing agent is selected from the group consisting of: a peptide-MHC complex, an agent that binds the peptide-MHC binding molecule outside of the target binding domain thereof (preferably protein A). In a preferred embodiment the unbound or bound peptide-MHC binding molecule may be a bi- or multifunctional molecule which comprises at least one further functional entity, preferably selected from the group consisting of an effector entity, an entity that extends serum half-life, and / or a toxic entity as described herein. In a preferred embodiment the at least one further functional entity is selected from an entity that extends serum half-life, preferably a FC domain, and / or a toxic entity.

[0039] In a preferred embodiment, the capturing agent (CA) is bound to a solid matrix.

[0040] In a preferred embodiment, the step of enriching the peptide-MHC binding molecule (i.e. step c)) involves releasing the same from the capturing agent.

[0041] In a preferred embodiment, the biological sample is, or comprises, at least one element of the list consisting of: serum, plasma, tissue, tumor sample; or combinations thereof.

[0042] In a preferred embodiment, the target of interest to be identified may be an unbound (“free”) peptide-MHC binding molecule, and the capturing agent (CA) may be a capturing agent to which the peptide-MHC binding molecule specifically binds, the capturing agent comprising a given target peptide (TP).

[0043] In a preferred embodiment, the target of interest to be identified may be a bound or unbound peptide-MHC binding molecule, and the capturing agent (CA) may be a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof. The method may further comprise adding, to the sample, of an unbound (“free”) stable isotope labelled (SIL) variant of the peptide-MHC binding molecule.

[0044] In a preferred embodiment, the target of interest to be identified may be a bound peptide-MHC binding molecule, and the capturing agent (CA) may be a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof, and the method may further comprise adding, to the sample, of peptide-MHC complexes to which the peptide-MHC binding molecule specifically binds, wherein at least a part of said peptide-MHC complex is isotopically labelled.

[0045] In a preferred embodiment, the target of interest to be identified may be an unbound (“free”) peptide-MHC binding molecule, and the capturing agent (CA) may be a peptide-MHC complex to which the peptide-MHC binding molecule binds.

[0046] Brief description of the Figures

[0047] Fig. 1 A: Representative LC / MS fragment ion traces showing specificity and selectivity of the method. The pMHC binding molecule is a TCER® as explained elsewhere herein.

[0048] Fig. IB: Calibration curve depicting the ratio of titrated, unlabeled TCER® to labeled SIL TCER® using enrichment via pMHC immunoprecipitation. The calibration / titration curve shows linearity across a dynamic range of over 4 orders of magnitude. Note that pHLA as used in this Figure is synonymously used to pMHC as used elsewhere herein.

[0049] Fig. 1C: Calibration curve depicting the ratio of titrated, unlabeled TCER® to labeled SIL TCER® using enrichment via pMHC vs protein A immunoprecipitation. Three (3) different concentrations of the unlabeled TCER® molecule are displayed at a theoretical amount of 5, 24 and 118 firnol unlabeled TCER® injected into the LC / MS system. For each of the conditions, seven (7) different Lys-C released TCER® peptides are displayed. Notably, pMHC (binding TCR domain) and protein A (binding IgGl domain) bind the TCER® molecules at different sites, yet recovery of the unlabeled TCER® vs the SIL TCER® is virtually identical. Note that pHLA as used in this Figure is synonymously used to pMHC as used elsewhere herein.

[0050] Peptides 1 to 7 are proteolytic fragments (LysC proteolysis) of a bispecific peptide-MHC binding molecule comprising a target binding domain of a T cell receptor (TCER® format) characterized as follows:

[0051] Fig. 2: Quantification of TCER® molecules which had been applied to two different in vivo mice models engrafted with a human melanoma tumor transplant. Protein lysate from the two melanoma models was subsequently subjected to protein A- (Fig. 2A) or pMHC-mediated (Fig. 2B) immunoprecipitation paired with a SIL TCER® spike for normalization and absolute quantification. Each bar depicts absolute peptide amount of the seven TCER®-specific peptides of interest. Averaging the abundance of all seven peptides shows high quantitative precision with a CV of 10.01% and 13.24%, respectively.

[0052] Fig. 3: Illustration of the complex binding reaction between a TCR or TCRmAb, MHC and the target peptide presented by the latter.

[0053] Fig. 4: Shows a preferred embodiment of the present invention in which a) the target of interest (IPOI) to be identified is an unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a peptide-MHC complex to which the peptide-MHC binding molecule specifically binds, the peptide-MHC complex comprising a given target peptide (TP).

[0054] The peptide-MHC complex presents the target peptide (TP) which, when presented by MHC, is recognized by the peptide-MHC binding molecule. The peptide-MHC binding molecule shown in the figure comprises (i) the target binding domain of e.g. a T cell receptor, (ii) additionally, a T cell engaging entity, e.g., a binder that binds to CD3 or a binder that binds to the constant or variable domain of a TCR alpha or beta chain, and (iii) an antibody Fc domain that is optionally effector function silenced so as to no longer evoke Antibody Dependent Cell mediated Cytotoxicity (ADCC) reactions.

[0055] These three limitations shown in Fig. 4 and other figures shall not be construed as limiting regarding the scope and / or disclosure of the present invention, yet show, illustratively, embodiments only.

[0056] In any case, however, when capturing a peptide-MHC binding molecule that comprises such T cell engaging entity, entities bound by said T cell engaging entity can be co-precipitated and then be analyzed as well. Hence, if, e.g., the T cell engaging entity is an anti CD3 antibody domain, CD3 bound thereto can be co-precipitated. If the T cell engaging entity is an antibody that binds to the constant or variable domain of a TCR alpha or beta chain, TCR alpha or beta chains bound thereto can likewise be co-precipitated.

[0057] Optionally, a stable isotope labelled (SIL) variant of the peptide / MHC binding molecule can be added prior to or spiked in after enrichment, for quantification by means of mass spectrometry.

[0058] Experimental support for this embodiment is provided, inter alia, in Fig. 1 A (calibration curve using pMHC - exemplary ion traces), Fig. IB (calibration curve using pMHC - ratio unlabeled vs SIL TCER), Fig. 1C - pMHC enrichment vs protein A enrichment and Fig 2 (ex vivo data - quantification via pMHC enrichment).

[0059] While the peptide-MHC binding molecule binds preferably to its specific target peptide when presented by MHC, there is also the likelihood that it may bind, with lower preference, to nonspecific target peptides, e.g., analogues of the specific target peptide which share some degree of sequence similarity therewith. As a result, in this method, non-specific pMHC can also be enriched and analyzed.

[0060] Fig. 5 : Shows a preferred embodiment of the present invention in which a) the target of interest (IPOI) to be identified is a bound or unbound (“free”) peptide-MHC binding molecule, and (b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof. For the bound peptide-MHC binding molecule, the bound peptide-MHC complex is shown in parentheses.

[0061] Further, c) an unbound (“free”) stable isotope labelled (SIL) variant of the peptide-MHC binding molecule is added to the sample prior to enrichment to capture unbound peptide-MHC complexes that present the target peptide (TP). These SIL variants of the peptide-MHC binding molecule are likewise captured by the capturing agent (CA).

[0062] While the peptide-MHC binding molecule binds preferably to its specific target peptide when presented by MHC, there is also the likelihood that it may bind, with lower preference, to nonspecific target peptides, e.g., analogues of the specific target peptide which share some degree of sequence similarity therewith. As a result, in this method, non-specific pMHC can also be enriched and analyzed.

[0063] Experimental support for this embodiment is provided, inter alia, in Fig. 1C (pMHC enrichment vs protein A enrichment), Fig. 2A (ex vivo data - quantification via protein A enrichment).

[0064] Fig. 6: Shows a preferred embodiment of the present invention in which a) the target of interest (IPOI) to be identified is a bound or unbound peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof. For the bound peptide-MHC binding molecules, the bound peptide / MHC complex is shown in parentheses.

[0065] In this preferred embodiment, all peptide-MHC binding molecules (i.e., bound and unbound) are isolated- and with them, all peptide-MHC complexes that are bound thereto. Peptide-MHC complexes within the sample that are not bound by peptide-MHC binding molecules are not isolated.

[0066] In a preferred embodiment of this method, the bound peptide-MHC binding molecules can later be identified and quantified by using, as an internal standard, a SIL variant of the target peptide (TP) bound by the peptide-MHC binding molecule. By quantifying said target, due to the 1 : 1 stochiometry, bound peptide-MHC binding molecules can likewise be quantified.

[0067] While the peptide-MHC binding molecule binds preferably to its specific target peptide when presented by MHC, there is also the likelihood that it may bind, with lower preference, to nonspecific target peptides, e.g., analogues of the specific target peptide which share some degree of sequence similarity therewith. As a result, in this method, non-specific pMHC can also be enriched and analyzed.

[0068] Fig. 7 : Shows a preferred embodiment of the present invention in which a) the target of interest (IPOI) to be identified is a bound peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof.

[0069] Further, (c) peptide-MHC complexes are added to the sample to which the peptide-MHC binding molecule specifically binds, wherein at least a part of said peptide-MHC complex - for example, the target peptide (TP) - is SIL labelled.

[0070] The specificity is due to the target peptide (TP) presented by peptide-MHC complexes that are added. In this context, a “part of said peptide-MHC complex” means either that (i) the peptide presented by the peptide-MHC complex or (ii) the MHC, or fragments thereof is isotopically labeled.

[0071] This preferred embodiment is suitable to identify, in a sample that comprises excess "free" peptide-MHC binding molecule (e.g., in a patient sample who was before treated with a molecule that is or comprises a T cell receptor or a TCR mimic antibody, or a fragment thereof, wherein the sample comprises a large share of blood or plasma), the share of bound peptide- MHC binding molecule, relative to the “free” peptide-MHC binding molecule.

[0072] While the peptide-MHC binding molecule binds preferably to its specific target peptide when presented by MHC, there is also the likelihood that it may bind, with lower preference, to nonspecific target peptides, e.g., analogues of the specific target peptide which share some degree of sequence similarity therewith. As a result, in this method, non-specific pMHC can also be enriched and analyzed.

[0073] The following table summarizes the embodiments discussed above.

[0074] Table 1: Overview of some embodiments of the invention

[0075] The following table shows some examples for the generic features used above.

[0076] Table 2: Some examples for the generic features used above

[0077] Fig. 8:

[0078] Figures shows the simultaneous quantification of target peptide, HLA and beta-2- microglobulin using the method of the invention as compared to art-known methods which require the use of three different set-ups.

[0079] A)

[0080] Shows the quantification of relative target peptide abundance (along with co-precipitated HLA)

[0081] Method 1 : “Method of the present invention, co-precipitation and quantitation of pHLA Method 2: Proprietary AbsQuant® platform for highly sensitive pHLA quantitation B)

[0082] Shows the simultaneous quantification of the protein HLA comparing the method of the present invention (method 1) and the gold standard known in the prior art (method 2) quantifying the target peptide as shown in Fig 8a.

[0083] C)

[0084] Shows the simultaneous quantification of beta-2-microglobulin (b2m) comparing the method of the present invention (method 1) and the gold standard known in the prior art (method 2) quantifying the target peptide as shown in Fig 8a.

[0085] Fig. 9

[0086] Shows that the quantification using the method of the present invention is highly selective and can quantify the molecule of interest at very high accuracy

[0087] Fig. 10

[0088] Shows quantification of TCER molecule detected in xenograft tissue. The quantification of the method of the present invention shows consistent values in experimental replicates and correlates with the gold standard method AbsQuant (method 2 in the example section) as indicated by the copies per cell (CpC) values determined with AbsQuant for the same samples.

[0089] Fig. H

[0090] Shows the accuracy of the method of the invention (method B) and the comparison to an art- known ECL based immunoassay (method A)

[0091] Fig. 12

[0092] Shows quantification of the pMHC -binding molecule (TCER) in various body compartments. Different normal organ tissues from tumor-transplanted mouse models treated with TCER were collected and analysed using the method of the present invention. A) TCER amount per g tissue in Mouse 1, B) TCER amount per g tissue in Mouse 2; C) direct comparison of the results of Mouse 1 and Mouse 2 Fig. 13

[0093] Shows the quantification of a TCER in serum samples and tumor biopsies of human patients receiving TCER for cancer treatment. The method of the present invention provides well correlated measurements for the serum and tumor samples of the clinical patients

[0094] Detailed Description of the Invention

[0095] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the embodiments described or process steps of the methods described as such embodiments and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting the scope of the invention. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

[0096] It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said another embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

[0097] Furthermore, the content of the state of the art documents referred to herein is incorporated by reference. This refers, particularly, to state of the art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions. In the context of the present invention the “target of interest” encompasses short peptides, polypeptides as well as proteins (including immune proteins, e.g. peptide-MHC binding molecules). In another preferred embodiment wherein the target of interest is a polypeptide or protein, multiple peptides thereof are identified and / or quantified for improved accuracy and precision.

[0098] According to a first aspect of the invention, 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:

[0099] (i) binds a target of interest, and / or

[0100] (ii) is the target of interest c) enriching the peptide-MHC -binding molecules from said sample to obtain a test sample; d) treating the test sample with a proteolytic enzyme to obtain proteolytic fragments of inter alia the one or more target(s) of interest (IPOI) e) identifying one or more of said proteolytic fragments by means of mass spectrometry, thereby identifying the one or more target(s) of interest.

[0101] Peptide-MHC binding molecules bind, by definition, to a peptide-MHC (pMHC) complex, i.e. an MHC presenting a peptide.

[0102] Typically, a given peptide-MHC binding molecule (e.g., a molecule comprising, or consisting of, a T cell receptor or a fragment thereof, or a TCR mimic antibody or a fragment thereof) has a preference for a specific combination of MHC subtype and specific peptide.

[0103] As discussed above, different subtypes of MHC exist which bind T cell epitopes with varying likelihood, due to sequence preferences of the different MHC subtypes. Due to structural differences of these different MHC subtypes, different subgroups of peptide-MHC binding molecule exist which are adapted to the different MHC subtypes.

[0104] Within a subgroup of peptide-MHC binding molecules, different members exist which bind with preference to a peptide-MHC complex comprising the respective MHC subtype and a defined cell epitope. As discussed above, a T cell epitope that is preferred by the respective peptide-MHC binding molecule, when presented by the respective MHC subtype, is called “target peptide” (TP) herein.

[0105] As discussed, a “target peptide” is a peptide to which, when presented by MHC, the target binding part of a TCR mimic antibody or a T cell receptor can bind.

[0106] Typically, a TCR mimic antibody or a T cell receptor has a preferred target peptide (also called specific target peptide), i.e. a peptide to which, when presented by MHC, it binds with preference.

[0107] For example such “specific target peptide” is a peptide which, when presented by the respective MHC subtype, is bound by the peptide-MHC binding molecule (e.g., a molecule comprising, or consisting of, a T cell receptor or a fragment thereof, or a TCR mimic antibody or a fragment thereof) with an affinity (Kd) of <10 nM, preferably, <1 nM and more preferably <0.1 nM. The term “KD”, as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of kd to kaand is expressed as a molar concentration (M).

[0108] A peptide that, when presented by the respective MHC subtype, is still bound by the peptide / MHC binding molecule, yet with an affinity (Kd) of > 10 nM, may hence be called a “non-specific target peptide” herein.

[0109] El Amrani, et al (Ther Drug Monit 2019;41 :640-647) describe a method of therapeutic drug monitoring of tumor necrosis factor alpha (TNF-a) inhibitors such as adalimumab (ADM) and infliximab (IFX). The method is based on LC-MS / MS of human plasma or serum and uses a biotinylated TNF-a coated on a streptavidin 96-well plate. In LC-MS stable isotope labelled (SIL) variants of ADM and IFX were introduced as internal standard before sample purification. However, this reference does not mention the analysis of a target of interest in a biological sample comprising one or more peptide-MHC binding molecules, like e.g. TCRs or TCR mimicking antibodies. Further, the reference does not mention that the method can also be used to quantify the binding molecule’s target (like e.g. pMHC). A similar disclosure with the same deficiencies can be found in Ladwig et al (Clin Vaccine Immunol. 2017 May 5;24(5)) and in Neubert et al (Clin Chem. 2020 Feb l;66(2):282-301).

[0110] Furthermore, as shown in Fig. 3, the binding reaction between a peptide-MHC binding molecule and its target is more complex than the simple antibody-target interaction. While regarding the latter, only two elements are involved (antibody and target), the binding reaction between a peptide-MHC binding molecule and its target involves three elements, namely the binding molecule (e.g. a TCR or TCRmAb), the MHC and the peptide presented by the MHC.

[0111] According to a preferred embodiment of the invention, identifying the one or more target(s) of interest (IPOI) encompasses quantifying the one or more target(s) of interest.

[0112] According to a preferred embodiment of the invention, the method further comprises adding to the biological sample and / or test 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 bearing a SIL target peptide (TP), the complex being capable of specifically binding to the peptide / MHC binding molecule.

[0113] Peptides labelled with stable, non-radioactive isotopes are increasingly used for convenient detection in research. Isotope-labelled, or ‘heavy’ amino acids, are derived from natural amino acids by substitution of certain atoms (N, C, H) with their ‘heavy isotope’ variant. The most frequently used stable isotopes are13C (carbon- 13),15N (nitrogen- 15), and2H (deuterium).

[0114] SIL-peptides display identical physiochemical properties and chemical reactivity as their nonlabelled counterparts. However, under certain conditions labelled and unlabelled peptides behave differently. This constitutes the basis for using SIL-peptides in a variety of absolute quantification applications. For example, such SIL variants of proteins or peptides may be used as internal standards in LC-MS based proteomics. Details and protocols of using SIL variants of proteins or peptides in LC-MS based proteomics are for example disclosed in Faria et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 15; 1001 : 156-68).

[0115] According to a preferred embodiment, Arg and Lys residues within the SIL variant of the MHC- binding proteins are labelled with stable isotopes.

[0116] Using stable isotope labelled (SIL) variants of the peptide-MHC -binding molecule of interest as internal standard presupposes that these variants fold identically and exhibit same enrichment efficiency as unlabelled peptide-MHC -binding molecules of interest.

[0117] As used herein, the term “stable isotope labelled (SIL) variant” of the peptide-MHC -binding molecule relates to a variant of the peptide-MHC -binding molecule that is identical in sequence thereto, yet are labelled with stable, non-radioactive isotopes. Contrary to small peptides which are chemically synthesized, peptide-MHC -binding molecules are being produced in recombinant expression systems, which reduces the degrees of freedom available for the application of labeling isotypes, relative to chemical synthesis.

[0118] Furthermore, in case unstable isotopes are used in either the peptide-MHC complex or the stable isotope labelled (SIL) variant of the peptide-MHC -binding molecule, radioactive decay can possibly result in non-stable masses, which are the primary readout by MS, and hence affect the measurement.

[0119] According to a preferred embodiment of the invention, identifying one or more proteolytic fragments by means of mass spectrometry encompasses at least one method selected from the group consisting of mass spectrometry (MS) tandem mass spectrometry (MS / MS) liquid chromatography coupled with mass spectrometry (LC-MS, LC-MS / MS) These methods are well known to the skilled artisan. See, e.g. Coon et al (Biotechniques. 2005 Apr;38(4):519, 521, 523), Yadav et al. (Nature. 2014 Nov 27;515(7528):572-6) or Fritsche et al (Proteomics. 2018 Jun;18(12):el700284), the contents of which are incorporated herein by reference for enablement purposes.

[0120] According to a preferred embodiment of the invention, the biological sample is obtained from a human or animal subject. According to another preferred embodiment of the invention, the human or animal subject has been treated with the peptide-MHC binding molecule prior to obtaining the sample.

[0121] According to a preferred embodiment of the invention, the one or more target(s) of interest (IPOI) is, comprises or is comprised in, at least one entity selected from the group consisting of

[0122] • an unbound (“free”) or bound peptide-MHC binding molecule, or a target binding fragment thereof,

[0123] • peptide-MHC complex (pMHC) and / or

[0124] • a target peptide (TP).

[0125] The pMHC or target peptide may be specifically bound by the peptide-MHC binding molecule or may be bound with lower specificity than another pMHC or target peptide (e.g. so called off targets).

[0126] As used herein, the term “bound peptide-MHC binding molecule” relates to a peptide-MHC binding molecule that, before or after being captured, binds, is bound to or has bound a peptide-MHC complex.

[0127] As used herein, the term “unbound (“free”) peptide-MHC binding molecule” relates to a peptide-MHC binding molecule that is not bound to a peptide-MHC complex.

[0128] According to a preferred embodiment of the invention, the unbound or bound peptide-MHC binding molecule or target binding fragment thereof is at least one of a T cell receptor (TCR) or a target binding fragment thereof, and / or TCR mimic antibody (TCRmAb), or a target binding fragment thereof.

[0129] According to a preferred embodiment of the invention, the MHC molecule within the peptide- MHC complex is MHC class I.

[0130] According to a preferred embodiment of the invention, the MHC molecule within the peptide- MHC complex is HLA.

[0131] According to a preferred embodiment of the invention, the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule within the target binding domain thereof.

[0132] According to a preferred embodiment of the invention, the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof.

[0133] A capturing agent that binds to a domain of the peptide-MHC binding molecule within the target binding domain thereof elates to a capturing agent that is recognized by the peptide-MHC binding molecule, by means of its target binding domain. Such capturing agent can be, for example, the peptide-MHC binding molecule’s target, i.e., the peptide-MHC complex which it specifically binds. Therefore, such capturing agent would preferentially capture one specific peptide-MHC binding molecule.

[0134] A capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof relates to a capturing agent that binds a larger number of different peptide-MHC binding molecules. Examples for such capturing agents are provided herein elsewhere. According to a preferred embodiment of the invention, the unbound or bound peptide-MHC binding molecule comprises at least one further functional entity selected from the group consisting of

[0135] • an effector entity

[0136] • an entity that extends serum half-life, and / or

[0137] • a toxic entity

[0138] Such peptide-MHC binding molecule is sometimes also called bi- or multispecific, or bi- or multifunctional.

[0139] Features of such bi- or multispecific peptide-MHC binding molecules are disclosed in Jones HF, Molvi Z, Klatt MG, Dao T, Scheinberg DA. Front Immunol. 2021 Jan 25; 11 :585385, the content of which is incorporated herein by reference for enablement purposes. Therein, the multispecific peptide-MHC binding molecules in the meaning of the present invention are called “BiTE”, “TCRmmAb”, “TCRmBiTE” or “ImmTAC”.

[0140] Some selected commercial examples are shown in the following table:

[0141] Table 3: Selected commercial examples of bi- or multispecifc peptide-MHC binding molecules In case the bi- or multispecific peptide-MHC binding molecule comprises, e.g., a second binder (e.g., an anti CD3 antibody, an anti TCR antibody), as discussed below, or a second reactive entity (e.g., an Fc domain or an albumin binding domain), as discussed below, said second binder or reactive entity can be used as a target for capturing the peptide-MHC binding molecule, e.g., with a CD3 domain, a TCR domain, Protein A or albumin as a capturing agent (CA), immobilized, e.g. on beads or a column. In such case, bound and unbound peptide-MHC binding molecules can be captured, because the peptide-MHC binding molecule’s target binding domain would not be involved in the capturing reaction. This allows not only quantification of the peptide-MHC binding molecule alone, yet also quantification of bound peptide-MHC complexes and / or target peptides.

[0142] On the other hand, if a peptide-MHC complex is used as a capturing agent (CA), which binds a peptide-MHC binding molecule via its target binding domain, the second binder (e.g., an anti CD3 antibody, an anti TCR antibody), as discussed below, or the second reactive entity (e.g., an Fc domain or an albumin binding domain), as discussed below, can be used to quantify the respective molecules bound by said second binder or second reactive entity.

[0143] According to a preferred embodiment of the invention, the capturing agent (CA) binds to at least one functional entity selected from the group consisting of

[0144] • an effector entity

[0145] • an entity that extends serum half-life, and / or

[0146] • a toxic entity within the peptide-MHC binding molecule.

[0147] According to a preferred embodiment of the invention, the effector entity is a T cell engaging entity (also called “T cell recruiting entity”). According to other preferred embodiments of the invention, the T cell engaging entity may be a binder that binds to CD3 or a binder that binds to the constant or variable domain of a TCR alpha or beta chain. In principle, the T cell engaging entity may be any entity that binds to any kind of T cell surface antigen, like, e.g., CD2, CD 16, or TRGV9 / Null (Vy9 / Null), though. According to preferred embodiments of the invention, the T cell engaging entity is for example an antibody selected from the group consisting of

[0148] • 0KT3 or target binding fragments or derivatives thereof (Wong et al., Transplantation.

[0149] 1990 Oct;50(4):683-9)

[0150] • BlinCD3 or target binding fragments or derivatives thereof (Dorken et al., United States patent US 7575923 B2. 2009. Aug 18)

[0151] • UCHT1 or target binding fragments or derivatives thereof (Macdonald et al., Clin Exp Immunol. 1982 Jul;49(l): 123-8)

[0152] • Foralumab or target binding fragments or derivatives thereof (Giuffrida & Sabatino. Pharmacological Research. 159: 105040, 2020)

[0153] • Teplizumab or target binding fragments or derivatives thereof (Alegre et al., Transplantation 57(11), 1537-1543 (1994))

[0154] • BMA031 or target binding fragments or derivatives thereof (Borst et al., Hum Immunol. 1990 Nov;29(3): 175-88)

[0155] In a preferred embodiment the T cell engaging activity comprised within the peptide-MHC binding molecule is BMA031.

[0156] In a preferred embodiment the T cell engaging activity comprised within the peptide-MHC binding molecule is UCHT1 or more preferably humanUCHTl (i.e. hUCHTl).

[0157] The International Nonproprietary Name (INN) of OKT3 is muromonab-CD3. OKT3 was approved by the U.S. Food and Drug Administration (FDA) in 1985, making it the first monoclonal antibody to be approved anywhere as a drug for humans. Muromonab-CD3 is a murine antibody provided in the IgG format and targets CD3s. It is approved for the therapy of acute, glucocorticoid-resistant rejection of allogeneic renal, heart and liver transplants. It has also been investigated for use in treating T-cell acute lymphoblastic leukemia.

[0158] BlinCD3 is an antibody that is described in Drugbank, Accession Number DB09052, amino acid (AA) residues 256-498, the content of which is incorporated herein by reference. BlinCD3 is derived from OKT3 by humanization ((Dorken et al., United States patent US 7575923 B2.

[0159] 2009. Aug 18)) and has about 93% sequence identity therewith in the variable domains.

[0160] BlinCD3, 0KT3 and UCHT3, Teplizumab and Foralumab are examples for binders that bind to CD3.

[0161] The monoclonal antibody BMA031 is reported to be specific for the common determinant on the TCR alpha / beta / CD3 complex, and does not bind to the gamma / delta TCR. BMA031 is hence an example of a binder that binds to the constant or variable domain of a TCR alpha or beta chain. BMA031 is highly immunosuppressive and is capable of inducing apoptosis of activated T cells via a mechanism of activation-induced cell death (AICD) (Wesselborg et al. (May 1993) J. Immunol. 150(10): 4338-4345).

[0162] As used herein, the term “derivatives thereof’ is meant to relate to modified variants of the respective reference TCR or antibody which have retained or even improved properties relative thereto. This encompasses solubility, stability, immunogenicity or target binding properties. All these modifications are subject to routine technologies readily available to the skilled artisan. In particular, the term encompasses chimerized or humanized variants of the reference antibody, as well as affinity maturated variants and soluble fragments like (Fab)2, Fab, scFv, diabodies and the like, as well as combinations thereof.

[0163] As used herein, the term “target binding fragments thereof’ is meant to relate to modified variants of the respective reference TCR or antibody which typically comprise at least the CDRs and / or variable domains of the antibody or TCR, and thus retain its target binding properties. Such fragments encompass soluble fragments like (Fab)2, Fab, scFv, diabodies and the like. Oftentimes, such fragments are being fused to one or more further functional entities so as to create a bi- or multispecific or bi- or multifunctional molecule.

[0164] According to a preferred embodiment of the invention, the entity that extends serum half-life is at least one selected from the group consisting of

[0165] • antibody Fc domain or fragment thereof albumin or a functional analogue thereof, and / or albumin binding entity

[0166] Albumin (oftentimes nicknamed HSA = human serum albumin) has a serum half-life of 3 weeks in humans. This feature can be used to improve the pharmacokinetics of shorter-lived biologies. For instance, an albumin-binding entity can be used to recruit patient intrinsic albumin. A prerequisite for such design is that the ABD-albumin interaction does not interfere with pH-dependent binding of albumin to the human neonatal Fc receptor (FcRn), as FcRn acts as the principal regulator of the half-life of albumin. Alternatively, albumin or a functional analogue thereof can be fused to the peptide-MHC binding molecule directly.

[0167] In like manner, in particular if the peptide-MHC binding molecule is a soluble fragment of a TCR or a TCR mimic antibody, such molecule can be fused to an antibody Fc domain. Generally, the pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. Mutated variants of the antibody Fc domain, which for example have the substitutions Q311R / M428L, M252Y / S254T / T256E (oftentimes called “YTE”) or M428L / N434S (oftentimes called “LS”), may extend the serum half-life even further (Ko, et al., Exp Mol Med 54, 1850-1861 (2022)).

[0168] According to preferred embodiments of the invention, the serum albumin binding entity is for example one selected from the group consisting of

[0169] • AlbuBinder 1 (a small-molecule noncovalent HSA binder (Vantourout et al., Bioconjug Chem. 2021 Feb 17;32(2):279-289)

[0170] • albumin-binding DARPin domain (Steiner et al., Protein Engineering, Design and Selection, Volume 30, Issue 9, September 2017, pages 583-591)

[0171] • serum albumin specific repebody (Kim et al., Journal of Controlled Release. 315. 2019)

[0172] • serum albumin specific VHH (van Faassen et al., The FASEB Journal. 2020; 34: 8155- 8171.) According to a preferred embodiment of the invention, the capturing agent (CA) is at least one selected from the group consisting of

[0173] • protein A

[0174] • pMHC

[0175] • anti-Fc domain antibody

[0176] • CD3

[0177] • anti-CD3 binder

[0178] • constant or variable domain of a TCR alpha or beta chain,

[0179] • binder that binds to a binder to the constant or variable domain of a TCR alpha or beta chain

[0180] • antiidiotypic binder, and / or

[0181] • albumin or a fragment thereof.

[0182] In a preferred embodiment the capturing agent (CA) is pMHC.

[0183] An anti CD3 binder is for example an antibody that binds to an anti-CD3 antibody (e.g., to Okt3). With such binder, bispecific peptide-MHC binding molecules comprising an anti CD3 antibody as a T cell engager can be captured.

[0184] Albumin or a fragment thereof can be used as a capturing agent (CA) if the peptide-MHC binding molecule is bi- or multispecific and comprises an albumin binding domain for serum half-life extension, as discussed elsewhere herein.

[0185] A binder that binds to a binder to the constant or variable domain of a TCR alpha or beta chain is for example an antibody that binds to an antibody against the common determinant on the TCR alpha / beta / CD3 complex (e.g., to BMA031). With such binder, bispecific peptide-MHC binding molecules comprising a respective T cell engager can be captured. Protein A binds to antibody Fc domains, which are for example comprised in full length IgG antibodies, or in peptide-MHC binding molecules that comprise an Fc-domain, e.g., for serum half-life extension, as discussed elsewhere herein.

[0186] In a preferred embodiment the capturing agent (CA) is protein A.

[0187] As used herein, the term “antiidiotypic” binder relates to a binder that binds to the variable region of another binder (the idiotype). Each of the binders can be e.g. an antibody, or a functional fragment thereof, or a TCR, or a functional fragment thereof.

[0188] According to a preferred embodiment of the invention, the capturing agent (CA) is bound to a solid matrix. The solid matrix can for example be selected from the group consisting of beads, preferably magnetic beads, and / or a column.

[0189] In preferred embodiments according to the invention, the capturing agent (CA) is bound to the solid matrix by biotin / streptavidin. In such preferred embodiment, the solid matrix, e.g. the magnetic beads, may for example carry a streptavidin moiety, while the capturing agent (CA) may carry a biotin moiety. The streptavidin labelled solid matrix, e.g. the magnetic beads, are incubated with the biotin labelled capturing agents to associate them thereto.

[0190] According to a preferred embodiment of the invention, the step of enriching the peptide-MHC binding molecules involves releasing the same from the capturing agent.

[0191] According to a preferred embodiment of the invention, the proteolytic enzyme is at least one selected from the group consisting of

[0192] • AspN

[0193] • Trypsin

[0194] • LysC, and / or

[0195] • GluC

[0196] The following table shows some characteristics of these enzymes. Table 4: Examples for enzymes that can be used for proteolytic digestion prior to MS

[0197] While the above mentioned enzymes are particularly useful in the context of the present invention, many other proteolytic enzymes can be used to generate respective protein-derived fragments for subsequent analysis via Mass Spectrometry (MS). As an alternative, proteins can also be degraded by non-enzymatic methods, e.g. chemically-induced proteolytic cleavage at low pH.

[0198] In general, methods are preferred which lead to an efficient digestion and which further lead to release of a defined set of peptides to avoid signal dilution. This is best achieved by using specific proteases as listed in Table 4, which cleave N- or C-terminal to a narrow set of defined amino acids. Further, proteases are preferred which produce peptides containing at least 2 isotopically labelled amino acids. Since metabolic incorporation efficiency is usually at -99% during expression of stable isotope labelled (SIL) variants of, e.g., the peptide-MHC binding molecule in recombinant host cells (e.g., a bispecific molecule comprising a target binding fragment of a TCR), a small portion of the SIL variants remain unlabelled. This can lead to false identifications of the unlabelled peptide-MHC binding molecule signal and should be avoided. If the peptide of interest contains at least 2 labelled amino acids (e.g., Lys and / or Arg), this false unlabelled signal is further diluted to 0.01%, and appropriate selection of the SIL variant abundance further avoids that unlabelled peptide-MHC binding molecule signals would be detected and quantified, which are derived from the SIL variant. Since LysC can produce peptides carrying multiple Lys and Arg residues and also shows high cleavage efficiency, it was selected as protease of choice here. According to a preferred embodiment of the invention, the method further comprises a step of disulfide bond reduction and / or thiol alkylation of the biological sample or test sample prior to treating the same with a proteolytic enzyme.

[0199] Disulfide bond reduction serves to denature the immune protein or peptide of interest, so as to facilitate subsequent analysis.

[0200] Such disulfide bond reduction can for example be carried out with TCEP. TCEP (Tris(2- carboxyethyl)phosphine-hydrochloride) is a potent, versatile, non-volatile, odourless, thiol-free reducing agent with broad application in reduction of disulfide bonds. It is easily soluble and very stable in many aqueous solutions.

[0201] Thiol Alkylation converts free Cystein thiols into thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of protein fragments comprising Cys residues. Such thiol alkylation can for example be carried out with CAA. CAA (chloroacetaldehyde) is a metabolite of ifosfamide, and is capable of alkylating free thiols.

[0202] According to a preferred embodiment of the invention, the method further comprises homogenization and / or cell lysis of the sample.

[0203] According to a preferred embodiment of the invention, the method further comprises determination of the whole protein content or concentration in the sample

[0204] In a preferred embodiment, the BCA protein assay is used for that purpose. The BCA protein assay combines the reduction of Cu2+to Cu1+by proteins in an alkaline medium with the highly sensitive and selective colorimetric detection of copper cations (Cu1+) by bicinchoninic acid (BCA). The first step consists of chelating copper with protein in an alkaline environment to form a light blue complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a coloured chelate complex with copper ions in an alkaline environment containing sodium potassium tartrate. According to a preferred embodiment of the invention, at least part of the amino acid sequence of the one or more target(s) of interest (IPOI) is known upfront.

[0205] According to a preferred embodiment of the invention, the specificity of the peptide-MHC- binding molecule of interest to a peptide-MHC-complex comprising a given target peptide (TP) is known upfront.

[0206] According to a preferred embodiment of the invention, the biological sample is, or comprises, at least one element comprised in the list consisting of:

[0207] • serum sample

[0208] • plasma sample

[0209] • tissue sample

[0210] • tumor sample or combinations thereof.

[0211] The term "sample”, as used herein, refers to a sample obtained from a human or animal subject. The sample may be of any biological tissue or fluid. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, urine, peritoneal fluid, and pleural fluid, liquor cerebrospinalis. tear fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof. In a preferred embodiment the biological sample is fluid or solid (e.g. the method of the present invention is suitable for exercising with fluid and solid biological samples).

[0212] The sample may have been obtained by aspiration or punctuation, excision or by any other surgical method. Such a biological sample may comprise cells obtained from a patient. The cells may be found in a cell "smear" in solid tumor material, in a lavage fluid, or in a body fluid. The sample may be a processed sample, e.g. a sample, which has been frozen, fixed, embedded or the like. A preferred type of sample is a formaline fixed paraffin embedded (FFPE) sample. Preparation of FFPE samples are standard medical practice and these samples can be conserved for long periods of time.

[0213] According to a preferred embodiment of the invention, a) the target of interest (IPOI) to be identified is an unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a peptide-MHC complex to which the peptide-MHC binding molecule specifically binds, the peptide-MHC complex comprising a given target peptide (TP).

[0214] The peptide-MHC complex presents the target peptide (TP) which, when presented by MHC, is recognized by the peptide-MHC binding molecule.

[0215] As used herein, the term “peptide-MHC complex to which the peptide-MHC binding molecule specifically binds” relates to peptide-MHC complexes as defined elsewhere herein, with the proviso that they present a peptide which, in combination with the MHC presenting it, is bound (or detected, or recognized) by the peptide-MHC binding molecule.

[0216] In this preferred embodiment, a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule may be spiked in after enrichment as internal standard for the quantification of the peptide-MHC binding molecule, e.g., in mass spectrometry (MS).

[0217] Internal standards are widely used in mass spectrometry not only to correct for sample preparation variations during extraction and chemical derivatization but also to compensate for variability in signal intensity due to ion-suppression caused by matrix components that may influence the efficiency of ionization. In the field of proteomics, internal standards allow the absolute quantification of proteins with tandem mass spectrometry. In most cases, stableisotope labelled (SIL) peptides are used as internal standards for the quantification of signature peptides obtained from the enzymatic digestion of the protein. As an alternative, stable-isotope labelled (SIL) variants of the full protein can be used and then be subjected to identical enzymatic digestion. See, for example, Foque et al., Anal Chem. 2018 Dec 18;90(24): 14126- 14130), the comment of which is enclosed herein by reference for enablement purposes. According to a preferred embodiment of the method of the invention, a) the target of interest (IPOI) to be identified is a bound or unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof, wherein the method of the invention further comprises c) addition, to the sample, of an unbound (“free”) stable isotope labelled (SIL) variant of the peptide-MHC binding molecule

[0218] As discussed above, such capturing agent (CA) may bind to at least one functional entity selected from the group consisting of

[0219] • an effector entity

[0220] • an entity that extends serum half-life, and / or

[0221] • a toxic entity within the peptide-MHC binding molecule.

[0222] It should be understood that the further embodiments discussed above in the context of capturing agents apply to these embodiments as well.

[0223] In this embodiment, bound and unbound (“free”) peptide-MHC binding molecules are not the only proteins or peptides of interest that can be identified. Isolating “bound” peptide-MHC binding molecules implies that the peptide-MHC complexes to which the peptide-MHC binding molecules specifically bind are isolated as well and can hence be identified.

[0224] Also, the addition of unbound (“free”) stable isotope labelled (SIL) variant of the MHC -binding protein to the test sample contributes to also isolate the peptide-MHC complexes within the sample that were so far not bound by the unlabeled variants of the peptide-MHC binding molecules.

[0225] For quantification of the co-precipitated peptide-MHC complexes (“bycatch”), a stable isotope labelled (SIL) variant of the target peptide (TP), MHC or beta 2 microglobulin (b2m) can be spiked in after enrichment as an internal standard for mass spectrometry (MS). See, e.g., WO2022184832 Al, the content of which is incorporated herein by reference for enablement purposes, for protocols and methods on how to quantify MHC molecules.

[0226] According to a preferred embodiment of the invention, a) the target of interest (IPOI) to be identified is a bound or unbound peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof.

[0227] In this embodiment, all peptide-MHC binding molecules (i.e., bound and unbound) are isolated and with them, all peptide-MHC complexes that are bound thereto. Peptide-MHC complexes within the sample that are not bound by peptide-MHC binding molecules are not isolated.

[0228] In a preferred embodiment of this method, the bound peptide-MHC binding molecules can later be identified and quantified by using, as an internal standard, a SIL variant of the target peptide (TP) bound by the peptide / MHC binding molecule. By quantifying said target, due to the 1 :1 stoichiometry, bound peptide-MHC binding molecules can likewise be quantified.

[0229] For quantification of the co-precipitated peptide-MHC complexes (“bycatch”), a stable isotope labelled (SIL) variant of the target peptide (TP), MHC or beta 2 microglobulin (b2m) can be spiked in after enrichment as an internal standard for the MS. See , e.g., WO2022184832 Al, the content of which is incorporated herein by reference for enablement purposes, for protocols and methods on how to quantify MHC molecules.

[0230] According to a preferred embodiment of the method of the invention, a) the target of interest (IPOI) to be identified is a bound peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof, wherein the method of the invention further comprises c) addition, to the sample of peptide-MHC complexes to which the peptide-MHC binding molecule specifically binds, wherein at least a part of said peptide-MHC complex is isotopically labelled.

[0231] In this context, a “part of said peptide-MHC complex” means that (i) the target peptide (TP) presented by the peptide-MHC complex and / or (ii) the MHC, or fragments thereof is isotopically labeled.

[0232] This embodiment is suitable to identify, in a sample that comprises excess "free" peptide-MHC binding molecule (e.g., in a sample from a patient who was treated before with a molecule that is or comprises a T cell receptor or a TCR mimic antibody, or a fragment thereof, wherein the sample comprises a large share of blood or plasma), the share of bound peptide-MHC binding molecule, relative to the “free” peptide-MHC binding molecule.

[0233] According to a preferred embodiment of the invention, the unbound (“free”) or bound peptide- MHC binding molecule is, or comprises, at least one entity selected from the group consisting of

[0234] • T cell receptor (TCR) or a target binding fragment thereof,

[0235] • TCR mimic antibody (TCRmAb), or a target binding fragment thereof

[0236] According to a preferred embodiment of the invention, a) the target of interest (IPOI) to be identified is an unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a peptide-MHC complex to which the peptide-MHC binding molecule binds

[0237] In case the peptide-MHC complex presents the specific target peptide (STP; e.g., a peptide that, when presented by the respective MHC subtype, is bound by the peptide-MHC binding molecule with high affinity), such binding is a specific binding. In case the peptide-MHC complex presents a non-specific target peptide (OTP) such binding is less specific or even absent. In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is the target of interest, wherein the capturing agent binds the peptide-MHC binding molecule within a target binding domain thereof (preferably wherein the CA is a peptide-MHC complex specifically bound by the peptide-MHC binding molecule), optionally a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule is added to the biological sample and the target of interest is quantified.

[0238] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is one target of interest, wherein the pMHC (i.e. the target peptide and the MHC) specifically bound by the peptide-MHC binding molecule is another target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide-MHC binding molecule, if present), optionally a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule is added to the biological sample and the target of interest is quantified. In a preferred alternative embodiment the pMHC contains not the specific target peptide, but a non-specific target peptide (OTP).

[0239] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is the target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide- MHC binding molecule, if present), optionally a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule is added to the biological sample and the target of interest is quantified.

[0240] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is one target of interest, wherein the target peptide comprised in the pMHC specifically bound by the peptide- MHC binding molecule is another target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide-MHC binding molecule, if present), optionally a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule is added to the biological sample and the target of interest is quantified.

[0241] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is one target of interest, wherein the pMHC (i.e. the target peptide and the MHC) specifically bound by the peptide-MHC binding molecule is another target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide-MHC binding molecule, if present).

[0242] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is the target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide- MHC binding molecule, if present).

[0243] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is one target of interest, wherein the target peptide comprised in the pMHC specifically bound by the peptide- MHC binding molecule is another target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide-MHC binding molecule, if present).

[0244] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is one target of interest, wherein the pMHC (i.e. the target peptide and the MHC) specifically bound by the peptide-MHC binding molecule is another target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide-MHC binding molecule, if present), optionally a stable isotope labelled (SIL) variant of the peptide-MHC complex is added to the biological sample and the target of interest is quantified.

[0245] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is the target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide- MHC binding molecule, if present), optionally a stable isotope labelled (SIL) variant of the peptide-MHC complex is added to the biological sample and the target of interest is quantified.

[0246] In a preferred embodiment, the peptide-MHC binding molecule is a bispecific binding molecule (preferably additionally comprising a binding domain of an antibody) and is one target of interest, wherein the target peptide comprised in the pMHC specifically bound by the peptide- MHC binding molecule is another target of interest, wherein the capturing agent binds the peptide-MHC binding molecule outside of a target binding domain thereof (preferably wherein the CA binds to a FC domain of the peptide-MHC binding molecule, if present), optionally a stable isotope labelled (SIL) variant of the peptide-MHC complex is added to the biological sample and the target of interest is quantified.

[0247] The present invention also pertains to the following items.

[0248] 1. A method of identifying one or more immune proteins or peptides of interest (i.e. the targets of interest) in a biological sample comprising one or more peptide-MHC binding molecules, which method comprises the steps of a) obtaining a biological sample b) adding to the sample a capturing agent (CA) that binds the peptide-MHC binding molecule, wherein the peptide-MHC binding molecule

[0249] (i) binds the immune protein or peptide of interest, or

[0250] (ii) actually is the immune protein or peptide of interest c) enriching peptide-MHC -binding molecules from said sample to obtain a test sample d) treating the sample or test sample with a proteolytic enzyme to obtain proteolytic fragments of inter alia the immune protein or peptide of interest (IPOI) e) identifying one or more of said proteolytic fragments by means of mass spectrometry (MS), thereby identifying the immune protein or peptide of interest.

[0251] 2. The method according to any one of the aforementioned items, wherein identifying the immune protein or peptide of interest (IPOI) encompasses quantifying the immune protein or peptide of interest.

[0252] 3. The method according to any one of the aforementioned items, further comprising adding at least one of a) a stable isotope labelled (SIL) variant of the peptide-MHC binding molecule to the test sample, and / or b) a peptide-MHC complex bearing a stable isotope labelled target peptide (TP), the complex being capable of binding to the peptide-MHC binding molecule.

[0253] 4. The method according to any one of the aforementioned items, wherein identifying one or more proteolytic fragments by means of MS encompasses at least one method selected from the group consisting of

[0254] • mass spectrometry (MS)

[0255] • tandem mass spectrometry (MS / MS)

[0256] • liquid chromatography coupled with mass spectrometry (LC-MS, LC-MS / MS)

[0257] 5. The method according to any one of the aforementioned items, wherein the biological sample is obtained from a human or animal subject. 6. The method according to any one of the aforementioned items, wherein the human or animal subject has been treated with the peptide-MHC binding molecule prior to obtaining the sample.

[0258] 7. The method according to any one of the aforementioned items, wherein the immune protein or peptide of interest is, or comprises, at least one selected from the group consisting of

[0259] • an unbound (“free”) or bound peptide-MHC binding molecule, or a target binding fragment thereof,

[0260] • peptide-MHC complex (pMHC) and / or

[0261] • a target peptide (TP).

[0262] 8. The method according to item 7, wherein the unbound or bound peptide-MHC binding molecule or target binding fragment thereof is at least one of

[0263] • a T cell receptor (TCR) or a target binding fragment thereof, and / or

[0264] • TCR mimic antibody (TCRmAb), or a target binding fragment thereof.

[0265] 9. The method according to any one of the aforementioned items, wherein the MHC molecule within the peptide-MHC complex is MHC class I, optionally is HLA.

[0266] 10. The method according to any one of the aforementioned items, wherein the capturing agent (CA) a) is a capturing agent that binds to a domain of the peptide-MHC binding molecule within the target binding domain thereof, or b) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof.

[0267] 11. The method according to any one of the aforementioned items, wherein the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof. 12. The method according to any one of the aforementioned items, wherein the unbound or bound peptide-MHC binding molecule is a bi- or multifunctional molecule which comprises at least one further functional entity selected from the group consisting of

[0268] • an effector entity

[0269] • an entity that extends serum half-life, and / or

[0270] • a toxic entity

[0271] 13. The method according to any one of the aforementioned items, wherein at least one of: a) the capturing agent (CA) binds to at least one functional entity selected from the group consisting of

[0272] • an effector entity

[0273] • an entity that extends serum half-life, and / or

[0274] • a toxic entity within the peptide-MHC binding molecule, b) the effector entity is a T cell engaging entity, c) the entity that extends serum half-life is at least one selected from the group consisting of

[0275] • an antibody Fc domain or fragment thereof

[0276] • albumin or a functional analogue thereof, and / or

[0277] • an albumin binding entity and / or d) the capturing agent is at least one selected from the group consisting of

[0278] • protein A

[0279] • pMHC

[0280] • anti-Fc domain antibody

[0281] • CD3

[0282] • anti-CD3 binder

[0283] • constant or variable domain of a TCR alpha or beta chain,

[0284] • binder that binds to a binder to the constant or variable domain of a TCR alpha or beta chain,

[0285] • antiidiotypic binder, and / or albumin or a fragment thereof

[0286] 14. The method according to any one of the aforementioned items, wherein the effector entity is a T cell engaging entity.

[0287] 15. The method according to any one of the aforementioned items, wherein the entity that extends serum half-life is at least one selected from the group consisting of

[0288] • an antibody Fc domain or fragment thereof

[0289] • albumin or a functional analogue thereof, and / or

[0290] • an albumin binding entity

[0291] 16. The method according to any one of the aforementioned items, wherein the capturing agent is at least one selected from the group consisting of

[0292] • protein A

[0293] • pMHC

[0294] • an anti-Fc domain antibody

[0295] • CD3

[0296] • an anti-CD3 binder

[0297] • the constant or variable domain of a TCR alpha or beta chain,

[0298] • a binder that binds to a binder to the constant or variable domain of a TCR alpha or beta chain,

[0299] • a antiidiotypic binder, and / or

[0300] • albumin or a fragment thereof.

[0301] 17. The method according to any one of the aforementioned items, wherein the capturing agent (CA) is bound to a solid matrix.

[0302] 18. The method according to any one of the aforementioned items, wherein the solid matrix is at least one selected from the group consisting of

[0303] • beads, preferably magnetic beads, and / or

[0304] • column 19. The method according to any one of the aforementioned items, wherein the step of enriching the peptide-MHC binding molecules involves releasing the same from the capturing agent.

[0305] 20. The method according to any one of the aforementioned items, wherein the proteolytic enzyme is at least one selected from the group consisting of

[0306] • AspN

[0307] • Trypsin

[0308] • LysC, and / or

[0309] • GluC

[0310] 21. The method according to any one of the aforementioned items, which method further comprises a step of disulfide bond reduction and / or thiol alkylation of the biological sample or test sample prior to treating the same with a proteolytic enzyme.

[0311] 22. The method according to any one of the aforementioned items, which method further comprises homogenization and / or cell lysis of the sample.

[0312] 23. The method according to any one of the aforementioned items, which method further comprises determination of the whole protein content or concentration in the sample

[0313] 24. The method according to any one of the aforementioned items, wherein a) at least part of the amino acid sequence of the immune protein or peptide of interest (IPOI) is known upfront, and / or b) the specificity of the peptide-MHC -binding molecule of interest to a peptide-MHC- complex comprising a given target peptide (TP) is known upfront.

[0314] 25. The method according to any one of the aforementioned items, wherein the specificity of the peptide-MHC -binding molecule of interest to a peptide-MHC-complex comprising a given target peptide (TP) is known upfront. 26. The method according to any one of the aforementioned items, wherein the biological sample is, or comprises, at least one element comprised in the list consisting of

[0315] • Serum sample

[0316] • Plasma sample

[0317] • Tissue sample

[0318] • Tumor sample or combinations thereof.

[0319] 27. The method according to any one of the aforementioned items, wherein a) the immune protein or peptide of interest (IPOI) to be identified is an unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent to which the peptide-MHC binding molecule specifically binds, the capturing agent comprising a given target peptide (TP).

[0320] 28. The method according to any one of the aforementioned items, wherein a) the immune protein or peptide of interest (IPOI) to be identified is a bound or unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof, which method further comprises c) addition, to the sample, of an unbound (“free”) stable isotope labelled (SIL) variant of the peptide-MHC binding molecule

[0321] 29. The method according to any one of the aforementioned items, wherein a) the immune protein or peptide of interest (IPOI) to be identified is a bound peptide-MHC binding molecule, and b) the capturing agent (CA) is a capturing agent that binds to a domain of the peptide-MHC binding molecule outside of the target binding domain thereof, which method further comprises c) addition, to the sample, of peptide-MHC complexes to which the peptide-MHC binding molecule specifically binds, wherein at least a part of said peptide-MHC complex is isotopically labelled.

[0322] 30. The method according to any one of the aforementioned items, wherein the unbound (“free”) or bound peptide-MHC binding molecule is, or comprises, at least one entity selected from the group consisting of

[0323] • T cell receptor (TCR) or a target binding fragment thereof,

[0324] • TCR mimic antibody (TCRmAb), or a target binding fragment thereof

[0325] 31. The method according to any one of the aforementioned items, wherein a) the immune protein or peptide of interest (IPOI) to be identified is an unbound (“free”) peptide-MHC binding molecule, and b) the capturing agent (CA) is a peptide-MHC complex to which the peptide-MHC binding molecule binds.

[0326] Examples

[0327] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0328] Though being written in present tense, the experiments have actually been carried out by the inventors. Results are shown in the figures.

[0329] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus.

[0330] Sample lysis and determination of protein concentration

[0331] Methods used in the following are disclosed in WO2022184832, example 2, the content of which is incorporated herein by reference for enablement purposes.

[0332] Tissue samples taken from subjects who received a peptide-MHC (pMHC) binding molecule are homogenized. The pMHC binding molecule is a TCER®, i.e., a T cell engaging receptor comprising a) a single chain fragment comprising the target binding domain of a T cell receptor (TCR), b) a single chain comprising the variable domains of the monoclonal antibody BMA031

[0333] (Borst et al., Hum Immunol. 1990 Nov;29(3): 175-88.), which binds to the constant or variable domain of a TCR alpha or beta chain, as a T cell engager, and c) an antibody Fc domain that is effector function-silenced so as to no longer evoke Antibody Dependent Cell mediated Cytotoxicity (ADCC) reactions (Reiter et al., Stabilization of the FvFragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions. Biochemistry, 1994, 33, 5451 - 5459).

[0334] Fig 4 shows a schematic of the pMHC binding molecule with elements a) to c).

[0335] Tissue samples are lysed, and the protein concentration of cell lysates is determined with the BC A- Assay . The lysate of the respective sample is thawed and dilute to 5 pg / pL to ensure a final protein content of 100 pg per lysate.

[0336] Coupling of biotinylated pMHC monomer to magnetic streptavidin-coated beads and enrichment of TCER® molecules in tissue samples

[0337] Vortex carefully or invert the beads to obtain a uniform suspension. Transfer the appropriate amount of beads slurry into a 1.5 mL LoBind Eppendorf tube. Place the tube in the magnetic stand, wait for 10 seconds (s) and remove the supernatant (storage buffer). The respective amount of wash buffer is added to the beads (Table 5). Mix the beads carefully and place them on the magnetic stand, wait for 10 s and remove the bin / wash buffer from the tube.

[0338] Add the biotinylated pMHC monomer to the beads and add wash buffer. Incubate the sample in the thermomixer C at 1 , 100 rpm for 1 h. After the incubation, put the sample on the magnetic stand for 10 s and remove the supernatant. Add the appropriate amount of wash buffer (Table 5) to the beads and mix the tube for 5 min in the thermomixer C at 1,100 rpm. Place the tube on the magnetic stand for 10 s and remove the supernatant. Repeat the wash steps two times more for a total of 3 washes. After the washing steps, the beads are absorbed in wash / binding buffer according to Table 5.

[0339] Label new 0.5 mL LoBind Eppendorf tubes with the respective sample name. Add 20 pL diluted lysate (5 pg / pL, resulting in 100 pg total protein amount) and 10 pL SIL TCER® spike into a new 0.5 mL centrifuge LoBind tubes. Transfer 5 pL coupled bead-MRF mixture to the samples. Caution: mix the bead solution well before transferring to the samples! Put the samples in the thermomixer C and incubate for 1 h at 1,100 rpm at room temperature. After the incubation, put the sample on the magnetic stand for 10 s and remove the supernatant. Add 100 pL wash buffer to the beads and mix the tube for 10 min in the thermomixer C at 1,100 rpm. Place the tube on the magnetic stand for 10 s and remove the supernatant. Repeat the wash steps two times more for a total of 3 washes.

[0340] With the following steps the enriched TCER® on the pMHC -biotin beads will be eluted for further quantification. Therefore, add 20 pL elution buffer to the samples and incubate for 10 min in the thermomixer C at 1,100 rpm. The samples are then placed on the magnetic stand. When the beads are migrated to the tube walls (incubate at least 10 s), carefully transfer the supernatant to a new 0.5 mL LoBind centrifuge tube. Add 2 pL neutralization buffer to the eluted sample. At this point, the workflow can be paused, and the samples can be stored at -20 °C in the freezer.

[0341] Coupling of biotinylated protein A to magnetic streptavidin-coated beads and enrichment of TCER® molecules in serum samples

[0342] Table 6: Overview of number of samples and amount of volume serum samples Vortex carefully or invert the beads to obtain a uniform suspension. Transfer the appropriate amount of beads slurry into a 1.5 mL LoBind Eppendorf centrifuge tube. Place the tube in the magnetic stand for 10 s and remove the supernatant (storage buffer). The respective amount of wash buffer is added to the beads (Table 6). Mix the beads carefully and place them on the magnetic stand for 10 s. Remove the wash buffer from the tube.

[0343] Add the respective volume of biotinylated protein A (see, Table 6, concentration of 0.5 pg / pL) to the beads and add wash buffer. Incubate the samples in the thermomixer C at 1,100 rpm for 1 h. After the incubation put the sample on the magnetic stand for 10 s and remove the supernatant. Add the appropriate amount of wash buffer to the beads (Table 6) and mix the tube for 5 min in the thermomixer C at 1,100 rpm. Place the tube on the magnetic stand, wait for 10 s and remove the supernatant. Repeat the wash steps two times more for a total of 3 washes. After washing of the bead-protein A mixture, the beads are absorbed in wash / binding buffer according to Table 6.

[0344] Label new 0.5 mL LoBind Eppendorf tubes with the respective sample name. Add 20 pL diluted lysate (5 pg / pL, resulting in 100 pg total protein amount) and 10 pL SIL TCER® spike into the prepared tubes. Transfer 5 pL bead-protein A mixture to the samples. Put the samples in the thermomixer C and incubate for 1 h at 1,100 rpm at room temperature. After the incubation put the sample on the magnetic stand for 10 s and remove the supernatant. Add the appropriate amount of wash buffer to the beads (Table 6) and mix the tube for 10 min on the thermomixer C at 1,100 rpm. Place the tube on the magnetic stand for 10 s and remove the supernatant. Repeat the wash steps two times more for a total of 3 washes.

[0345] With the following steps the enriched TCER® on the protein A-biotin beads will be eluted for further quantification. Therefore, add 20 pL elution buffer the samples and incubate the tubes for 10 min on the thermomixer C at 1,100 rpm. The samples are then placed on the magnetic stand. When the beads are migrated to the tube walls (incubate at least 10 s), carefully transfer the supernatant to a new 0.5 mL LoBind centrifuge tube. Add 2 pL neutralization buffer to the eluted sample. Further quantification of the target peptide (TP) can be achieved by adding SIL- peptide to a final concentration of 100 firnol. At this point, the workflow can be paused, and the samples can be stored at -20 °C in the freezer.

[0346] In-solution digestion after enrichment

[0347] After enrichment of the TCER® with either pMHC or protein A, the samples are enzymatically digested (in-solution digestion, (Doellinger et al., Molecular & Cellular Proteomics, Volume 19, Issue 1, 2020). First add 10 pL of labeled DIGESTIF protein standard and 10 pL of AmBic buffer pH 8.5 to each samples. For reduction of the proteins, add 4 pL of 60 mM TCEP followed by 4 pL of the alkylating agent 240 mM CAA. Vortex the sample and incubate for 10 minutes at 70 °C in the thermomixer C or thermocycler. Add 4 pL ProteaseMax (0.5%) and incubate the samples for 5 min at 37 °C in the thermomixer C. For the digestion add 4 pL LysC (0.02 pg / pL) and incubate the samples in the thermomixer C at 37°C for 3 h at 1,000 rpm. To stop the digestion, add 4 pL 99.9% FA to the samples. Vortex each sample, label each tube with the respective label and transfer the samples to the MS Team for subsequent LC / MS analysis.

[0348] Calibration curves

[0349] TCER®-negative tumor lysate from ovarian cancer was being used as tissue surrogate matrix and different amounts of unlabeled TCER® were spiked in, while the amount of the stable isotope labelled (SIL) TCER® internal standard (IS) was kept constant. Following immunoprecipitation using biotin-pMHC & proteolytic digestion, a sample share equivalent to the spiked TCER® amount as shown in brackets was injected into the LC / MS system. As an example, MS / MS ion traces of peptide-3 are shown at different concentrations of the spiked TCER®. Respective fragment ions used for ion extraction and quantification are further depicted as a legend. Bottom plots display the fragment ion signal of the SIL TCER® version, which was kept at a constant concentration. Top plots show signals of the titrated at either 1 firnol or 118 firnol (middle and right) or without an unlabeled TCER® spike. Results are shown in Fig. 1A

[0350] Unlabeled TCER® was titrated at 7 different concentrations while SIL TCER® was kept constant. 7 different peptides released via proteolytic digestion by protease Lys-C are displayed. The peptides show comparable ratios towards their isotopically labeled counterpart and provide a robust metric for accurate quantitation of unlabeled TCER® molecules using enrichment via pMHC down to a theoretical amount of ~0.1 fmol on column. Results are shown in Fig. IB and 1C.

[0351] Quantification of TCER® molecules

[0352] Tumor sample after TCER® injection was cut out, homogenized and lysed in CHAPS buffer. Protein lysate from the two melanoma models was subsequently subjected to protein A- (Fig. 2A) or pMHC-mediated (Fig. 2B) immunoprecipitation paired with a SIL TCER® spike for normalization and absolute quantification. After proteolytic cleavage via LysC, samples were analyzed using targeted mass spectrometry of peptide ion signals of interest. Each bar depicts absolute peptide amount of the seven TCER®-specific peptides of interest. The far right bar depicts average abundance of all seven peptides showing high quantitative accuracy with a CV of 10.01% and 13.24%, respectively. Results are shown in Fig. 2.

[0353] Simultaneous Quantitation of TCER and target pHLA

[0354] Xenograft tumors (cell line Hs 695T) grown in mice and partially treated with TCER molecules were excised and lysed in CHAPS buffer supported by ultrasonification. The lysate was subsequently split and stored at -80°C.

[0355] Method 1 is the method of the present invention used here for simultaneous quantitation of the pHLA peptide of interest, plus HLA and beta-2-microgloibulin (b2m) chains. This was achieved via coprecipitation using biotinylated protein A as capture agent, spike-in of SIL TCER® variant and subsequent acid-mediated protein elution (see figure 5 for overview of setup). The results of the below example are depicted in figures 8 to 10. Of note the method of the present invention allows the simultaneous measurement of multiple parameters, whereas the art known methods require multiple different set ups, i.e. b2m and HLA as bound by the TCER can only be determined with the method of the present invention and not the art-known method 2. This results in substantial reduction of effort. Furthermore, the method of the present invention requires substantially less material than the art known method 2 without a sacrifice in accuracy or specificity.

[0356] The eluted protein sample was subsequently reduced and alkylated via addition of TCEP and chloroacetamide, and subsequently cleaved using LysC. Analysis via LC / MS was performed on an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific) on-line coupled to an Evosep One (Evosep, Odense, Denmark) in PRM mode in the orbitrap. Relative abundance of HLA, b2m and the pHLA peptide was determined in Skyline software. Absolute abundance of the applied TCER molecule was determined using a set of selected TCER-specific peptides and normalized against the spiked-in isotopically labeled TCER counterpart.

[0357] For comparison quantitation via method 2 (AbsQuant) was performed, whereby one aliquot was subjected to BB7.2-mediated immunoprecipitation of HLA-A*02 complexes. Peptides were eluted from antibody-resin by acid treatment and purified by ultrafiltration. pHLA abundance for the peptide of interest was determined using the AbsQuant method (disclosed in US10545154B2) based on the number of cells within the investigated tissue, total amount of the isolated peptide, and target-specific pHLA isolation efficiency. Hereby, each of these three parameters was determined experimentally. The number of cells was determined on the basis of quantitation of DNA content in the investigated tissue sample. DNA was isolated using the QIAamp DNA Mini Kit (QIAGEN) from lysate aliquot, which was sampled during the isolation of HLA-restricted peptides from primary tissue. The DNA yield was quantified using the Qubit dsDNA High Sensitivity (HS) Assay Kit (Applied Biosystems / Thermo Fisher Scientific), and the number of cells was interpolated from DNA content using a standard curve derived from peripheral blood mononuclear cells. For absolute quantitation, a series of targeted nanoLC- MS / MS measurements was performed on an Orbitrap mass spectrometer (Thermo Fisher Scientific).

[0358] Two differently isotopically labeled peptide equivalents were synthesized. One of the isotopically labeled equivalents was used as an absolute quantity reference and was spiked into retention vials of each xenograft sample, which was used for absolute quantitation. The other isotopically labeled equivalent was used to generate the peptide-specific standard curve. Thereby, one of the isotopically labeled equivalents was titrated, and the other one was used as mentioned before as an absolute quantity reference. The MS / MS spectra were acquired using parallel reaction monitoring restricting to labeled peptide masses for the analysis of standard curves and labeled and native peptide masses for the analysis of tumor and normal tissue samples. The MS / MS signals of selected fragment ions were extracted using Skyline 4.2.0 and translated into an absolute peptide amount using peptide-specific standard curves. The efficiency of pHLA isolation was established by spiking of refolded pHLA complex of the investigated peptide into tissue lysate during the peptide isolation procedure and subsequent detection and quantitation of the peptide by nanoLC -MS / MS as described above for quantitation of natively presented peptide.

[0359] Accuracy and comparison to electrochemiluminescent (ECL) based immunoassay (MesoScaleDiscovery ® (MSD) immunoassay)

[0360] Absolute quantitation of the TCER molecules via LC / MS using the method of the invention (method B in Fig 11) in human serum samples was achieved via precipitation using biotinylated pHLA as capture agent, spike-in of SIL TCER® variant and subsequent acid-mediated protein elution (see figure 4 for general set-up). The eluted protein sample was subsequently reduced and alkylated via addition of TCEP and chloroacetamide, and subsequently cleaved using LysC. Analysis via LC / MS was performed on an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific) on-line coupled to an Evosep One (Evosep, Odense, Denmark) in PRM mode in the orbitrap. Absolute abundance of the applied TCER molecule was determined using a set of selected TCER-specific peptides and normalized against the spiked-in isotopically labeled TCER counterpart.

[0361] For absolute TCER quantitation using method A, streptavidin-coated immunoassay plates enabling ECL-based detection were blocked with 3% BSA (bovine serum albumin in PBS). Following washing steps with Wash Buffer (0.05% Tween20 in PBS), MSD plates were incubated with biotinylated TCER capture agent (i.e. pHLA). After further washing steps, the MSD plates were blocked again with 3% BSA and washed. TCER was incubated and following the washing steps with Wash Buffer, the plates were incubated with SULFO-TAG conjugated specific anti-idiotype antibody. After the next washing steps with Wash Buffer, Read Buffer B was added and the resulting electrochemiluminescence (ECL) signal was read on the ECL plate reader.

[0362] The results obtained with the method of the invention (method B) and the art known ECL-based immunoassay (method A) is depicted in figure 11 and demonstrates very high correlation (R2=0.9846).

[0363] Quantitative Analysis of TCER® in various body compartments (example: xenograft mouse model)

[0364] The method of the present invention has also the advantage that input material from various sources can be used, whereas typically art-known methods are limited to individual sample types such as serum / plasma. In any case use of different sources requires a different set-up and further optimization for the art-known methods, which is not required for the method of the present invention. The results of the below disclosed experiment is depicted in figure 12.

[0365] A selection of various organs from tumor-transplanted mice were excised and collected prior to lysis using CHAPS buffer, ultra sonification and storage at -80°C. Subsequently, the lysate was precipitated using biotinylated protein A as capture reagent with an additional spike-in of the matching SIL TCER® variant followed by acid-mediated protein elution (see figure 5 for general set-up). The eluted protein sample was subsequently reduced and alkylated via addition of TCEP and chloroacetamide, and subsequently cleaved using LysC. Analysis via LC / MS was performed on an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific) on-line coupled to an Evosep One (Evosep, Odense, Denmark) in PRM mode in the orbitrap. Absolute abundance of the applied TCER molecule was determined using a set of selected TCER-specific peptides and normalized against the spiked-in isotopically labeled TCER counterpart. Total TCER content was normalized against the respective organ sample input weight.

[0366] Serum vs Tumor tissue

[0367] TCER content was analyzed in serum samples and matching tumor samples, sampled at a similar timeframe, from human subj ects previously treated with the TCER molecule of interest. A total of 10 pL serum and ~ 10 mg fresh-frozen tumor tissue (stored in an RNA-preserving agent) was used as sample input.

[0368] Tumor tissue was washed with PBS prior to lysis in CHAPS, followed by ultra sonification. TCER content in serum samples was analyzed by using biotinylated pHLA as the immunoprecipitation capture agent (see figure 4 for general set up), whereas total TCER content in tissue samples was analyzed by utilizing biotinylated protein A as a capture agent and adding the isotopically labeled SIL TCER variant prior to acid-mediated protein elution (see figure 5 for the general set up).

[0369] Subsequently, the eluted protein sample was reduced and alkylated via addition of TCEp and chloroacetamide, and subsequently cleaved using LysC. Analysis via LC / MS was performed on an Orbitrap Fusion Tribrid instrument (Thermo Fisher Scientific) on-line coupled to an Evosep One (Evosep, Odense, Denmark) in PRM mode in the orbitrap. Absolute abundance of the applied TCER molecule was determined using a set of selected TCER-specific peptides and normalized against the spiked-in isotopically labeled TCER counterpart. Total TCER content was normalized against the respective organ sample input weight or serum volume.

Claims

What is claimed is:

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. 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. 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. 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. 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. 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. The method according to any one of the preceding claims, wherein the biological sample is a human or animal sample.

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. 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. 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. 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. 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. 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. 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.

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