WO2025125477A1

WO2025125477A1 - Mhc class i molecule matched negative control reagents

Info

Publication number: WO2025125477A1
Application number: PCT/EP2024/086005
Authority: WO
Inventors: Thomas Holberg BLICHER; Liselotte Brix
Original assignee: Immudex ApS
Current assignee: Immudex ApS
Priority date: 2023-12-12
Filing date: 2024-12-12
Publication date: 2025-06-19
Anticipated expiration: 2026-06-12

Abstract

The present disclosure relates peptide pools and MHC class I/peptide multimers comprising peptides from said peptide pools. These pools and multimers are useful as MHC class I allele-matched negative control reagents for identifying peptide-unspecific binding of immune cells, such as T cells. The disclosure further provides compositions, nucleic acids and vectors comprising or encoding said pools or multimers, and methods for their use or production.

Description

MHC class I molecule matched negative control reagents

Technical field

The present disclosure relates to peptide pools and MHC class l/peptide multimers comprising peptides from said peptide pools. These pools and multimers are useful as MHC class I allele-matched negative control reagents for identifying peptide-unspecific binding of immune cells, such as T cells.

Background

It is essential to use negative control reagents in peptide-MHC (pMHC) class I multimer based assays for detection of T cells. Negative control reagents for pMHC multimer based assays have previously been described, see e.g. WO 2009/003492.

The type of negative control reagents disclosed in the prior art comprises allele-specific MHC class l/peptide multimers, wherein each MHC comprises the same nonsense peptide. Nonsense peptides are peptides that interact efficiently with the MHC protein but are expected to not support specific binding of the MHC class l/peptide multimer to the T cell receptor (TCR) in question.

Examples of prior art nonsense peptides for HLA-A*0201 include GLAGDVSAV (SEQ ID NO: 1 ) and ALIAPVHAV (SEQ ID NO: 2) (WO 2009/003492). This type of nonsense peptides has only been developed for a few alleles by a trial-and-error approach. Development of such nonsense peptides for each allele is a very time-consuming process, making development of nonsense peptides for all alleles a technical and timeconsuming challenge.

There is thus an unmet need for MHC class I allele-specific negative control reagents, as well as standardized methods for generation of these, which can easily be applied to all types of MHC class I alleles.

Summary

The present disclosure provides a standardized method for generation of negative control reagents, which can easily be applied to all types of MHC class I alleles. This method can be used to formulate allele-matched peptide pools that can be used as negative controls when comprised in e.g. MHC class l-peptide multimers to identify peptide-unspecific binding of immune cells.

In some aspects of the present disclosure is provided a pool of MHC class I molecule- matched negative control peptides, wherein said pool comprises or consists of a plurality of peptides, wherein each peptide of said plurality of peptides comprises or consists of an amino acid sequence comprising the binding motif of said MHC class I molecule, and wherein said pool comprises at least 5 different peptides.

In some aspects of the present disclosure is provided an MHC class l/peptide multimer comprising a plurality of monomers, each monomer comprising a single peptide complexed to an MHC class I molecule, wherein at least two, such as at least 3, such as at least 4, such as at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45 of said peptides of said multimer are different, or such as wherein each of said peptides of said multimer are different.

As a plurality of different peptides are present in the same multimer, each peptide is thus present only once or very few times. Even immune cell receptors specific to one of the included complexed peptides will have too low avidity to bind the multimer, due to their very few numbers in the multimer. The multimers of the present disclosure therefore control for immune cells with receptors that peptide-independently bind to the MHC class l-peptide complexes, e.g. because of binding directly to the MHC class I molecule itself and not to at least part of the peptide, and the multimers therefore function as effective MHC class I allele-matched negative controls.

In some embodiments, each of said peptides is selected from a pool of peptides as disclosed elsewhere herein.

In some aspects of the present disclosure is provided a nucleic acid encoding the MHC class l/peptide monomer and/or the MHC class l/peptide multimer as disclosed elsewhere herein.

In some aspects of the present disclosure is provided a vector comprising the nucleic acid as disclosed elsewhere herein. In some aspects of the present disclosure is provided a composition comprising the pool of peptides as disclosed elsewhere herein and/or an MHC class l/peptide multimer as disclosed elsewhere herein.

In some aspects of the present disclosure is provided a method for producing a pool of peptides as disclosed elsewhere herein, said method comprising the steps of: a) identifying the binding motifs of one or more MHC class I molecules, alleles or supertypes; b) producing and formulating a set of peptides, said set comprising a plurality of different peptides, each peptide comprising or consisting of an amino acid sequence comprising the binding motif of at least one of said one or more MHC class I molecules, alleles or supertypes, thereby producing said pool of peptides.

In some aspects of the present disclosure is provided a method for producing the MHC class l/peptide multimer as disclosed elsewhere herein, said method comprising the steps of: a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class l/peptide multimer, optionally wherein said multimer is defined as disclosed elsewhere herein except that said multimer does not comprise said complexed peptides, whereby a plurality of peptides of said pool of peptides is complexed with said multimer, thereby producing said MHC class l/peptide multimer, or a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class I molecule, optionally wherein said MHC class I molecule is defined as disclosed elsewhere herein except that said MHC class I molecule does not comprise said complexed peptide, whereby a peptide of said pool of peptides is complexed with said MHC class I molecule thereby forming an MHC class l/peptide monomer; and b) multimerizing a plurality of said monomers to produce said MHC class l/peptide multimer, wherein at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as each of said monomers of said plurality of monomers are different. In some aspects of the present disclosure is provided a method for measuring the unspecific background binding of one or more peptide-unspecific immune cells, such as peptide-unspecific T cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells, such as T cells; b) providing one or more MHC class l/peptide multimers as disclosed elsewhere herein or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) measuring the binding of said immune cells, such as T cells, to said MHC class l/peptide multimers, thereby measuring said unspecific background binding.

In some aspects of the present disclosure is provided a method for isolation of one or more peptide-unspecific immune cells, such as peptide-unspecific T cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells, such as T cells; b) providing one or more MHC class l/peptide multimers as disclosed elsewhere herein or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) isolating said immune cells, such as T cells, specific for said MHC class l/peptide multimers.

Description of Drawings

Figure 1. FACS plots showing staining of PBMC samples from two donors (D179 and D191 ) with negative control MHC Dextramer reagents. The cells shown are live, single, CD3+ lymphocytes. Each plot shows staining with anti-CD8 antibody versus Dextramer staining. Skewing of the distribution of the CD8+T lymphocytes is observed with the nonsense peptide but not the peptide pool for D179 suggesting weak binding of the nonsense peptide; indicated with dotted circle and arrow. Dotted circles indicate the same region in all plots. Detailed description

Definitions

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise. Thus, for example, reference to “MHC molecule” includes a plurality of MHC molecules.

By the term “one or more” as used herein is intended to include one and a plurality i.e. more than one.

TCR: T-cell receptor

MHC: Major Histocompatibility Complex

Cell population: The term refers to one or more cells, such as two or more cells, such as multiple cells, such as a sample comprising cells. Said cells need not share any defining characteristics, such as cell cycle stage or tissue origin and can thus be any collection of multiple cells.

Conjugated: The term conjugated refers to two molecules that have been joined, such as through covalent or non-covalent interaction. For example a marker, such as an antibody, may be conjugated to a fluorescent tag or a DNA oligonucleotide through a linker, such as a stretch of amino acids.

As used herein, the terms “DNA barcode oligonucleotide” and “oligonucleotide barcode” are used interchangeably and refer to unique oligo-nucleotide sequences typically ranging from 10 to more than 50 nucleotides. The barcode may have shared amplification sequences in the 3’ and 5’ ends, and a unique sequence in the middle. This sequence can be revealed by sequencing and can serve as a specific barcode for a given molecule.

The term “oligonucleotide label” refers to a nucleic acid label that may comprise or consist of DNA, RNA, and/or artificial nucleotides such as PLA or LNA. The oligonucleotide label may comprise one or more of the following components: a 5’ first primer region (forward), a barcode region (e.g. an oligonucleotide barcode), 3’ second primer region (reverse), random nucleotide region, connector molecule, stabilityincreasing components, short nucleotide linkers in between any of the above- mentioned components, adaptors for sequencing and annealing region. Preferably the oligonucleotide label comprises at least a barcode region surrounded by primer regions, where the barcode region comprises a sequence of consecutive nucleic acids. In one embodiment the oligonucleotide label comprises or consists of DNA, RNA, artificial nucleic acids and/or Xeno nucleic acid (XNA).

Unless otherwise indicated, the terms “backbone molecule” and “multimerization domain” are used interchangeably herein.

A “MHC Class I molecule” as used everywhere herein is used interchangeably with MHC I molecule and is defined as a molecule which comprises 1-3 subunits, including a MHC I heavy chain, a MHC I heavy chain combined with a MHC I beta2microglobulin chain, a MHC I heavy chain combined with MHC I beta2microglobulin chain through a flexible linker, a MHC I heavy chain combined with an antigenic peptide, a MHC I heavy chain combined with an antigenic peptide through a linker, a MHC I heavy chain/ MHC I beta2microglobulin dimer combined with an antigenic peptide, and a MHC I heavy chain/MHC I beta2microglobulin dimer combined with an antigenic peptide through a flexible linker to the heavy chain or beta2microglobulin. The MHC I molecule chains can be changed by substitution of single or by cohorts of native amino acids, or by inserts, or deletions to enhance or impair the functions attributed to said molecule.

MHC Class I like molecules (including non-classical MHC Class I molecules) include CD1d, HLA E, HLA G, HLA F, HLA H, MIC A, MIC B, ULBP-1 , ULBP-2, and ULBP-3. The MHC molecule may suitably be a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule. Such MHC complexes from different species have different names. E.g. in humans, MHC complexes are denoted HLA. The person skilled in the art will readily know the name of the MHC class I complexes from various species.

In general, the term “MHC class I molecule” is intended to include all class I alleles. By way of example, in humans e.g. HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and HLA-H alleles are of interest shall be included, and in the mouse system, H-2 alleles are of interest shall be included. Likewise, in the rat system RT1 -alleles, in the porcine system SLA-alleles, in the bovine system BoLA, in the avian system e.g. chicken-B alleles, are of interest and shall be included.

By the terms “MHC class I complexes” as used herein are meant such complexes and multimers thereof, which are capable of performing at least one of the functions attributed to said complex. The terms include both classical and non-classical MHC class I complexes. The meaning of “classical” and “non-classical” in connection with MHC class I complexes is well known to the person skilled in the art. Non-classical MHC class I complexes are subgroups of MHC-like complexes. The term “MHC class I complex” includes MHC Class I molecules as well as MHC-like molecules (Class I), including the subgroup non-classical MHC Class I molecules.

The term “MHC class I superfamily” refers to MHC molecules with a shared peptide- binding motif. In the context of the present invention the term “superfamily” may be used interchangeably with the term “supertype”.

The term “binding motif” as used herein in relation to MHC class I molecules or supertypes refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC molecule or supertype. The binding motif may also be known as the consensus sequence or anchor sequence for binding.

Non-covalent: The term non-covalent bond as used herein is a type of chemical bond that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions.

Unless otherwise indicated, the term “MHC class I molecule” refers to an MHC class I molecule without a complexed peptide. An MHC class I molecule complexed with a peptide is referred to as an “MHC class l/peptide monomer”.

Unless otherwise indicated, the term “any amino acid” may refer to any of the 20 standard amino acids: Alanine (A), Arginine (R), Asparagine (N), Aspartic acid (D), Cysteine (C), Glutamine (Q), Glutamic acid (E), Glycine (G), Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Proline (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), and Valine (V).

The term non-standard, non-natural or modified amino acids refer to any non- proteogenic amino acid, which is characterized by the presence of an amino and a carboxylic acid functional group connected by one or more carbon atoms. Such nonstandard amino acids may carry the equivalent of a side chain as found in the 20 standard amino acids. The side chain may be chemically or structurally similar to that of any of the 20 natural amino acids, it may be a derivative of any such side chain, or it may have a chemical composition completely distinct from that of the 20 natural amino acids. All non-standard amino acids may be considered for synthesis of peptides, either alone or in combination with the 20 natural amino acids.

Peptide pool

The present disclosure provides peptide pools that may be matched to a single MHC class I supertype, such as to a single MHC class I allele, i.e. a single MHC class I molecule. The pools may be designed such that all peptides in a single pool comprise a sequence containing the binding motif of the MHC class I supertype or molecule to which they are matched. In theory, all or nearly all of the peptides in a single pool should therefore be able to bind MHC class I molecules of the matched supertype or the MHC class I molecule to which said pool is matched. In some embodiments, the peptide pools contain a plurality of different peptides that are able to bind MHC class I molecules of the matched supertype or the MHC class I molecule to which said pool is matched.

In some aspects of the present disclosure is provided a pool of MHC class I molecule- matched negative control peptides, wherein said pool comprises a plurality of peptides, wherein each peptide of said plurality of peptides comprises or consists of an amino acid sequence comprising the binding motif of said MHC class I molecule, and wherein said pool comprises at least 5 different peptides.

In some embodiments of the present disclosure is provided a pool of MHC class I molecule-matched negative control peptides, wherein said pool consists of a plurality of peptides, wherein each peptide of said plurality of peptides comprises or consists of an amino acid sequence comprising the binding motif of said MHC class I molecule, and wherein said pool comprises at least 10 different peptides.

In some embodiments, the pool comprises at least 10, such as at least 25, such as at least 50, such as at least 75, such as at least 100, such as at least 150, such as at least 200, such as at least 300, such as at least 500, such as at least 1000, such as at least 2500, such as at least 5000, such as at least 10.000, such as at least 25.000, such as at least 50.000, such as at least 75.000, such as at least 100.000, such as at least 250.000, such as at least 750.000, such as at least 1 .000.000, such as at least 5.000.000, or such as at least 10.000.000 different peptides.

The pool may be designed to match any given MHC class I superfamily or may be designed to match any given specific MHC class I molecule, such as any given MHC class I allele.

The MHC class I molecules or superfamilies according to the present disclosure are in one embodiment of human origin and/or animal origin. In one embodiment, the MHC class I molecules or superfamilies according to the present disclosure is of mammal origin (e.g., macaque origin, rodent origin, such as mouse or rat origin). In another embodiment the MHC class I molecules or superfamilies according to the present disclosure is of non-mammalian vertebrate origin (such as from fish, bird, insect, amphibian, and/or reptile origin). In some embodiments, the MHC molecule or superfamily according to the present disclosure is selected from the group consisting of human, non-human primates, Gorilla gorilla, Pan troglodytes, Macacca mulatta, Orangutang, Rodents, Mus musculus, Rattus norvegicus and Lagomorpha, such as rabbits and hares.

Thus, in some embodiments of the present disclosure, the MHC class I molecule or superfamily is from a gorilla. In some embodiments of the present disclosure, the MHC class I molecule or superfamily is from a chimpanzee. In some embodiments of the present disclosure, the MHC class I molecule or superfamily is from a rhesus macaque. In some embodiments of the present disclosure, the MHC class I molecule or superfamily is from a rodent. In some embodiments of the present disclosure, the MHC class I molecule or superfamily is from a mouse. In some embodiments of the present disclosure, the MHC class I molecule or superfamily is from a rat. In some embodiments, the MHC class I molecule or superfamily is from a human. Human MHC class I supertypes and their binding motifs are generally known by the person skilled in the art, see e.g. Lund et al., 2004, and Sidney et al., 2008, which are hereby incorporated by reference in their entirety.

In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

In some embodiments, the MHC class I molecule is a HLA-A molecule. In some embodiments, the MHC class I molecule is a HLA-B molecule. In some embodiments, the MHC class I molecule is a HLA-C molecule. In some embodiments, the MHC class I molecule is a HLA-E molecule. In some embodiments, the MHC class I molecule is a HLA-F molecule. In some embodiments, the MHC class I molecule is a HLA-G molecule.

In some embodiments, MHC class I molecule is a member of an HLA supertype selected from the group consisting of A01 , A02, A03, A24, A26, B07, B08, B27, B39, B44, B58, and B62.

The term “MHC class I superfamily” refers to a group of MHC molecules with a shared peptide-binding motif and a superfamily comprises different alleles as described in the literature e.g., in Lund et al., 2004 and elsewhere herein.

The A01 superfamily includes MHC class I alleles, which shares binding motif with HLA-A*01 :01 . Similarly, the A02 superfamily includes MHC class I alleles, which shares binding motif with HLA-A*02:01 , the A03 superfamily includes MHC class I alleles, which shares binding motif with HLA-A*03:01 , the A24 superfamily includes MHC class I alleles, which shares binding motif with HLA-A*24:02, the A26 superfamily includes MHC class I alleles, which shares binding motif with HLA-A*26:01 , the B07 superfamily includes MHC class I alleles, which shares binding motif with HLA-B*07:02, the B08 superfamily includes MHC class I alleles, which shares binding motif with HLA- B*08:01 , the B27 superfamily includes MHC class I alleles, which shares binding motif with HLA-B*27:01 , the B39 superfamily includes MHC class I alleles, which shares binding motif with HLA-B*39:01 , the B44 superfamily includes MHC class I alleles, which shares binding motif with HLA-B*44:02, the B58 superfamily includes MHC class I alleles, which shares binding motif with HLA-B*58:01 and the B62 superfamily includes MHC class I alleles, which shares binding motif with HLA-B*15:01 .

As an example, the superfamily A024 comprises alleles such as HLA-A*23:01 , HLA- A*23:03, HLA-A*23:05, which shares binding motif with HLA-A*24:02. The superfamily B44 includes HLA-B*44:03 and HLA-B*44:04, which shares binding motif with HLA- B*44:02. The MHC class I alleles comprised in each superfamily can be found in literature e.g., Sidney et al., 2008.

In some embodiments, MHC class I molecule is a member of the A01 superfamily.

In some embodiments, MHC class I molecule is a member of the A02 superfamily.

In some embodiments, MHC class I molecule is a member of the A03 superfamily.

In some embodiments, MHC class I molecule is a member of the A24 superfamily.

In some embodiments, MHC class I molecule is a member of the A26 superfamily.

In some embodiments, MHC class I molecule is a member of the B07 superfamily.

In some embodiments, MHC class I molecule is a member of the B08 superfamily.

In some embodiments, MHC class I molecule is a member of the B27 superfamily.

In some embodiments, MHC class I molecule is a member of the B39 superfamily.

In some embodiments, MHC class I molecule is a member of the B44 superfamily.

In some embodiments, MHC class I molecule is a member of the B58 superfamily.

In some embodiments, MHC class I molecule is a member of the B62 superfamily.

Without being bound by theory, peptides with lengths from 8 to 13 amino acids are able to bind to and complex with MHC class I molecules.

In some embodiments, each peptide of said plurality of peptides in said pool is from 8 to 13 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is from 9 to 13 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is from 9 to 12 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is from 10 to 12 amino acids in length.

Said peptides of said plurality of peptides in said pool may be the same length or different lengths. In some embodiments, each peptide of said plurality of peptides in said pool is 8 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is 9 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is 10 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is 11 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is 12 amino acids in length. In some embodiments, each peptide of said plurality of peptides in said pool is 13 amino acids in length.

For pools matched to human MHC class I molecule supertypes, the peptides of each pool may comprise one of the binding motifs according to Table 1 , below, in their sequence. For illustration, X[T][D/E]XXXX[Y] in Table 1 , below, indicates that the binding motif for 8-mers to HLA supertype A01 molecules has any amino acid in position 1 , a threonine (T) in position 2, an aspartic acid (D) in position 3, any amino acid in position 4, any amino acid in position 5, any amino acid in position 6, any amino acid in position 7, and a tyrosine (Y) in position 8.

The minimum length of each peptide is preferable 8 amino acids. In Table 1 are indicated human MHC class I superfamily binding motifs for peptides of this length. For longer peptides, additional amino acids (any amino acid) are added immediately prior to the last listed amino acid in Table 1 . E.g. for HLA supertype A01 9-mers the following consensus binding motif applies: X[T][D/E]XXXXX[Y], the underlined amino acid indicating the additional amino acid compared to Table 1 . Similarly, for HLA supertype A01 10-mers the following consensus binding motif applies: X[T1[D/E1XXXXXX[Y1, underlined amino acids indicating the additional amino acids compared to Table 1.

Table 1 - Human MHC class I supertype binding motifs

In some embodiments, the amino acid of the second or fifth position, and the last position of the binding motif of an MHC class I molecule is selected from specific amino acids. I.e, in some embodiments, the amino acid of the second or fifth position, and the last position of the binding motif of an MHC class I molecule is not variable, hence is not any amino acid (‘X’).

Thus, in some embodiments, each of said plurality of peptides of the pool comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein i. a) X₂ is an amino acid at the second position of said binding motif of said MHC class I molecule; b) X13 is defined as an amino acid at the last position of said binding motif of said MHC class I molecule; c) Xi and X3-X7 are each independently any amino acid; and d) X₈-Xi2 are each independently any amino acid or absent; or ii. a) X₅ is an amino acid at the fifth position of said binding motif of said MHC class I molecule; b) X13 is defined as the amino acid at the last position of said binding motif of said MHC class I molecule; c) X1-X4 and X₆-X₇ are each independently any amino acid; and d) X₈-Xi2 are each independently any amino acid or absent. In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype A01 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is T; c) X₃ is D or E; d) X4-X7 are each independently any amino acid; e) X₈-Xi2 are each independently any amino acid or absent; and f) X13 is Y.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype A02 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X2 is M or L; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) Xi₃ is L or V.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype A03 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is T or L; c) X₃ is F; d) X4-X7 are each independently any amino acid; e) X₈-Xi2 are each independently any amino acid or absent; and f) X is K or R.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype A24 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is Y or F; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is F or L.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype A26 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any standard amino acid; b) X₂ is T or V; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is Y or M.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B07 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is P; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) Xi₃ is L or F.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B08 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence Xi-X2-X3-X4-X₅-X6-X7-X₈-X9-Xio-Xn-Xi2-Xi3, wherein a) X1-X4 is any amino acid; b) X₅ is R; c) X₆-X₇ are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is L.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B27 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence Xi-X2-X3-X4-X₅-X6-X7-X₈-X9-Xio-Xn-Xi2-Xi3, wherein a) Xi is any amino acid; b) X₂ is R; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is L.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B39 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is H, R, or Q; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is L.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B44 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence Xi-X2-X3-X4-X₅-X6-X7-X₈-X9-Xio-Xn-Xi2-Xi3, wherein a) Xi is any amino acid; b) X2 is D or E; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is I, L, F, Y, or W.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B58 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence Xi-X2-X3-X4-X₅-X6-X7-X₈-X9-Xio-Xn-Xi2-Xi3, wherein a) Xi is any amino acid; b) X₂ is S or A; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is W.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype B62 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence Xi-X2-X3-X4-X₅-X6-X7-X₈-X9-Xio-Xn-Xi2-Xi3, wherein a) Xi is any amino acid; b) X₂ is M or Q; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is F.

In some embodiments, the pool is matched to HLA-A*01 :01 . In some embodiments, the pool is matched to HLA-A*02:01 . In some embodiments, the pool is matched to HLA- A*03:01 . In some embodiments, the pool is matched to HLA-A*24:02. In some embodiments, the pool is matched to HLA-A*26:01 . In some embodiments, the pool is matched to HLA-B*07:02. In some embodiments, the pool is matched to HLA-B*08:01. In some embodiments, the pool is matched to HLA-B*27:01. In some embodiments, the pool is matched to HLA-B*39:01 . In some embodiments, the pool is matched to HLA- B*44:02. In some embodiments, the pool is matched to HLA-B*58:01. In some embodiments, the pool is matched to HLA-B*15:01 .

HLA-C superfamilies can be defined in analogy with the well-known HLA-A and B superfamilies. As for the HLA-A and B superfamilies, each HLA-C superfamily comprises HLA-C alleles with a shared peptide-binding motif, and each superfamily can be represented by a specific allele, which binds peptides adhering to the corresponding motif.

In some embodiments, MHC class I molecule is a member of an HLA supertype selected from the group consisting of A01 , A02, A03, A24, A26, B07, B08, B27, B39, B44, B58, B62, C01 , C02, C04, C07, C08, C14 and C15.

In one embodiment, the invention includes MHC class l-binding motifs for HLA-C superfamilies and their representative HLA alleles, such as those listed in Table 2.

The minimum length of each peptide is preferable 8 amino acids. In Table 2 are indicated human MHC class I superfamily binding motifs for peptides of this length. For longer peptides, additional amino acids (any amino acid) are added immediately prior to the last listed amino acid in Table 2, the same way as for Table 1 .

Table 2

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C01 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X2 is A or L; c) X₃ is P; d) X4-X7 are each independently any amino acid; e) X₈-Xi2 are each independently any amino acid or absent; and f) X13 is L or I.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C02 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is A; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is F, L, M or Y.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C04 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is F, W or Y; c) X₃ is D; d) X4-X7 are each independently any amino acid; e) X₈-Xi2 are each independently any amino acid or absent; and f) X13 is F, I, L or M.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C07 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is R or Y; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is F, I, L, V, M or Y.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C08 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is A or S; c) X₃ is D; d) X4-X7 are each independently any amino acid; e) X₈-Xi2 are each independently any amino acid or absent; and f) X13 is F, I, L or M.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C14 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is Y or F; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is F, L, M or Y.

In some embodiments, the pool is matched to an MHC class I molecule that is an HLA supertype C15 MHC class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is A, S or T; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is V, L or I.

In some embodiments, the pool is matched to a murine H2 allele. In some embodiments, the H2 allele is Db. In some embodiments, the H2 allele is Dd. In some embodiments, the H2 allele is Kd. In some embodiments, the H2 allele is Kk. In some embodiments, the H2 allele is Ld.

In Table 3, below, are indicated murine MHC class I allele binding motifs for peptides with a length of 8 amino acids. For longer peptides, additional amino acids (any amino acid) are added immediately prior to the last listed amino acid in Table 3, corresponding to position 8 in the below table.

Table 3 - Murine MHC class I allele binding motifs

In some embodiments, the pool is matched to an MHC class I molecule that is a murine H2-Db class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is S; c) X3-X4 are each independently any amino acid; d) X₅ is N; e) X₆-X₇ are each independently any amino acid; f) X₈-Xi2 are each independently any amino acid or absent; and g) X13 is L or I. In some embodiments, the pool is matched to an MHC class I molecule that is a murine H2-Dd class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is S; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X is L or F.

In some embodiments, the pool is matched to an MHC class I molecule that is a murine H2-Kb class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is S; c) X₃ is Y or F; d) X4-X7 are each independently any amino acid; e) X₈-Xi2 are each independently any amino acid or absent; and f) X is L or M.

In some embodiments, the pool is matched to an MHC class I molecule that is a murine H2-Kd class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is Y; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is I or L.

In some embodiments, the pool is matched to an MHC class I molecule that is a murine H2-Kk class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is E; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X13 is I.

In some embodiments, the pool is matched to an MHC class I molecule that is a murine H2-Ld class I molecule, and each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid; b) X₂ is P; c) X3-X7 are each independently any amino acid; d) X₈-Xi2 are each independently any amino acid or absent; and e) X is L or F.

In some embodiments, the amino acids in the positions of the peptides, which may be selected from any amino acid, are selected from any standard amino acid, i.e. from the group consisting of alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), and Valine (V).

Two different peptide pools according to the present disclosure may also be mixed together to provide a pool covering two or MHC class I molecules or supertypes, i.e. to create a pool matched to more than one MHC class I molecule or supertype.

In some embodiments, the pool comprises a) a first set of peptides, each of the peptides of said first set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a first MHC class I molecule; and b) a second set of peptides, each of the peptides of said second set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a second MHC class I molecule, wherein said first and second MHC class I molecules are different.

In some embodiments, the pool comprises a) a first set of peptides, each of the peptides of said first set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a first MHC class I molecule; and b) a second set of peptides, each of the peptides of said second set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a second MHC class I molecule, wherein the supertype of said first and second MHC class I molecules are different.

In some embodiments, the pool comprises a) a first set of peptides, each of the peptides of said first set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a first MHC class I molecule, wherein said first set of peptides is defined as disclosed elsewhere herein; and b) a second set of peptides, each of the peptides of said second set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a second MHC class I molecule, wherein said second set of peptides is defined as disclosed elsewhere herein, wherein said first and second MHC class I molecules are different.

In some embodiments, the pool comprises a) a first set of peptides, each of the peptides of said first set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a first MHC class I molecule, optionally wherein said first set of peptides is defined as disclosed elsewhere herein; and b) a second set of peptides, each of the peptides of said second set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a second MHC class I molecule, optionally wherein said second set of peptides is defined as disclosed elsewhere herein, wherein the supertype of said first and second MHC class I molecules are different.

MHC class l/peptide multimer

MHC class I multimers complexed with peptides (MHC class l/peptide multimers), the peptides being specific for the binding motif of one or more MHC class I molecules, such as one or more MHC class I superfamilies, may be useful as negative controls for peptide-unspecific binding of immune cells, such as T cells.

It is important to note that the peptides of each multimer do not necessarily need to comprise the full consensus sequence or binding motif of the matched MHC class I molecule or superfamily. For the purposes of the present disclosure, it is sufficient that the peptides are able to successfully bind the MHC class I molecules of the multimer, whereby a MHC class l/peptide multimer can be produced comprising a minimum number of different peptides.

In one aspect of the present disclosure is provided an MHC class l/peptide multimer comprising a plurality of monomers, each monomer comprising a single peptide complexed to an MHC class I molecule, wherein at least two, such as at least 3, such as at least 4, such as at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45 of said peptides of said multimer are different, or such as wherein each of said peptides of said multimer are different, such as different to each other.

The term “MHC class l/peptide monomer” refers to an MHC class I molecule with a complexed peptide according to the present disclosure, and the terms “monomer” and “MHC class l/peptide monomer” are therefore used interchangeably herein, unless specifically otherwise indicated. The term “MHC class l/peptide multimer” refers to a complex comprising multiple MHC class l/peptide monomers according to the present disclosure associated by covalent and/or noncovalent bonds. For example, an MHC class l/peptide multimer includes, but is not limited to, an MHC class l/peptide dimer, trimer, tetramer, pentamer, hexamer, heptamer or octamer or any higher valency multimer, e.g., comprising 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more than 24 MHC class l/peptide monomers.

MHC class l/peptide multimers are described in detail in W002072631 , W02008116468, W02009003492 and W02020127222, which are incorporated herein by reference.

In some embodiments of the present disclosure is provided an MHC class l/peptide multimer comprising a plurality of monomers, each monomer comprising a single peptide complexed to an MHC class I molecule, wherein at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% of said peptides of said multimer are different, such as different to each other. In some embodiments, each of the peptides of said multimer is selected from a pool of peptides as described elsewhere herein.

A multimer may thus be matched to a single MHC class I molecule or superfamily. In some embodiments, each of the peptides of a multimer are selected from a peptide pool specific for a single MHC class I molecule supertype. In some embodiments, each of the peptides of a multimer are selected from a peptide pool specific for an MHC class I molecule, such as for a single HLA allele.

Some MHC class I molecules, such as HLA-I alleles, are defined with an intermediary supertype, e.g. A01/A03 or A01/A24, see e.g. Sidney et al., 2008. Multimers made from a peptide pool comprising peptides specific for at least two different MHC molecules may thus also be useful as negative controls for these alleles.

A multimer may thus be matched to more than one single MHC class I molecule or superfamily. In some embodiments, each of the peptides are selected from a peptide pool specific for at least two different MHC class I molecule supertypes, such as for two or more different HLA supertypes. In some embodiments, each of the peptides are selected from a peptide pool specific for at least two different MHC class I molecules, such as for two or more different HLA alleles.

In some embodiments, the monomer exhibits native-like properties in terms of recognition by TCRs and/or other MHC class I complex-engaging receptors. In some embodiments, said monomer thus exhibits native-like properties in terms of recognition by TCRs and/or other MHC class I complex-engaging receptors.

In some embodiments, said peptide complexed to said MHC class I molecule exhibits native-like properties in terms of T-cell recognition, NK cell recognition and/or recognition of other immune cells.

The MHC class I molecules in a multimer according to the present disclosure can be identical MHC class I molecules or substantially identical MHC class I molecules. It may also be desirable that the multimers are matched to more than one MHC class I molecule or superfamily. Thus, in some embodiments, at least two of the MHC class I molecules in a multimer are different.

In some embodiments of the present disclosure, the MHC class l/peptide multimer is a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer or an octamer or any higher valency multimer, e.g., comprising 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more than 24 monomers according to the present disclosure.

In some embodiments of the present disclosure, the MHC class l/peptide multimer comprises at least two different MHC class l/peptide monomers according to the present disclosure. In some embodiments of the present disclosure, the MHC class l/peptide multimer comprises at least 3, such as at least 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 different MHC class l/peptide monomers.

Each MHC class l/peptide monomer of the MHC class l/peptide multimer can be associated with one or more multimerization domains such as a multimerization domain selected from the group consisting of IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, dextran, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs that can bind MHC class I molecules.

The term “multimerization domain” as used herein can be any type of molecule that is directly or indirectly associated with one or more MHC class l/peptide monomers and/or MHC molecules. A multimerization domain is a molecule, a complex of molecules, or a solid support, to which one or more MHC class l/peptide monomers and/or MHC class I molecules can be attached. A multimerization domain can consist of one or more carriers and/or one or more scaffolds and may also contain one or more linkers connecting carrier to scaffold, carrier to carrier, and/or scaffold to scaffold. The multimerization domain may also contain one or more linkers that can be used for attachment of the the MHC class l/peptide monomers, MHC class I molecules and/or other molecules to the multimerization domain. In this disclosure, a multimerization domain will in one embodiment refer to a functionalized polymer (e.g., dextran) that is capable of reacting with MHC class l/peptide monomers and/or MHC class I molecules, thus covalently attaching the MHC class l/peptide monomer and/or MHC class I molecule to the multimerization domain, or that is capable of reacting with scaffold molecules (e.g., streptavidin), thus covalently attaching streptavidin to the multimerization domain; the streptavidin then may bind the MHC class l/peptide monomers and/or MHC class I molecules. Multimerization domains include IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, dextran, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs that can bind MHC class l/peptide monomers, MHC class I molecules and other molecules, such as identified in detail herein elsewhere.

Non-limiting examples of suitable multimerization domain(s) are polysaccharides including dextran molecules, carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextran lactone, and cyclodextrins, pullulans, schizophyllan, scleroglucan, xanthan, gellan, O-ethylamino guaran, chitins and chitosans indlucing 6- O- carboxymethyl chitin and N-carboxymethyl chitosan, derivatised cellolosics including carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, hydroxy- ethyl cellulose, 6-amino-6-deoxy cellulose and O-ethyl- amine cellulose, hydroxylated starch, hydroxypropyl starch, hydroxyethyl starch, carrageenans, alginates, and agarose, synthetic polysaccharides including ficoll and carboxy-methylated ficoll, vinyl polymers including poly (acrylic acid), poly (acryl amides), poly (acrylic esters), poly (2-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly (maleic acid), poly (maleic anhydride), poly (acrylamide), poly (ethyl-co- vinyl acetate), poly (methacrylic acid), poly (vinyl- alcohol), poly (vinyl alcohol-co-vinyl chloroacetate), aminated poly (vinyl alcohol), and co block polymers thereof, poly ethylene glycol (PEG) or polypropylene glycol or poly (ethylene oxide-co-propylene oxides) comprising polymer backbones including linear, comb-shaped or StarBurst dendrimers, poly amino acids including polylysines, polyglutamic acid, polyurethanes, poly (ethylene imines), pluriol, proteins including peptides, polypeptides, antigen binding peptides, albumins, immunoglobulins, coiled-coil helixes e.g. Fos-Jun or Fos-Jun like or coiled-coiled dimers/trimers/ tetramers/pentamers, streptavidin, avidin, streptactin, T-cell receptors, other protein receptors and virus- like proteins (VLP), and polynucleotides, DNA, RNA, PNA, LNA, oligonucleotides and oligonucleotide dendrimer constructs and small organic molecules including but not limited to steroids, peptides, linear or cyclic structures, aromatic structures, aliphatic structures. The term “Dextran” as used herein is a complex, branched polysaccharide made of glucose molecules joined into chains of varying lengths. The straight chain consists of a1->6 glycosidic linkages between glucose molecules, while branches begin from a1- >3 linkages (and in some cases, a1->2 and a1->4 linkages as well).

In one embodiment of the present disclosure, the MHC class l/peptide multimer comprises at least 2 MHC class l/peptide monomers, such as at least 3 MHC class l/peptide monomers, such as at least 4 MHC class l/peptide monomers, such as at least 5 MHC class l/peptide monomers, such as at least 6 MHC class l/peptide monomers, such as at least 7 MHC class l/peptide monomers, such as at least 8 MHC class l/peptide monomers, such as at least 9 MHC class l/peptide monomers, such as at least 10 MHC class l/peptide monomers, such as at least 11 MHC class l/peptide monomers, such as at least 12 MHC class l/peptide monomers, such as at least 13 MHC class l/peptide monomers, such as at least 14 MHC class l/peptide monomers, such as at least 15 MHC class l/peptide monomers, such as at least 16 MHC class l/peptide monomers, such as at least 17 MHC class l/peptide monomers, such as at least 18 MHC class l/peptide monomers, such as at least 19 MHC class l/peptide monomers, or such as at least 20 MHC class l/peptide monomers.

In another embodiment of the present disclosure, the MHC class l/peptide multimer comprises 2 to 50 MHC class l/peptide monomers, such as 10 to 20 MHC class l/peptide monomers, such as 2 to 4 MHC class l/peptide monomers, such as 4 to 5 MHC class l/peptide monomers, such as 5 to 6 MHC class l/peptide monomers, such as 6 to 8 MHC class l/peptide monomers, such as 8 to 10 MHC class l/peptide monomers, such as 10 to 12 MHC class l/peptide monomers, such as 12 to 14 MHC class l/peptide monomers, such as 14 to 16 MHC class l/peptide monomers, such as 16 to 18 MHC class l/peptide monomers, such as 18 to 20 MHC class l/peptide monomers, such as 20 to 25 MHC class l/peptide monomers, such as 25 to 30 MHC class l/peptide monomers, such as 30 to 40 MHC class l/peptide monomers, such as 40 to 50 MHC class l/peptide monomers of the present disclosure, or any combination of these intervals.

In some embodiments of the present disclosure, the MHC class l/peptide multimer comprises no more than 50 MHC class l/peptide monomers in total, such as no more than 45 MHC class l/peptide monomers, such as no more than 40 MHC class l/peptide monomers, such as no more than 35 MHC class l/peptide monomers, such as no more than 30 MHC class l/peptide monomers, such as no more than 25 MHC class l/peptide monomers, such as no more than 20 MHC class l/peptide monomers, such as no more than 15 MHC class l/peptide monomers, or no more than 10 MHC class l/peptide monomers in total.

In one embodiment of the present disclosure, the MHC class l/peptide multimer comprises at least 2 MHC class l/peptide monomers, such as at least 3 MHC class l/peptide monomers such as at least 4 MHC class l/peptide monomers, such as at least 5 MHC class l/peptide monomers, such as at least 6 MHC class l/peptide monomers, such as at least 7 MHC class l/peptide monomers, such as at least 8 MHC class l/peptide monomers, such as at least 9 MHC class l/peptide monomers, such as at least 10 MHC class l/peptide monomers, such as at least 11 MHC class l/peptide monomers, such as at least 12 MHC class l/peptide monomers, such as at least 13 MHC class l/peptide monomers, such as at least 14 MHC class l/peptide monomers, such as at least 15 MHC class l/peptide monomers, such as at least 16 MHC class l/peptide monomers, such as at least 17 MHC class l/peptide monomers, such as at least 18 MHC class l/peptide monomers, such as at least 19 MHC class l/peptide monomers, or such as at least 20 MHC class l/peptide monomers of the present disclosure.

In another embodiment the MHC class l/peptide multimer of the present disclosure comprises 2 to 50 MHC class l/peptide monomers, such as 10 to 20 MHC class l/peptide monomers, such as 4 to 6 MHC class l/peptide monomers, such as 6 to 8 MHC class l/peptide monomers, such as 8 to 10 MHC class l/peptide monomers, such as 10 to 12 MHC class l/peptide monomers, such as 12 to 14 MHC class l/peptide monomers, such as 14 to 16 MHC class l/peptide monomers, such as 16 to 18 MHC class l/peptide monomers, such as 18 to 20 MHC class l/peptide monomers, such as 20 to 25 MHC class l/peptide monomers, such as 25 to 30 MHC class l/peptide monomers, such as 30 to 40 MHC class l/peptide monomers, such as 40 to 50 MHC class l/peptide monomers of the present disclosure, or any combination of these intervals.

In some embodiments of the present disclosure, the MHC class l/peptide multimer comprises no more than 50 MHC class l/peptide monomers, such as no more than 45 MHC class l/peptide monomers, such as no more than 40 MHC class l/peptide monomers, such as no more than 35 MHC class l/peptide monomers, such as no more than 30 MHC class l/peptide monomers, such as no more than 25 MHC class l/peptide monomers, such as no more than 20 MHC class l/peptide monomers, such as no more than 15 MHC class l/peptide monomers, or no more than 10 MHC class l/peptide monomers.

In some embodiments of the present disclosure, the MHC class l/peptide multimer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 MHC class l/peptide monomers of the present disclosure, respectively.

In a specific embodiment of the present disclosure, the MHC class l/peptide multimer has exactly 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 MHC class l/peptide monomers of the present disclosure, respectively.

In some embodiments, some of the MHC class l/peptide monomers or all of the MHC class l/peptide monomers comprised in an MHC class l/peptide multimer comprise different peptides, such as different peptides compared to the other peptides of said multimer. In one embodiment the MHC class l/peptide multimer of the present disclosure comprises at least 2, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 MHC class l/peptide monomers each of which comprise different peptides, such as different peptides compared to the other peptides of said multimer.

In one embodiment, the MHC class l/peptide multimer of the present disclosure comprises one or more labels. In one embodiment the MHC class l/peptide multimer of the present disclosure comprises at least two labels. These labels can all be different or identical, or some the labels can be identical and some different. In some embodiments of the present disclosure, all labels are different or at least two of the labels are different. In some embodiments of the present disclosure, all labels are identical or at least two of the labels are identical. In one embodiment of the present disclosure, the one or more labels comprise at least one fluorescent label and/or at least one oligonucleotide label.

In a specific embodiment of the present disclosure, the at least one oligonucleotide label comprises one or more of: a 5’ first primer region (forward), a barcode region, 3’ second primer region (reverse), random nucleotide region, connector molecule, stability-increasing components, short nucleotide linkers in between any of the above- mentioned components, adaptors for sequencing and annealing region.

In one embodiment the one or more labels are directly attached to the MHC class l/peptide multimer. In one embodiment the one or more labels are indirectly attached to the MHC class l/peptide multimers, such as via one or more marker molecules carrying one or more labels.

The one or more labels may be used for combinatorial use of labelling. The one or more labels may result in positive selection of said MHC class l/peptide multimer or alternatively in negative selection of said MHC class l/peptide multimer. The one or more labels may comprise one or more covalently attached labels and/or one or more non-covalently attached labels. The one or more labels may be covalently attached to “polypeptide a” of the MHC class I molecule and/or MHC class l/peptide monomer, covalently attached to “polypeptide b” of the MHC class I molecule and/or MHC class l/peptide monomer, covalently attached to the peptide and/or covalently attached to the one or more multimerization domains. Alternatively, the one or more labels may be non-covalently attached to “polypeptide a” of the MHC class I molecule and/or MHC class l/peptide monomer, non-covalently attached to “polypeptide b” of the MHC class I molecule and/or MHC class l/peptide monomer, non-covalently attached to the peptide and/or non-covalently attached to the one or more multimerization domains. In another embodiment of the present disclosure, the one or more labels may be covalently and/or non-covalently attached to the multimerization domain via a molecule, wherein the molecule e.g., may be selected from the group consisting of an antibody, an aptamer, a protein, a sugar residue, and a nucleotide such as DNA. In a specific embodiment the one or more labels are attached to the MHC class l/peptide multimer via a streptavidinbiotin linkage. In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label may be identical.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label may be different.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each oligonucleotide label may be identical.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each oligonucleotide label may be different.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label and each oligonucleotide label may be identical.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label and each oligonucleotide label may be different.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label may be identical and each oligonucleotide label may be different.

In embodiments of the present disclosure, wherein multimers comprising at least two MHC class l/peptide monomers comprise both at least a fluorescent label and at least an oligonucleotide label, each fluorescent label may be different and each oligonucleotide label may be identical. The term “Label” is used interchangeable with labeling molecule. Label as described herein is an identifiable substance that is detectable in an assay and that can be attached to a molecule creating a labeled molecule. The behavior of the labeled molecule can then be studied. Labels may be organic or inorganic molecules or particles. Examples of labels include, but are not limited to, polymers, nucleic acids, DNA, RNA, oligonucleotides, peptides, fluorescent labels, phosphorescent labels, enzyme labels, chemiluminescent labels, bioluminescent labels, haptens, antibodies, dyes, nanoparticle labels, elements, metal particles, heavy metal labels, isotope labels, radioisotopes, stable isotopes, chains of isotopes and single atoms, or combination thereof. The labelling compound may suitably be selected from fluorescent labels such as 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)- carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeston Red, Green fluorescent protein (GFP) and analogues thereof, and conjugates of R-phycoerythrin or allophycoerythrin and e.g. Cy5 or Texas Red, and inorganic fluorescent labels based on semiconductor nanocrystals (like quantum dot and Qdot™ nanocrystals), and time-resolved fluorescent labels based on lanthanides like Eu³⁺ and Sm³⁺.

In one embodiment, an MHC class I molecule, MHC class l/peptide monomer and/or MHC class I multimer according to the present disclosure comprises at least one oligonucleotide label, such as a nucleotide label, for example an oligonucleotide label. Such nucleic acids labels are disclosed in WO 2015/188839 and WO 2015/185067 (which are hereby incorporated by reference).

In a particular embodiment the label is an oligonucleotide, such as a nucleic acid molecule comprising or consisting of DNA, RNA, and/or artificial nucleotides such as PLA or LNA. In one embodiment the oligonucleotide label comprises one or more of the following components: a 5’ first primer region (forward), a barcode region, 3’ second primer region (reverse), random nucleotide region, connector molecule, stability-increasing components, short nucleotide linkers in between any of the above- mentioned components, adaptors for sequencing and annealing region. Preferably the oligonucleotide label comprises at least a barcode region surrounded by primer regions, where the barcode region comprises a sequence of consecutive nucleic acids. In one embodiment the oligonucleotide label comprises or consists of DNA, RNA, artificial nucleic acids and/or Xeno nucleic acid (XNA).

In one embodiment at least two different labels are attached to an MHC class I molecule, an MHC class l/peptide monomer, and/or an MHC class l/peptide multimer according to the present disclosure, such as one or more fluorescent labels and an oligonucleotide label.

In some embodiments, the MHC class l/peptide multimer according to the preset disclosure comprises one or more fluorescent labels. In some embodiments, the MHC class l/peptide multimer according to the preset disclosure comprises one or more fluorescent labels selected from the group of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, 2-4'- maleimidylanilino)naphthalene-6- sulfonic acid sodium salt, 5-((((2- iodoacetyl)amino)ethyl)amino), naphthalene-1 -sulfonic acid, Pyrene-1 -butanoic acid, AlexaFluor 350 (7-amino-6-sulfonic acid-4-methyl coumarin-3-acetic acid, AMCA (7- amino-4-methyl coumarin-3-acetic acid), 7-hydroxy-4-methyl coumarin-3-acetic acid, Marina Blue (6,8-difluoro-7-hydroxy-4-methyl coumarin-3-acetic acid), 7- dimethylamino-coumarin-4-acetic acid, Fluorescamin-N-butyl amine adduct, 7-hydroxy- coumarine-3-carboxylic acid, CascadeBlue (pyrene-trisulphonic acid acetyl azide), Cascade Yellow, Pacific Blue (6,8 difluoro-7-hydroxy coumarin-3-carboxylic acid), 7- diethylamino-coumarin-3-carboxylic acid, N-(((4-azidobenzoyl)amino)ethyl)- 4-amino- 3 ,6-disu Ifo- 1 ,8-naphthalimide, dipotassium salt), Alexa Fluor 430, 3- perylenedodecanoic acid, 8-hydroxypyrene-1 ,3,6-trisulfonic acid trisodium salt, 12-(N- (7-nitrobenz-2-oxa-1 ,3- diazol-4-yl)amino)dodecanoic acid, N,N'-dimethyl-N- (iodoacetyl)-N'-(7-nitrobenz-2- oxa-1 ,3-diazol-4-yl)ethylenediamine, Oregon Green 488 (difluoro carboxy fluorescein), 5-iodoacetamidofluorescein, propidium iodide-DNA adduct, Carboxy fluorescein, fluor dyes, Pacific Blue™, Pacific Orange™, Cascade Yellow™, AlexaFluor®(AF), AF350, AF405, AF430, AF488,AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800, Quantum Dot based dyes, QDot® Nanocrystals (Invitrogen, MolecularProbs), Qdot®525, Qdot®565, Qdot®585, Qdot®605, Qdot®655, Qdot®705, Qdot®800, DyLight™ Dyes (Pierce) (DL); DL549, DL649, DL680, DL800, Fluorescein (Flu) or any derivate of that, such as FITC, Cy-Dyes, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, 7-AAD, TO-Pro-3, fluorescent Proteins, R-Phycoerythrin (RPE), Phycobili Proteins, Allophycocyani (APC), PerCp, B-Phycoerythrin, C-Phycocyanin, APC, fluorescent proteins, Green fluorescent proteins; GFP and GFP derivated mutant proteins; BFP,CFP, YFP, DsRed, DSred-2, T1 , Dimer2, mRFP1 ,MBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, Tandem dyes, RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-AlexaFluor® tandem conjugates; RPE-Alexa610, RPE- TxRed, Tandem dyes with APC, APC-Aleca600, APC-Alexa610, APC-Alexa750, APC- Cy5, APC-Cy5.5, multi fluorochrome assemblies, FRET-based dyes (Fluorescence resonance energy transfer), ionophors; ion chelating fluorescent props, props that change wavelength when binding a specific ion, such as Calcium, props that change intensity when binding to a specific ion, such as Calcium, Calcium dyes, lndo-1 -Ca²⁺, lndo-2-Ca²⁺.

The one or more labels are in specific embodiments selected from the group consisting of APC, APC-Cy7, ABC-H7, APC-R700, Alexa Flours™ 488, Alexa Flours™555, Alexa Flours™647, Alexa Flours™700, AmCyan, BB151 , BB700, BUV395, BUV496, BUV563, BUV615, BUV661 , BUV737, BUV805, BV421 , BV480, BV510, BV605, BV711 , BV750, BV786, FITC, PE, PE-CF594, PE-Cy5, PE-CY5.5, PE-cy7, Pasific Blue, PERCP, pPerCp-Cy5.5, PE, R718, RY586, V450 and V500 (wherein in BV means Brilliant violet, wherein BLIV means Brilliant ultra violet and PE means R- Phycoerythrin). In another embodiment the one or more labels can be selected from the group consisting of cFluor®B515, cFluor®B532, cFluor®B548, cFluor®B675, cFluor®B690, cFluor®BY575, cFluor®BY610, cFluor®BY667, cFluor®BY710, cFluor®BY750, cFluor®BY781 , cFluor®B250, cFluor®R659, cFluor®R668, cFluor®R685, cFluor®R720, cFluor®R780, cFluor®R840, cFluor®v420, cFluor®v547, cFluor®v450, cFluor®v610 and cFluor®YG610.

In one embodiment, the MHC class l/peptide multimer of the present disclosure comprises one or more chemiluminescent labels, such as one or more labels selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In one embodiment the MHC class l/peptide multimer of the present disclosure comprises one or more bioluminescent labels, such as one or more labels selected from the group consisting of luciferin, luciferase and aequorin.

In one embodiment the MHC class l/peptide multimer of the present disclosure comprises one or more enzyme labels, such as one or more enzyme labels selected from the group peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

In one embodiment the MHC class l/peptide multimer of the present disclosure comprises one or more chromophore labels.

In one embodiment the MHC class l/peptide multimer of the present disclosure comprises one or more metal labels.

In one embodiment the MHC class l/peptide multimer of the present disclosure comprises one or more radioactive labels such as one or more labels selected from the group consisting of a radionuclide, an isotope, a label comprising a rays, a label comprising rays or a label comprising y rays.

Any of the above embodiments regarding labels can be combined in any order.

Nucleic acids and vectors

In some aspects, the present disclosure provides a nucleic acid encoding the MHC class l/peptide monomer and/or the MHC class l/peptide multimer as disclosed elsewhere herein.

In some embodiments is provided a nucleic acid encoding the MHC class l/peptide monomer as disclosed elsewhere herein. In some embodiments is provided a nucleic acid encoding the MHC class l/peptide multimer as disclosed elsewhere herein. In some embodiments, said nucleic acid comprises or consists of DNA. In some embodiments, said nucleic acid comprises or consists of RNA.

In some aspects of the present disclosure is provided a vector comprising the nucleic acid as disclosed elsewhere herein. In some embodiments, the vector may comprise a promoter operably linked to said nucleic acid.

In some aspects of the present disclosure is provided a composition comprising the pool of peptides as disclosed elsewhere herein and/or an MHC class l/peptide multimer as disclosed elsewhere herein.

In some aspects, is provided a composition comprising the nucleic acid as disclosed elsewhere herein and/or the vector as disclosed elsewhere herein.

In some embodiments of the present disclosure is provided a composition comprising the pool of peptides as disclosed elsewhere herein. In one embodiment the composition comprises different peptides such as at least least 10, such as at least 25, such as at least 50, such as at least 75, such as at least 100, such as at least 150, such as at least 200, such as at least 300, such as at least 500, such as at least 1000, such as at least 2500, such as at least 5000, such as at least 10.000, such as at least 25.000, such as at least 50.000, such as at least 75.000, such as at least 100.000, such as at least 250.000, such as at least 750.000, such as at least 1 .000.000, such as at least 5.000.000, or such as at least 10.000.000 different peptides.

In some embodiments of the present disclosure is provided a composition comprising the MHC class l/peptide multimer as disclosed elsewhere herein.

In some embodiments of the present disclosure is provided a composition comprising the nucleic acid as disclosed elsewhere herein. In some embodiments of the present disclosure is provided a composition comprising the vector as disclosed elsewhere herein.

In some embodiments of the present disclosure, the composition comprises at least 1 , such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 5000, 10.000, 50.000, 100.000 or 500.000 MHC class l/peptide multimers according to the present disclosure.

In some embodiments of the present disclosure, the composition comprises at least 2, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 5000, 10.000, 50.000, 100.000 or 500.000 different MHC class l/peptide multimers according to the present disclosure, such as wherein each of said MHC class l/peptide multimers are different to each other.

In some embodiments of the present disclosure, the composition further comprises one or more pH-stabilizing (buffer) components, such as one or more pH-stabilizing (buffer) components selected from the group consisting of Tris, MES, MOPS, phosphate, carbonate, Bis-tris and HEPES.

In some embodiments of the present disclosure, the composition further comprises one or more salts, such as one or more salts selected from the group consisting of NaCI, CaCh, and L-arginine.

In some embodiments of the present disclosure, the composition further comprises one or more stabilizers, such as one or more stabilizers selected from the group consisting of glycerol, PEG and BSA.

In specific embodiments of the present disclosure, the composition comprises one or more pH-stabilizing (buffer) components, one or more salts and one or more stabilizers.

Methods of production

In some embodiments of the present disclosure is provided a method for producing a pool of peptides as disclosed elsewhere herein, said method comprising the steps of: a) identifying the binding motifs of one or more MHC class I molecules; b) producing and formulating a set of peptides, said set comprising a plurality of different peptides, each peptide comprising or consisting of an amino acid sequence comprising the binding motif of said one or more MHC class I molecules, thereby producing said pool of peptides.

In some aspects of the present disclosure is provided a method for producing a pool of peptides as disclosed elsewhere herein, said method comprising the steps of: a) identifying the binding motifs of one or more MHC class I supertypes; b) producing and formulating a set of peptides, said set comprising a plurality of different peptides, each peptide comprising or consisting of an amino acid sequence comprising the binding motif of at least one of said one or more MHC class I supertypes, thereby producing said pool of peptides.

Without being bound by theory, multimers as disclosed elsewhere herein except not comprising already complexed peptides, may spontaneously bind and complex free peptides that come into contact with the MHC class I molecules of the multimer.

The MHC class l/peptide multimer of the present disclosure may thus be produced by mixing peptides of a pool beforehand and letting these bind randomly to MHC multimers not comprising already complexed peptides. The MHC class l/peptide multimer of the present disclosure may also be produced by first producing MHC class l/peptide monomers, and then letting these bind randomly to the multimer backbone as disclosed elsewhere herein.

The MHC class l/peptide multimer may also be produced from specific, selected MHC class l/peptide monomers that are multimerized by binding to the backbone as disclosed elsewhere herein. In some aspects of the present disclosure is thus provided a method for producing the MHC class l/peptide multimer as disclosed elsewhere herein, said method comprising the steps of: a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class l/peptide multimer, optionally wherein said multimer is defined as disclosed elsewhere herein except that said multimer does not comprise said complexed peptides, whereby a plurality of peptides of said pool of peptides is complexed with said multimer, thereby producing said MHC class l/peptide multimer, or a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class I molecule, optionally wherein said monomer is defined as disclosed elsewhere herein except that said monomer does not comprise said complexed peptide, whereby a peptide of said pool of peptides is complexed with said monomer; and b) multimerising a plurality of said monomers to produce said MHC class l/peptide multimer, wherein at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as each of said monomers of said plurality of monomers are different, such as are different to each other.

In some embodiments, the present disclosure provides a method for producing the MHC class l/peptide multimer as disclosed elsewhere herein, said method comprising the step of: a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class l/peptide multimer, whereby a plurality of peptides of said pool of peptides is complexed with said multimer, thereby producing said MHC class l/peptide multimer.

In some embodiments, the present disclosure provides a method for producing the MHC class l/peptide multimer as disclosed elsewhere herein, said method comprising the step of: a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class l/peptide multimer, wherein said multimer is defined as disclosed elsewhere herein except that said multimer does not comprise said complexed peptides, whereby a plurality of peptides of said pool of peptides is complexed with said multimer, thereby producing said MHC class l/peptide multimer.

In some embodiments, the present disclosure provides a method for producing the MHC class l/peptide multimer as disclosed elsewhere herein, said method comprising the steps of: a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class I molecule, whereby a peptide of said pool of peptides is complexed with said monomer; and b) multimerising a plurality of said monomers to produce said MHC class l/peptide multimer, wherein at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as each of said monomers of said plurality of monomers are different, such as different to each other.

Said multimerization may be performed by any useful methods known to the skilled person. For example, said plurality of monomers may be contacted with a backbone as disclosed elsewhere herein, whereby the MHC class l/peptide multimers as disclosed herein are produced.

In some embodiments, the present disclosure provides a method for producing the MHC class l/peptide multimer as disclosed elsewhere herein, said method comprising the steps of: a) contacting a pool of peptides as disclosed elsewhere herein with an MHC class I molecule, wherein said monomer is defined as disclosed elsewhere herein except that said monomer does not comprise said complexed peptide, whereby a peptide of said pool of peptides is complexed with said monomer; and b) multimerising a plurality of said monomers to produce said MHC class l/peptide multimer, wherein at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as each of said monomers of said plurality of monomers are different, such as different to each other. In some embodiments, the pool of MHC monomers is obtained using loadable MHC technology, where there MHC monomers are formulated in a peptide-receptive state allowing binding of an exogenously added peptide. Examples of loadable MHC technology include photo-cleavable peptides, chaperone-stabilized, empty MHC monomers, and monomers stabilized through weakly binding dipeptides or other stabilizing agents. All of these implementations of loadable MHC technology allow efficient loading of a peptide into an MHC monomer and represent an alternative to classical refolding of MHC monomers with peptide.

Methods of use

The presently provided negative control reagents are able to effectively identify immune cells that interact with MHC class l/peptide molecules and/or multimers in a peptide-unspecific manner.

The presently disclosed reagents may be tailored to specific MHC class I molecules or supertypes. In this way, the negative controls may be tailored to specific patient genetic backgrounds. For example, an HLA allele-matched negative control may be tailored to a specific HLA allele known to be expressed in a patient, thereby avoiding non-specific binding of patient immune cells to otherwise more standardized negative controls, which may be directed to alleles not present in the patient, for which the patient’s immune cells have not been selected against.

The MHC class l/peptide multimers of the present disclosure are thus useful as negative controls in all types of assays that measure peptide-specific binding of MHC class l/peptide molecules, such as MHC class l/peptide multimers by immune cells. In some embodiments, said assay is a cellular assay. In some embodiments, said assay is a biochemical assay. In some embodiments, said assay is an in vitro assay, such as a cellular in vitro assay or a biochemical in vitro assay.

In some aspects of the present disclosure is provided a method for measuring the unspecific background binding of one or more peptide-unspecific immune cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more MHC class l/peptide multimers as disclosed elsewhere herein or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) measuring the binding of said immune cells to said MHC class l/peptide multimers, thereby measuring said unspecific background binding.

In some embodiments of the present disclosure is provided a method for measuring the unspecific background binding of one or more peptide-unspecific T cells, said method comprising the steps of: a) providing a sample comprising a population of T cells; b) providing one or more MHC class l/peptide multimers as disclosed elsewhere herein or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) measuring the binding of said T cells to said MHC class l/peptide multimers, thereby measuring said unspecific background binding.

In some aspects of the present disclosure is provided a method for isolation of one or more peptide-unspecific immune cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells; b) providing one or more MHC class l/peptide multimers as disclosed elsewhere herein or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) isolating said immune cells specific for said MHC class l/peptide multimers.

In some aspects of the present disclosure is provided a method for isolation of one or more peptide-unspecific T cells, said method comprising the steps of: a) providing a sample comprising a population of T cells; b) providing one or more MHC class l/peptide multimers as disclosed elsewhere herein or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) isolating said T cells specific for said MHC class l/peptide multimers.

In some embodiments, the disclosure relates to reagents and tools for the depletion or detection of cells, such as immune cells, exhibiting peptide-unspecific binding to MHC class I molecules. These reagents and tools for depletion can consist of or comprise multimer or monomer MHC reagents comprising at least two different peptides according to the present disclosure, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 ,17 , 18, 19, 20 or more different peptides according to the present disclosure. In some embodiments, the reagents and tools for depletion consist of or comprise MHC class l/peptide multimers and/or monomers as described elsewhere herein.

In some embodiments, the immune cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, MAIT cells, gamma-delta T cells, NK cells, iNKT cells, B cells, innate lymphoid cells, granulocytes, macrophages, and dendritic cells.

In some embodiments, the immune cells are CD4+ T cells. In some embodiments, the immune cells are CD8+ T cells. In some embodiments, the immune cells are MAIT cells. In some embodiments, the immune cells are gamma-delta T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are iNKT cells (invariant natural killer T cell). In some embodiments, the immune cells are B cells. In some embodiments, the immune cells are MAIT cells (mucosal- associated invariant T cells). In some embodiments, the immune cells are innate lymphoid cells. In some embodiments, the immune cells are granulocytes. In some embodiments, the immune cells are macrophages. In some embodiments, the immune cells are dendritic cells.

Items

The invention may further be described by one or more of the following items. 1 . A pool of MHC class I molecule-matched negative control peptides, wherein said pool comprises or consists of a plurality of peptides, wherein each peptide of said plurality of peptides comprises or consists of an amino acid sequence comprising the binding motif of said MHC class I molecule, and wherein said pool comprises at least 5 different peptides.

2. The pool of peptides according to item 1 , wherein said pool comprises at least

10, such as at least 25, such as at least 50, such as at least 75, such as at least 100, such as at least 150, such as at least 200, such as at least 300, such as at least 500, such as at least 1000, such as at least 2500, such as at least 5000, such as at least 10.000, such as at least 25.000, such as at least 50.000, such as at least 75.000, such as at least 100.000, such as at least 250.000, such as at least 750.000, such as at least 1 .000.000, such as at least 5.000.000, or such as at least 10.000.000 different peptides.

3. The pool of peptides according to any one of the preceding items, wherein the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

4. The pool of peptides according to any one of the preceding items, wherein said MHC class I molecule is a member of an HLA supertype selected from the group consisting of A01 , A02, A03, A24, A26, B07, B08, B27, B39, B44, B58, and B62 or optionally selected from the group consisting of A01 , A02, A03, A24, A26, B07, B08, B27, B39, B44, B58, B62, C01 , C02, C04, C07, C08, C14 and C15

5. The pool of peptides according to any one of the preceding items, wherein each of said plurality of peptides comprises or consists of the sequence Xi-X₂-X₃-X₄- X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n i. a) X₂ is an amino acid at the second position of said binding motif of said MHC class I molecule; b) X13 is defined as an amino acid at the last position of said binding motif of said MHC class I molecule; c) Xi and X₃-X₇ are each independently any amino acid, such as any standard amino acid; and d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; or ii. a) X₅ is an amino acid at the fifth position of said binding motif of said MHC class I molecule; b) X13 is defined as the amino acid at the last position of said binding motif of said MHC class I molecule; c) X1-X4 and X₆-X₇ are each independently any amino acid, such as any standard amino acid; and d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent. The pool of peptides according to any one of the preceding items, wherein said MHC class I molecule is an HLA supertype A01 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence Xi-X2-X3-X4-X5-X6-X₇-X₈-X₉-Xio-Xii-Xi2-Xi3, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is T; c) X3 is D, or optionally wherein X₈is D or E; d) X₄-X₇ are each independently any amino acid, such as any standard amino acid; e) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and f) X13 is Y. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype A02 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X₃-X4-X5-X₆-X₇-X₈-X9-Xi 0-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X2 is M or L; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) Xi₃ is L or V.

8. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype A03 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X2 is T, or optionally wherein X2 is T or L; c) X₃ is F; d) X4-X7 are each independently any amino acid, such as any standard amino acid; e) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and f) X13 is K or optionally wherein X13 is K or R.

9. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype A24 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is Y or F; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is F or L.

10. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype A26 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X₃-X4-X5-X₆-X7-X₈-X9-Xi 0-X11 -Xi 2-X1₃, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is T or V; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is Y or M. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B07 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is P; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) Xi₃ is L or F. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B08 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X₃-X4-X5-X₆-X7-X₈-X9-Xi 0-X11 -Xi 2-X13, wh erei n a) X1-X4 is any amino acid, such as any standard amino acid; b) X₅ is R; c) X₆-X₇ are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is L. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B27 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is R; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is L. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B39 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is H, R, or Q; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is L. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B44 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is D or E; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is L, F, or Y or optionally wherein X13 is I, L, F, Y or W. 16. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B58 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is S or A; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is W.

17. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype B62 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n a) Xi is any amino acid, such as any standard amino acid; b) X₂ is M or Q; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is F.

18. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is HLA-A*0101 or HLA-A*0301 or optionally any of HLA- A*02:01 , HLA-A*24:02, HLA-A*26:01 , HLA-B*07:02, HLA-B*08:01 , HLA- B*27:01 , HLA-B*39:01 , HLA-B*44:02, HLA-B*58:01 or HLA-B*15:01.

19. The pool of peptides according to any one of items 1 to 2, wherein the MHC class I molecule is a murine H2 allele, such as a H2 allele selected from the group consisting of Db, Dd, Kb, Kd, Kk and Ld.

20. The pool of peptides according to any one of items 1 to 2 and 19, wherein said

MHC class I molecule is a murine H2-Db class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5- X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is S; c) X3-X4 are each independently any amino acid, such as any standard amino acid; d) X₅ is N; e) X₆-X₇ are each independently any amino acid, such as any standard amino acid; f) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and g) X13 is L or I. The pool of peptides according to any one of items 1 to 2 and 19, wherein said MHC class I molecule is a murine H2-Dd class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5- X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is S; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) Xi₃ is L or F. The pool of peptides according to any one of items 1 to 2 and 19, wherein said MHC class I molecule is a murine H2-Kb class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5- X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is S; c) X₃ is Y or F; d) X4-X7 are each independently any amino acid, such as any standard amino acid; e) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and f) X13 is L or M.

23. The pool of peptides according to any one of items 1 to 2 and 19, wherein said MHC class I molecule is a murine H2-Kd class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5- X6-X7-X8-X9-X10-X11-X12-X13, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is Y; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is I or L.

24. The pool of peptides according to any one of items 1 to 2 and 19, wherein said MHC class I molecule is a murine H2-Kk class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5- X₈-X7-X₈-Xg-Xio-Xi 1-X12-X13, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is E; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) X13 is I.

25. The pool of peptides according to any one of items 1 to 2 and 19, wherein said MHC class I molecule is a murine H2-Ld class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5- X₈-X7-X₈-Xg-Xio-Xi 1-X12-X13, wherein a) Xi is any amino acid, such as any standard amino acid; b) X₂ is P; c) X3-X7 are each independently any amino acid, such as any standard amino acid; d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; and e) Xi₃ is L or F. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C01 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n g) Xi is any amino acid; h) X2 is A or L; i) X₃ is P; j) X4-X7 are each independently any amino acid; k) X₈-Xi2 are each independently any amino acid or absent; and l) X13 is L or I. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C02 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n f) Xi is any amino acid; g) X₂ is A; h) X3-X7 are each independently any amino acid; i) X₈-Xi2 are each independently any amino acid or absent; and j) X13 is F, L, M or Y. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C04 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X₃-X4-X5-X₆-X7-X₈-X9-Xi 0-X11 -Xi 2-X1₃, wh erei n g) Xi is any amino acid; h) X₂ is F, W or Y; i) X₃ is D; j) X4-X7 are each independently any amino acid; k) X₈-Xi2 are each independently any amino acid or absent; and l) X13 is F, I, L or M. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C07 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n f) Xi is any amino acid; g) X₂ is R or Y; h) X3-X7 are each independently any amino acid; i) X₈-Xi2 are each independently any amino acid or absent; and j) X13 is F, I, L, V, M or Y. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C08 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n g) Xi is any amino acid; h) X₂ is A or S; i) X₃ is D; j) X4-X7 are each independently any amino acid; k) X₈-Xi2 are each independently any amino acid or absent; and l) X13 is F, I, L or M. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C14 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X₃-X4-X5-X₆-X7-X₈-X9-Xi 0-X11 -Xi 2-X13, wh erei n f) Xi is any amino acid; g) X₂ is Y or F; h) X3-X7 are each independently any amino acid; i) X₈-Xi2 are each independently any amino acid or absent; and j) X13 is F, L, M or Y. The pool of peptides according to any one of items 1 to 5, wherein said MHC class I molecule is an HLA supertype C15 MHC class I molecule, and wherein each of said plurality of peptides comprises or consists of the sequence X1-X2- X3-X4-X5-X6-X7-X8-X9-X10-X11 -Xi 2-X13, wh erei n f) Xi is any amino acid; g) X₂ is A, S or T; h) X3-X7 are each independently any amino acid; i) X₈-Xi2 are each independently any amino acid or absent; and j) X13 is V, L or I. The pool of peptides according to any one of the preceding items, wherein said pool comprises a) a first set of peptides, each of the peptides of said first set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a first MHC class I molecule, optionally wherein said first set of peptides is according to any one of the preceding items; and b) a second set of peptides, each of the peptides of said second set of peptides comprising or consisting of an amino acid sequence comprising the binding motif of a second MHC class I molecule, optionally wherein said second set of peptides is according to any one of the preceding items, wherein said first and second MHC class I molecules are different, such as wherein the supertype of said first and second MHC class I molecules are different. The pool of peptides according to any one of the preceding items, wherein each peptide of said plurality of peptides is from 8 to 13 amino acids in length, such as 8 amino acids, such as 9 amino acids, such as 10 amino acids, such as 11 amino acids, such as 12 amino acids, or such as 13 amino acids in length. The pool of peptides according to any one of the preceding items, wherein said MHC class I molecule is from an organism selected from the group consisting of human, gorilla, chimpanzee, rhesus macaque and rodent, such as mouse or rat. 36. The pool of peptides according to any one of the preceding items, wherein the MHC class I molecule is a human MHC class I molecule.

37. An MHC class l/peptide multimer comprising a plurality of monomers, each monomer comprising a single peptide complexed to an MHC class I molecule, wherein at least two, such as at least 3, such as at least 4, such as at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45 of said peptides of said multimer are different, or such as wherein each of said peptides of said multimer are different.

38. The MHC class l/peptide multimer according to item 37, wherein each of said peptides is selected from a pool of peptides according to any one of the preceding items.

39. The MHC class l/peptide multimer according to any one of items 37 to 38, wherein said monomer exhibits native-like properties in terms of recognition by TCRs and/or other MHC class I complex-engaging receptors.

40. The MHC class l/peptide multimer according to any one of items 37 to 39, wherein said monomer exhibits native-like properties in terms of T-cell recognition, NK cell recognition and/or recognition of other immune cells.

41. The MHC class l/peptide multimer according to any one of items 37 to 40, wherein said peptides are selected from a peptide pool specific for a single MHC class I molecule supertype, such as for an MHC class I molecule, such as for a single HLA allele.

42. The MHC class l/peptide multimer according to any one of items 37 to 40, wherein said peptides are selected from a peptide pool specific for at least two different MHC class I molecule supertypes, such as for at least two different MHC class I molecules, such as for two or more different HLA supertypes or alleles. The MHC class l/peptide multimer according to any one of items 37 to 42, wherein each of said MHC class I molecules are identical. The MHC class l/peptide multimer according to any one of items 37 to 43, wherein the MHC class l/peptide multimer comprises at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45, such as at least 50, such as from 5 to 50 MHC class l/peptide monomers. The MHC class l/peptide multimer according to any one of items 37 to 44, wherein said multimer comprises 5 to 50 of said monomers, such as 5 to 8, such as 8 to 10, such as 10 to 12, such as 12 to 14, such as 14 to 16, such as 16 to 18, such as 18 to 20, such as 20 to 25, such as 25 to 30, such as 30 to 40, such as 40 to 50, or such as 10 to 20 of said monomers, or any combination of these intervals. The MHC class l/peptide multimer according to any one of items 37 to 45, wherein said multimer comprises exactly 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 of said monomers. The MHC class l/peptide multimer according to any one of items 37 to 46, wherein the multimer comprises no more than 50 monomers in total, such as no more than 45 monomers, such as no more than 30 monomers, such as no more than 35 monomers, such as no more than 25 monomers, such as no more than 25 monomers, such as no more than 20 monomers, such as no more than 15 monomers, or no more than 10 monomers in total. The MHC class l/peptide multimer according to any one of items 37 to 47, wherein each of said monomers is associated with one or more multimerization domains, such as a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said MHC class l/peptide monomers. The MHC class l/peptide multimer according to any one of items 37 to 48, wherein said multimer comprises at least one DNA barcode oligonucleotide, said at least one DNA barcode oligonucleotide comprising a primer region, a DNA barcode sequence that specifies the MHC-peptide specificity, and a capture region. The MHC class l/peptide multimer according to any one of items 37 to 49, wherein said monomers are linked to a backbone molecule. The MHC class l/peptide multimer according to item 50, wherein the backbone molecule comprises polysaccharides, including glucans such as dextran. The MHC class l/peptide multimer according to any one of items 50 to 51 , wherein said backbone molecule comprises dextran. The MHC class l/peptide multimer according to item 50, wherein the backbone molecule is a protein, such as a streptavidin, a coiled-coil polypeptide or a streptactin. The MHC class l/peptide multimer according to any one of items 50 to 53, wherein the two or more of said monomers are coupled to the backbone covalently or non-covalently, such as through one or more connector molecules, including a streptavidin-biotin binding and/or avidin-biotin; or and/or via the MHC heavy chain and/or via light chain (B2M). The MHC class l/peptide multimer according to any one of items 50 to 54, wherein the at least one DNA barcode oligonucleotide is coupled to the backbone, covalently or non-covalently, such as through one or more connector molecules, including a streptavidin-biotin binding and/or a avidin-biotin binding. 56. The MHC class l/peptide multimer according to any one of items 37 to 55, wherein said multimer further comprises one or more labels.

57. The MHC class l/peptide multimer according to item 56, wherein said multimer comprises at least two labels.

58. The MHC class l/peptide multimer according to any one of items 56 to 57, wherein at least two of the labels are different, such as wherein all labels are different.

59. The MHC class l/peptide multimer according to any one of items 56 to 57, wherein at least two of the labels are identical, such as wherein all labels are identical.

60. The MHC class l/peptide multimer according to any one of items 56 to 59, wherein one or more labels comprise at least one fluorescent label.

61 . The MHC class l/peptide multimer according to any one of items 56 to 60, wherein one or more labels comprise at least one oligonucleotide label, such as a nucleic acid molecule comprising or consisting of DNA, RNA, and/or artificial nucleotides, such as PLA or LNA.

62. The MHC class l/peptide multimer according to any one of items 56 to 61 , wherein one or more labels comprise at least one fluorescent label and at least one oligonucleotide label.

63. The MHC class l/peptide multimer according to any one of items 56 to 62, wherein one or more labels are oligonucleotides comprising one or more component selected from the group consisting of: a. a barcode region; b. a 5’ first primer region (forward); c. a 3’ second primer region (reverse); d. a random nucleotide region; e. a connector molecule; f. a stability-increasing component; g. a short nucleotide linker in between any of the above-mentioned components; h. an adaptors for sequencing; and i. an annealing region.

64. The MHC class l/peptide multimer according to any one of items 56 to 63, wherein one or more labels comprise at least one fluorescent label selected from the group consisting of PE, 5-(and 6)-carboxyfluorescein, 5- or 6-carboxy- fluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeston Red and Green fluorescent protein (GFP).

65. The MHC class l/peptide multimer according to any one of items 56 to 64, wherein one or more labels are chemiluminescent labels, such as a label selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

66. The MHC class l/peptide multimer according to any one of items 56 to 65, wherein one or more labels are bioluminescent labels, such as a label selected from the group consisting of luciferin, luciferase and aequorin.

67. The MHC class l/peptide multimer according to any one of items 56 to 66, wherein one or more labels are enzyme labels, such as an enzyme label selected from the group consisting of peroxidases, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

68. The MHC class l/peptide multimer according to any one of items 56 to 67, wherein one or more labels are chromophore labels. The MHC class l/peptide multimer according to any one of items 56 to 68, wherein one or more labels are metal labels. The MHC class l/peptide multimer according to any one of items 56 to 69, wherein one or more labels are radioactive labels, such as a label selected from the group consisting of a radionuclide, an isotope, a label comprising a rays, a label comprising p rays or a label comprising y rays. A nucleic acid encoding the MHC class l/peptide monomer and/or the MHC class l/peptide multimer according to any one of items 37 to70. A vector comprising the nucleic acid according to item 71 . A composition comprising the pool of peptides according to any one of items 1 to 36 and/or an MHC class l/peptide multimer according to any one of items 37 to 70. The composition according to item 73, wherein the composition comprises at least two MHC class l/peptide multimers according to any one of items 37 to 70, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or 1000 MHC class l/peptide multimers. The composition according to item 74, wherein the composition comprises different MHC class l/peptide multimers, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500 or 1000 different MHC class l/peptide multimers. A method for producing a pool of peptides according to any one of items 1 to 36, said method comprising the steps of: a) identifying the binding motifs of one or more MHC class I molecules, alleles or supertypes; b) producing and formulating a set of peptides, said set comprising a plurality of different peptides, each peptide comprising or consisting of an amino acid sequence comprising the binding motif of at least one of said one or more MHC class I molecules, alleles or supertypes, thereby producing said pool of peptides. A method for producing the MHC class l/peptide multimer according to any one of items 37 to 70, said method comprising the steps of: a) contacting a pool of peptides according to any one of items 1 to 36 with an MHC class l/peptide multimer, optionally wherein said multimer is defined according to any one of items 37 to 70 except that said multimer does not comprise said complexed peptides, whereby a plurality of peptides of said pool of peptides is complexed with said multimer, thereby producing said MHC class l/peptide multimer, or a) contacting a pool of peptides according to any one of items 1 to 36 with an MHC class I molecule, optionally wherein said MHC class I molecule is defined according to any one of items 37 to 70 except that said MHC class I molecule does not comprise said complexed peptide, whereby a peptide of said pool of peptides is complexed with said MHC class I molecule thereby forming an MHC class l/peptide monomer; and b) multimerizing a plurality of said monomers to produce said MHC class l/peptide multimer, wherein at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as each of said monomers of said plurality of monomers are different. A method for measuring the unspecific background binding of one or more peptide-unspecific immune cells, such as peptide-unspecific T cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells, such as T cells; b) providing one or more MHC class l/peptide multimers according to any one of items 37 to 70 or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) measuring the binding of said immune cells, such as T cells, to said MHC class l/peptide multimers, thereby measuring said unspecific background binding.

79. A method for isolation of one or more peptide-unspecific immune cells, such as peptide-unspecific T cells, said method comprising the steps of: a) providing a sample comprising a population of immune cells, such as T cells; b) providing one or more MHC class l/peptide multimers according to any one of items 37 to 70 or a composition comprising one or more of said MHC class l/peptide multimers; c) contacting said MHC class l/peptide multimer or said composition with said sample; and d) isolating said immune cells, such as T cells, specific for said MHC class l/peptide multimers.

80. The method according to any of items 78 to 79, wherein the immune cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, MAIT cells, gamma-delta T cells, NK cells, iNKT cells, B cells, innate lymphoid cells, granulocytes, macrophages, and dendritic cells.

Examples

Example 1 - staining of human PBMC samples with MHC Dextramer negative control reagents.

This example describes how human PBMC samples were stained with relevant antibodies and one allele-matched, MHC Dextramer negative control reagent prepared with either a single nonsense peptide or a peptide pool and subsequently analysed on a flow cytometer using the protocol outlined below.

1 . PBMC samples were thawed and resuspended at a density of 1 x 10⁶ cells in 50 pL stain and wash buffer (PBS with 5% FCS). 2. An aliquot of 10 pL of MHC Dextramer® reagent was added to the 50 pL cell sample to a final concentration of 5 nM, the mixture was vortexed briefly and then incubated in the dark at room temperature for 30 minutes.

3. Anti-CD3 and -CD8 antibodies were added to each PBMC sample according to manufacturer’s instructions and the samples were incubated at room temperature in the dark for another 20 minutes.

4. PBMC samples were washed twice, each time by adding 2 mL stain and wash buffer, centrifuging at 300 x g for 5 min. and removing the supernatant.

5. Next, PBMC samples were stained for viability using FVS780 dye according to the manufacturer’s instructions and subsequently washed twice as under point 4. above.

6. The resulting cell pellets were resuspended in 130 pL stain and wash buffer and analysed on a flow cytometer.

7. Cells were gated using a lymphocyte gate in an FSC/SSC plot followed by a single-cell gate in an FSA/FSH plot. Single cells were further gated to be CD3+ and viable and finally visualized in a CD8/Dextramer plot.

Negative control MHC Dextramer reagents are generally used to establish the levels of unspecific binding to other MHC Dextramer reagents carrying peptide epitopes under investigation for specific T cell recognition. This way, peptide-specific interactions can be discerned from irrelevant interactions. The levels of peptide-unspecific binding of MHC complexes varies between MHC alleles as well as between individuals.

As can be seen in the plots in Figure 1 , negative control MHC Dextramer reagents carrying either a nonsense peptide or a peptide pool generally behaved similarly and gave little to no background binding of CD8+ T cells. For some combinations of MHC allele and PBMC donor, however, nonsense peptide negative controls exhibited a skewing of the distribution of the background CD8+ cell population indicating weak recognition of the nonsense peptide (donor 179; indicated with dotted circle and arrow), but not the peptide pool. In selected cases, this may negatively impact the detection of weakly binding epitopes under investigation. As can be seen in Table 4, skewing of the negative population distribution also appeared with the peptide pool negative control reagents for HLA-A*01 :01 and HLA-A*03:01 , which indicates weak but general, peptide-independent recognition of these alleles by CD8+ T cells. Thus, negative control MHC Dextramer reagents carrying a pool of peptides are well- behaved as negative control reagents and allow gauging of true peptide-unspecific binding. By design, they eliminate the risk of inducing peptide-specific T cell recognition and are therefore simpler to define for any MHC allele than a corresponding nonsense peptide reagent.

Table 4. FACS analysis of PBMC samples stained with negative control HLA Dextramer reagents loaded with either a single nonsense peptide or a peptide pool. Donors marked with a star carry both HLA-A*02:01 and HLA-B*07:02.

*An additional X may be inserted in the motif to indicate a 9-mer, as also explained above Table 1 , e.g., X[T][D/E]XXXXX[Y],

Sequence overview

References

Lund, O., Nielsen, M., Kesmir, C. etal. Definition of supertypes for HLA molecules using clustering of specificity matrices. Immunogenetics 55, 797-810 (2004). https://doi.Org/10.1007/S00251 -004-0647-4

Sidney, J., Peters, B., Frahm, N. etal. HLA class I supertypes: a revised and updated classification. BMC ImmunoIQ, 1 (2008). https://doi.org/10.1186/1471-2172-9- 1

Claims

1 . A pool of MHC class I molecule-matched negative control peptides, wherein said pool comprises or consists of a plurality of peptides, wherein each peptide of said plurality of peptides comprises or consists of an amino acid sequence comprising the binding motif of said MHC class I molecule, and wherein said pool comprises at least 5 different peptides.

2. The pool of peptides according to claim 1 , wherein said pool comprises at least 10, such as at least 25, such as at least 50, such as at least 75, such as at least 100, such as at least 150, such as at least 200, such as at least 300, such as at least 500, such as at least 1000, such as at least 2500, such as at least 5000, such as at least 10.000, such as at least 25.000, such as at least 50.000, such as at least 75.000, such as at least 100.000, such as at least 250.000, such as at least 750.000, such as at least

1 .000.000, such as at least 5.000.000, or such as at least 10.000.000 different peptides.

3. The pool of peptides according to any one of the preceding claims, wherein the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA- E, HLA-F, and HLA-G.

4. The pool of peptides according to any one of the preceding claims, wherein each of said plurality of peptides comprises or consists of the sequence X1-X2-X3-X4-X5-X6-X7- X8-X9-X10-X11-X12-X13, wherein i. a) X₂ is an amino acid at the second position of said binding motif of said MHC class I molecule; b) X13 is defined as an amino acid at the last position of said binding motif of said MHC class I molecule; c) Xi and X3-X7 are each independently any amino acid, such as any standard amino acid; and d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent; or ii. a) X₅ is an amino acid at the fifth position of said binding motif of said MHC class I molecule; b) X13 is defined as the amino acid at the last position of said binding motif of said MHC class I molecule; c) X1-X4 and X₆-X₇ are each independently any amino acid, such as any standard amino acid; and d) X₈-Xi2 are each independently any amino acid, such as any standard amino acid, or absent.

5. An MHC class l/peptide multimer comprising a plurality of monomers, each monomer comprising a single peptide complexed to an MHC class I molecule, wherein at least two, such as at least 3, such as at least 4, such as at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45 of said peptides of said multimer are different, or such as wherein each of said peptides of said multimer are different, and wherein said peptides are selected from a pool of peptides according to any of the preceding claims.

6. The MHC class l/peptide multimer according to claim 5, wherein each of said monomers is associated with one or more multimerization domains, such as a multimerization domain selected from the group consisting of proteins, peptides, albumins, immunoglobulins, coiled-coil helixes, polynucleotides, IgG, streptavidin, avidin, streptactin, micelles, cells, polymers, polysaccharides, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs capable of binding said MHC class l/peptide monomers.

7. The MHC class l/peptide multimer according to any one of claims 5 to 6, wherein said multimer comprises at least one DNA barcode oligonucleotide, said at least one DNA barcode oligonucleotide comprising a primer region, a DNA barcode sequence that specifies the MHC-peptide specificity, and a capture region.

8. The MHC class l/peptide multimer according to any one of claims 5 to 7, wherein said multimer further comprises one or more labels.

9. The MHC class l/peptide multimer according to any one of claims 5 to 8, wherein one or more labels comprise at least one fluorescent label.

10. The MHC class l/peptide multimer according to any one of claims 5 to 9, wherein one or more labels comprise at least one fluorescent label and at least one oligonucleotide label.

11 . A composition comprising the pool of peptides according to any one of claims 1 to 4 and/or an MHC class l/peptide multimer according to any one of claims 5 to 10.

12. A method for producing a pool of peptides according to any one of claims 1 to 4, said method comprising the steps of: a. identifying the binding motifs of one or more MHC class I molecules, alleles or supertypes; b. producing and formulating a set of peptides, said set comprising a plurality of different peptides, each peptide comprising or consisting of an amino acid sequence comprising the binding motif of at least one of said one or more MHC class I molecules, alleles or supertypes, thereby producing said pool of peptides.

13. A method for producing the MHC class l/peptide multimer according to any one of claims 5 to 10, said method comprising the steps of: a. contacting a pool of peptides according to any one of claims 1 to 4 with an MHC class l/peptide multimer, optionally wherein said multimer is defined according to any one of claims 5 to 10 except that said multimer does not comprise said complexed peptides, whereby a plurality of peptides of said pool of peptides is complexed with said multimer, thereby producing said MHC class l/peptide multimer, or a. contacting a pool of peptides according to any one of claims 1 to 4 with an MHC class I molecule, optionally wherein said MHC class I molecule is defined according to any one of claims 5 to 10 except that said MHC class I molecule does not comprise said complexed peptide, whereby a peptide of said pool of peptides is complexed with said MHC class I molecule thereby forming an MHC class l/peptide monomer; and b. multimerizing a plurality of said monomers to produce said MHC class l/peptide multimer, wherein at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as each of said monomers of said plurality of monomers are different.

14. A method for measuring the unspecific background binding of one or more peptide-unspecific immune cells, such as peptide-unspecific T cells, said method comprising the steps of: a. providing a sample comprising a population of immune cells, such as T cells; b. providing one or more MHC class l/peptide multimers according to any one of claims 5 to 10 or a composition comprising one or more of said MHC class l/peptide multimers; c. contacting said MHC class l/peptide multimer or said composition with said sample; and d. measuring the binding of said immune cells, such as T cells, to said MHC class l/peptide multimers, thereby measuring said unspecific background binding.

15. A method for isolation of one or more peptide-unspecific immune cells, such as peptide-unspecific T cells, said method comprising the steps of: a. providing a sample comprising a population of immune cells, such as T cells; b. providing one or more MHC class l/peptide multimers according to any one of claims 5 to 10 or a composition comprising one or more of said MHC class l/peptide multimers; c. contacting said MHC class l/peptide multimer or said composition with said sample; and d. isolating said immune cells, such as T cells, specific for said MHC class l/peptide multimers.