Mammalian MHC peptide display as an epitope selection tool for vaccine design

Abstract

The present invention relates to methods for identifying candidate peptides presented by the major histocompatibility complex (MHC) for vaccination, induction of immune tolerance, TCR blockade, MHC-mediated toxin delivery, and redirection of T cells using CARs for immunogenicity testing and other in vitro T cell reactivity testing. The present invention further relates to methods for determining the MHC binding affinity of candidate peptides. The present invention provides a method for identifying candidate peptides presented by the major histocompatibility complex (MHC), comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting reporter cells exhibiting surface expression of the MHC-peptide complex, and determining the sequence of the candidate peptide displayed on the cell surface.

Description

[Technical field]

[0001] The present invention relates to methods for identifying candidate peptides presented by the major histocompatibility complex (MHC) for in vivo and / or in vitro interventions including vaccination, induction of immune tolerance, TCR blockade and MHC-mediated toxin delivery for immunogenicity testing and other in vitro T cell reactivity studies. [Background technology]

[0002] T cells play a central role in the immune system. The function of T cells depends heavily on the recognition of epitopes contained in peptides presented by antigen-presenting cells (APCs). APCs can be almost any nucleated cell type, but professional APCs such as dendritic cells (DCs) are particularly efficient in presentation. There are different sets of T cells with different functions, in particular, CD4+ T cells or CD8+ T cells, which recognize MHCII-binding epitopes presented by professional APCs or MHC1-binding epitopes presented by most cell types, respectively. MHC molecules play a key role in presenting cellular antigens or epitopes in the form of short linear peptides to T cells.

[0003] MHC consists of alpha and beta chains and a peptide bound to a groove formed by these chains. The stability of this complex is highly dependent on peptide binding, so that empty MHC molecules are downregulated and degraded from the cell surface. Nevertheless, empty MHC molecules are present on the surface of cells and can bind extracellular peptides and present them to T cells. Furthermore, two different isomers of empty MHC have been described (Rabunowitz et al., Immunity, 9, 699-709 (1998)): an active isomer with lower stability and an inactive isomer with higher stability. An individual's MHC repertoire is polygenic, and MHC genes are highly polymorphic. Thus, the set of MHC that an individual can express is likely to be very different from that of another random individual.

[0004] MHC-bearing peptides interact with the T cell receptor (TCR) present on the surface of T cells, which leads to T cell activation. APCs, such as dendritic cells or macrophages, can internalize antigens by phagocytosis or by receptor-mediated endocytosis.

[0005] To control the activity of T cells, it is important to determine their specific peptide ligands and to control or modulate the interaction of MHC-peptide complexes with TCR. It is therefore important to determine peptide binding affinity to MHC. Appropriate peptides or MHC-peptide complexes can be used for various applications. They can be used, for example, to identify or enrich reactive T cells or for in vitro immunogenicity testing. They can also be used to induce immune tolerance to an epitope that would otherwise activate T cells specific for this epitope, to block TCR, to deliver toxins to T cells in vivo, or to redirect T cells using MHC-containing chimeric antigen receptors MHC-CARs (Jyothi et al., Nat. Biotech, 20, 1215-1220 (2002)). The development of vaccines, especially tumor-specific antigen (TSA)-based cancer vaccines to activate T cells for the elimination of cancer cells, is a major goal of modern medicine. Tumor-specific peptides, so-called neo-antigens, are present only in tumor cells and are completely absent in normal tissues. Such tumor-specific peptides are created by tumor-specific DNA changes that lead to the formation of novel protein sequences. Due to the genetic heterogeneity of cells within and between different tumors, the set of neo-antigens present in a patient's tumor is primarily specific to this patient, even if they originate from the same organ. Tumor-specific peptides are displayed on the surface of tumor cells and so-called antigen-presenting cells (APCs) in the context of MHC. When tumor-specific peptides are presented on the cell surface, they can be recognized and destroyed by the immune system. Thus, vaccines containing tumor-specific peptides can stimulate the immune system to detect and destroy cancer cells that present these molecules on their surface.

[0006] Recently, it has been shown that TSAs resulting from somatic mutations accumulating in tumors are frequently recognized by tumor-infiltrating lymphocytes (TILs) (Linnemann et al., Nat Med 1-7 (2014)). Exome sequencing of tumor DNA and comparison with DNA from healthy tissues allows the discovery of such somatic mutations specific to the patient's tumor cells. However, although many mutations are usually detected (Vormehr et al., Curr Opin Immunol 39,14-22 (2016)), only a few TSAs (i.e., tumor-specific peptides) should be mixed in one vaccine, not only because of technical issues but also to minimize the risk of autoimmunity. The selection of these TSAs is important, since antigen-presenting cells (APCs) do not present all possible peptides on their MHC molecules, but a subset that matches certain MHC alleles specific to the patient. The selection of peptides, especially tumor-specific peptides, that are efficiently presented by APCs is important for many applications involving T cells and remains a bottleneck in tumor vaccine design. To solve this problem, candidate peptides are usually ranked according to their MHC binding affinity predicted using bioinformatics analysis (Sahin et al., Nature 1-19 (2017); Ott et al., Nature 547, 217-221 (2017)). However, recent data show that bioinformatics prediction of peptide presentation performs extremely poorly, especially for MHC class II epitopes (Sofron et al., Eur J Immunol 46, 319-328 (2015)). A "wet lab" method has been disclosed that uses an antigen-presenting reporter cell line to measure binding of T cells (through their TCR) to MHC-peptide complexes; WO2016097334. The reporter cell line expresses a chimeric antigen receptor that contains an MHC-peptide complex that activates a reporter gene in the reporter cell line upon binding of the T cell to the TCR.Indirectly, the method also measures the stability of MHC-peptide complexes, since only sufficiently stable MHC-peptide complexes appear on the surface of the reporter cell line, interact with T cells, and ultimately activate the reporter. However, the method does not allow to specifically measure the stability of MHC-peptide complexes, since the activation of the reporter gene also depends on the affinity of the TCR to the peptide epitope and other immune regulators. There is therefore a need to provide means and methods for reliably determining which peptides, particularly TSAs, are efficiently presented by APCs. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jyothi et al., Nat. Biotech, 20, 1215-1220 (2002) [Non-Patent Document 2] Linnemann et al., Nat Med 1-7 (2014) [Non-Patent Document 3] Vormehr et al., Curr Opin Immunol 39,14-22(2016) [Non-Patent Document 4] Sahin et al., Nature 1-19 (2017) [Non-Patent Document 5] Ott et al., Nature 547, 217-221 (2017) [Non-Patent Document 6] Sofron et al., Eur J Immunol 46,319-328(2015) Summary of the Invention [Means for solving the problem]

[0008] The present invention now meets this need, in that it provides such means and methods, as more particularly defined in the claims and embodiments of the invention that follow.

[0009] In its main aspects, the present invention relates to:

[0010] 1. A method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), comprising expressing a recombinant MHC-peptide complex comprising a covalently linked candidate peptide in a reporter cell line, detecting reporter cells exhibiting surface expression of the MHC-peptide complex, and determining the sequence of the candidate peptide displayed on the cell surface.

[0011] 2. (i) generating a library containing candidate peptides cloned in-frame upstream of recombinant MHC beta and / or alpha chains; (ii) transducing such libraries together with the corresponding MHC alpha and / or beta chains into a suitable reporter cell line; (iii) detecting and isolating cells that express one or more MHC-peptide complexes on the cell surface; (iv) isolating DNA from cells that display one or more MHC-peptide complexes on their cell surface; (v) determining the sequence of candidate peptides encoded by vectors incorporating said DNA isolated from cells that display MHC-peptide complexes on their cell surface. 2. The method of embodiment 1, comprising:

[0012] 3. A method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), comprising expressing a recombinant MHC-peptide complex comprising a covalently linked candidate peptide in a reporter cell line, detecting reporter cells exhibiting surface expression of the MHC-peptide complex, and determining the level of surface expression of the MHC-peptide complex and the sequence of the candidate peptide presented on the cell surface.

[0013] 4. (i) generating a library containing candidate peptides cloned in frame upstream of recombinant MHC beta and / or alpha chains; (ii) transducing such libraries together with the corresponding MHC alpha and / or beta chains into a suitable reporter cell line; (iii) detecting and isolating cells that express one or more MHC-peptide complexes on the cell surface; (iv) determining the level of cell surface expression of said one or more MHC-peptide complexes. (v) isolating DNA from cells that display one or more MHC-peptide complexes on their cell surface; (vi) determining the sequence of candidate peptides encoded by vectors incorporating said DNA isolated from cells that display MHC-peptide complexes on their cell surface; 4. The method of embodiment 3, comprising:

[0014] 5. The method of any one of aspects 1 to 4, wherein said MHC molecule is an MHC class II molecule comprising an extracellular MHC class II alpha chain and a transmembrane domain and an extracellular MHC class II beta chain and a transmembrane domain.

[0015] 6. The method of any one of aspects 1-4, wherein said MHC molecule is an MHC class I molecule comprising the extracellular MHC class I alpha chain and transmembrane domain and beta-2 microglobulin.

[0016] 7. The method of any one of aspects 1 to 4, wherein said MHC-peptide complex is a fusion protein comprising said candidate peptide, beta-2 macroglobulin, said extracellular MHC class I alpha chain and a transmembrane domain.

[0017] 8. The method of embodiment 6 or 7, wherein the MHC alpha chain has a Y84A mutation.

[0018] 9. The method according to any one of aspects 1 to 8, wherein each chain of the MHC molecule comprises a transmembrane domain.

[0019] 10. The method of any one of aspects 1-9, wherein said transmembrane domain is the native transmembrane domain of said MHC molecule.

[0020] 11. The method of any one of aspects 1-9, wherein said transmembrane domain is the transmembrane domain of a heterologous molecule, such as TCR α / β, TCR γ / δ, CD3 γ / δ / ε / ζ, CD4 or CD8 α / β.

[0021] 12. The method of any one of aspects 1 to 11, wherein said reporter cell line is a mammalian cell line.

[0022] 13. The method of any one of aspects 1 to 12, wherein said reporter cell line is a cell line that lacks said MHC class II peptide loading machinery.

[0023] 14. The method of embodiment 13, wherein said reporter cell line is a T cell hybridoma.

[0024] 15. The method according to any one of aspects 1 to 14, wherein said reporter cell line is a cell line lacking functional TAP1, TAP2 and / or beta-2-microglobulin genes.

[0025] 16. The method of embodiment 15, wherein said reporter cell line is a T cell hybridoma having a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene.

[0026] 17. The method of any one of the preceding aspects, wherein said candidate peptides are tumor-specific peptides carrying individual tumor-derived mutations.

[0027] 18. The method of embodiment 17, wherein the mutation is an SNV.

[0028] 19. The method according to any one of aspects 1 to 16, wherein the candidate peptide is an antigen that elicits an immune response.

[0029] 20. The method according to any one of the preceding aspects, wherein the candidate peptide is a compound undergoing immunogenicity testing.

[0030] 21. The method of any one of the previous embodiments, wherein reporter cells that efficiently express MHC on their surface are enriched by FACS- or MACS-based cell sorting.

[0031] 22. The method of any one of the preceding aspects, wherein the sequence of the candidate peptide displayed on the cell surface is determined by PCR and sequencing.

[0032] 23. The method of any one of aspects 1-22, wherein said candidate peptide is for use as a vaccine.

[0033] 24. The method of embodiment 23, wherein the vaccine is a tumor-specific antigen (TSA)-based cancer vaccine.

[0034] 25. The method according to any one of aspects 1 to 22, wherein said candidate peptide is for use in inducing immune tolerance to at least one of said epitopes it comprises.

[0035] 26. The method of any one of aspects 1 to 22 or 25, wherein said candidate peptide is for use in blocking TCR in the context of an MHC molecule.

[0036] 27. The method of any one of aspects 1 to 22 or 25, wherein said candidate peptide is for use in MHC-mediated toxin delivery to cells, in particular to T cells.

[0037] 28. The method of any one of aspects 1-22 or 25, wherein said candidate peptide is for use in redirecting cells using an MHC-CAR.

[0038] 29. The method of any one of aspects 1 to 22, wherein said candidate peptide is for use in immunogenicity testing.

[0039] 30. The method of any one of aspects 1 to 22 or 29, wherein said candidate peptides are for use in a T cell reactivity test.

[0040] 31. A method for determining the MHC binding affinity of a candidate peptide, comprising expressing in a reporter cell line a recombinant MHC-peptide complex comprising a covalently bound candidate peptide, detecting reporter cells presenting said MHC-peptide complex on the surface of said reporter cells, and determining the level of such presentation / expression.

[0041] 32. (i) generating a library comprising candidate peptides cloned upstream of a recombinant MHC alpha or beta domain connected to at least one transmembrane region; (ii) transducing such libraries together with the corresponding MHC alpha or beta domains into a suitable reporter cell line; (iii) detecting cells that display one or more MHC-peptide complexes on their cell surface; (iv) determining the level of such expression / presentation. 32. The method of embodiment 31, comprising:

[0042] The present invention provides a method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), comprising expressing in a reporter cell line a recombinant MHC-peptide complex comprising a covalently linked candidate peptide, detecting reporter cells exhibiting surface expression of the MHC-peptide complex, and determining the sequence of the candidate peptide displayed on the cell surface.

[0043] The present invention is based, at least in part, on the surprising discovery that the surface expression level of recombinant peptide-MHC complexes in mammalian cell lines can be quantitatively measured directly. It is even more surprising that such direct measurement of peptide-MHC complexes was feasible without complex co-culture systems involving T cells and / or elaborate reporter systems, by standard laboratory techniques based on antibody staining and flow cytometry. Although it may be known to those skilled in the art that predictions based on bioinformatics approaches are not always very accurate, it remains surprising that state-of-the-art bioinformatics methods were not able to fully predict the binding of peptides to specific MHC variants as determined by the methods disclosed herein. It is even more surprising that detection of MHC contained in recombinant MCR (peptide-MHC-TCR chimera as disclosed in WO2016097334) complexes was possible with CD3e antibodies regardless of the specific peptide and / or MHC variant to be analyzed. Without being bound by theory, the methods disclosed herein allow to measure biological variations in peptide presentation by MHC primarily without technical variations that may be introduced when using different antibodies to detect different MHC variants.

[0044] Peptides can be covalently attached to MHC molecules to overcome the need for peptide processing / loading machinery.This approach has been used to produce stable MHC tetramers (Crawford et al., Immunity 8,675-682(1998)), in various MHC display methods (Crawford et al., PLoS Biol 2,e90(2004); Wen et al., Protein Eng Des Sel 24,701-709(2011)), to generate mice expressing single peptides on APC (Ignatowicz et al., Cell 84,521-529(1996)), or in CARs designed to redirect T cells to other T cells (Jyothi et al., Nat Biotechnol 20,1215-1220(2002)). In many cases, MHC molecules have had to be mutated to allow expression on the surface of yeast or insect cells (Wen et al., Protein Eng Des Sel 24, 701-709 (2011)), and in some studies steps have been taken to stabilize peptides in the MHC groove by incorporating additional disulfide bridges (Truscott et al., J. Immunol. 178, 6280-6289 (2007)).

[0045] However, in the context of the present invention, it has been shown that in cells incapable of MHC peptide loading, recombinant MHC molecules without tethered peptides are undetectable or unstable (detectable at low levels) on the cell surface, even when overexpressed (Figure 4).

[0046] Thus, the relatively stable isomer of empty MHC described by Rabunowitz (Rabunowitz et al., Immunity, 9, 699-709 (1998)) does not persist very long on the surface of non-professional antigen-presenting cells, even if it is present. Importantly, in the context of the present invention, it has been shown that not all peptides tethered to recombinant MHC lead to efficient cell surface expression of MHC complexes. In fact, some prevent expression on the surface (Figures 2a and b). Thus, a higher expression of recombinant MHC molecules with tethered candidate peptides on the surface of a suitable reporter cell line compared to uninfected reporter cells and / or reporter cells transduced with MHC without covalently bound peptide or unstable MHC-peptide complexes can be used as an indicator of efficient peptide presentation by MHC. In other words, reporter cells exhibiting surface expression of recombinant MHC-peptide complexes can be detected by comparing the surface expression of said MHC-peptide complexes with the background signal of uninfected reporter cells or reporter cells transduced with MHC without covalently bound peptide or unstable MHC-peptide complexes. Moreover, such measurement of the level of presentation / expression (Figures 2a and 7) allows the formation of a ranking of peptides according to their MHC binding ability. The detection and / or ranking of peptides presented by MHC can, whenever necessary, include one or more normalization steps, for example, normalization with the background signal of each MHC variant that does not have the candidate peptide covalently bound or that exerts unstable interactions with the candidate peptide or random peptides. Normalization and / or standardization methods well known in the art can, whenever necessary, be used to determine the binding affinity of the candidate peptide to MHC based on the measured surface expression of recombinant MHC-peptide complexes.

[0047] In certain embodiments, the ranking of candidate peptides and / or the determination of binding affinity of candidate peptides to MHC may be directly correlated with the surface expression of recombinant MHC-peptide complexes as determined by CD3 staining using anti-CD3e, anti-CD3δ or anti-CD3γ antibodies. Preferably, the MHC is included in the MCR for said correlation with said CD3 staining.

[0048] It has now been shown for the first time that appropriately designed MHC class II-peptide complexes can be used to identify candidate peptides that are efficiently presented by APCs.

[0049] Furthermore, within the scope of the present invention, when using MHC-TCR fusion proteins (MCRs) that are composed of peptides linked to the native extracellular domain of MHC fused to the transmembrane unit of TCR, it has been found that not all MHC class II peptide combinations are efficiently expressed on the surface of cells.For example, when various MHC-peptide combinations are tested, differential surface expression of influenza matrix protein 1 (MP1)-derived peptides is observed.Importantly, peptide binding prediction analysis did not show a similar pattern of MHC peptide binding affinity.

[0050] In various embodiments, the present invention provides a method for identifying a candidate peptide of the foregoing embodiments described herein, comprising the steps of: (i) generating a library comprising candidate peptides, in particular a library comprising candidate peptides cloned in-frame upstream of recombinant MHC beta and / or alpha chains; (ii) transducing such libraries together with the corresponding MHC alpha and / or beta chains into a suitable reporter cell line; (iii) detecting cells that display one or more MHC-peptide complexes on their cell surface; (iv) isolating DNA from cells that display one or more MHC-peptide complexes on their cell surface; (v) determining the sequence of the candidate peptide within the MHC-peptide complex displayed on the cell surface. The present invention relates to a method comprising the steps of:

[0051] As used herein, the term "Major Histocompatibility Complex (MHC)" refers to MHC class I and MHC class II genes and the proteins encoded thereby. The terms MHC-I, MHC-II, MHC-1 and MHC-2 are variously used herein to refer to these classes of molecules.

[0052] In certain embodiments, the present invention relates to a method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting reporter cells exhibiting surface expression of said MHC-peptide complex, and determining the level of surface expression of said MHC-peptide complex and the sequence of the candidate peptide displayed on the cell surface.

[0053] In various embodiments, the present invention provides a method for identifying a candidate peptide of the foregoing embodiments described herein, comprising the steps of: (i) generating a library comprising candidate peptides cloned in-frame upstream of recombinant MHC beta and / or alpha chains; (ii) transducing such libraries together with the corresponding MHC alpha and / or beta chains into a suitable reporter cell line; (iii) detecting and isolating cells that express one or more MHC-peptide complexes on the cell surface; (iv) determining the level of cell surface expression of said one or more MHC-peptide complexes. (v) isolating DNA from cells that display one or more MHC-peptide complexes on their cell surface; (vi) determining the sequence of candidate peptides encoded by vectors integrated into the DNA isolated from cells that display MHC-peptide complexes on their cell surface; The present invention relates to a method comprising the steps of:

[0054] In mammals, there are two types of MHC molecules designated class I and class II. MHC class I molecules are present on almost all nucleated cells in the body and are recognized by CD8+ cells. MHC class II molecules are expressed primarily on professional APCs of the immune system and are recognized by CD4+ cells. The molecules are further subdivided by antigenic subtypes. Human MHC class I molecules, also called human leukocyte antigens (HLA), are designated HLA-A, -B, and -C. Human MHC class II molecules are designated HLA-DR, -DQ, and -DP.

[0055] MHC class I molecules are found on almost every cell in the body. 2 Microglobulin (β 2 They are heterodimers composed of polymorphic heavy chains (α) non-covalently associated with monomorphic (human), non-MHC-encoded light (β) chain proteins of about 12 kDa, termed α(m). The heavy α chains each contain about 90 amino acids (α at the N-terminus). 1 , α 2 and α 3 It is a polymorphic transmembrane glycoprotein of approximately 45 kDa consisting of three extracellular domains, a membrane-distal domain, α 1 and α 2 The α domain forms a peptide-binding groove or cleft of sufficient size to bind peptides of 8–10 amino acids, whereas the α 3 The domain is proximal to the plasma membrane. The peptides presented are 1 and α 2 It is held together by a peptide-binding groove in the central region of the domain.

[0056] MHC class I molecules are found only on a few specialized cell types, particularly antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells. Class II MHC molecules contain two distinct polypeptide chains, a 33 kDa α chain and a 28 kDa β chain, which are associated by non-covalent interactions. Like class I MHC molecules, class II MHC molecules are membrane-bound glycoproteins that contain an extracellular domain, a transmembrane segment, and a cytoplasmic tail.

[0057] Each chain in these non-covalent heterodimeric complexes contains two extracellular domains, a transmembrane domain, and a cytoplasmic tail. The extracellular domains are α and β for the α and β chains, respectively. 1 and α 2 Domain and beta 1 and β 2 The membrane-distal domain of class II molecules is the α 1 and β 1 Class IIα is composed of domains that form a peptide-binding groove or cleft of sufficient size to bind peptides that are typically 13-18 amino acids in length, or alternatively 10-18 amino acids or longer. 2 and β 2 The membrane-proximal pair of HLA-DQ and HLA-DQ-α chains have structural similarity to Ig constant (C) domains. In humans, there are three pairs of class II α and β chain genes, known as HLA-DR, HLA-DP and HLA-DQ. The highest level of polymorphism has been documented for HLA-DR.

[0058] As used herein, a candidate peptide refers to a peptide having a length of 8-18 amino acids or up to 50 amino acids. The preferred length of a candidate peptide that binds to MHC class I is 8, 9 or 10 amino acids. The preferred length of a candidate peptide that binds to MHC class II is 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids. In particular, the candidate peptide is comprised in an MHC-peptide complex encoded by a recombinant nucleic acid.

[0059] In various embodiments, the invention relates to the method of any one of the preceding embodiments described herein, wherein said MHC molecule is an MHC class II molecule comprising an extracellular MHC class II alpha chain and a transmembrane domain and an extracellular MHC class II beta chain and a transmembrane domain.

[0060] In other embodiments, the MHC molecule is an MHC class I molecule comprising an extracellular MHC class I alpha chain and transmembrane domain and beta-2 microglobulin.

[0061] In yet another embodiment, the MHC-peptide complex is a fusion protein comprising the candidate peptide, beta-2 macroglobulin, an extracellular MHC class I alpha chain and a transmembrane domain.

[0062] For MHC class I molecules, the Y84A mutation in the alpha chain has been shown to open the peptide binding pocket, allowing better regulation of ligated peptides (Mitaksov, V. et al., Chem Biol 14, 909-922 (2007)).

[0063] Thus, in certain embodiments, the MHC alpha chain of the MHC class I molecule has a Y84A mutation. For example, the Y84A mutation can be introduced by site-directed mutagenesis.

[0064] In some embodiments, the invention relates to the method of any one of the preceding embodiments described herein, wherein said MHC molecule comprises one or two transmembrane domains.

[0065] In various embodiments, the invention relates to the method of any one of the preceding embodiments described herein, wherein the transmembrane domain is the native transmembrane domain of the MHC molecule.

[0066] As used herein, the term "native transmembrane domain" refers to an MHC transmembrane domain that maintains the structural properties of the transmembrane portion of the MHC molecule in its native state.

[0067] In certain embodiments, the transmembrane domain is the transmembrane domain of a heterologous molecule, including, but not limited to, TCR α / β, TCR γ / δ, CD3 γ / δ / ε / ζ, CD4, or CD8 α / β.

[0068] The reporter cell line for carrying out the method of the present invention can be any cell line or cell line derivative that can be used in biological applications.Suitable cell lines or their derivatives include, but are not limited to, human, mouse, rat, monkey, hamster, fish, frog or insect origin, or yeast, such as, but are not limited to, HEK, CHO, 3T3, hybridoma, HeLa, Sf9, Schneider 2 cells, Jurkat, HUVEC, HL-60, MCF-7, Saos-2 cells, IMR-90, Raw 264.7, PC3, Vero, Cos, GH3, PC12, dog MDCK, Xenopus A6 or zebrafish AB9.

[0069] In certain embodiments, the reporter cell line is a mammalian cell line.

[0070] Suitable mammalian cell lines include, but are not limited to, T cell hybridoma, T cell clone, Jurkat, BW5147 α-β fusion partner, myeloma, HEK, CHO, 3T3.

[0071] In particular, reporter cell lines, preferably mammalian cell lines, suitable for use in the present invention lack the ability to display peptides derived from endogenous proteins that are naturally present in the cells. Thus, the cells should only present MHC-peptide complexes with candidate peptides from the peptide library of interest. The processing of peptides and their loading into MHC molecules for display is a multi-step process involving multiple molecular species that constitute the processing and presentation machinery. MHC class I molecules are loaded with peptides via a transporter called TAP (transporter associated with antigen processing), which transfers peptides to the ER, where they bind to MHC class I molecules. The loading of MHC class II molecules occurs by phagocytosis or autophagy; MHC class II molecules are delivered to phagolysosomes, where peptides are loaded before moving to the cell surface. The peptide processing / loading machinery of MHC class II molecules is present only in APCs.

[0072] According to the above, in one embodiment, a reporter cell line suitable for carrying out the method of the present invention is one that lacks MHC class II peptide loading machinery. Preferably, the reporter cell line that lacks MHC class II peptide loading machinery is a mammalian cell line.

[0073] In certain embodiments, the reporter cell line is a T cell hybridoma.

[0074] In another embodiment, a suitable reporter cell line for carrying out the methods of the invention is one that lacks functional TAP1, TAP2 and / or beta-2-microglobulin genes.

[0075] In certain embodiments, the reporter cell line is a T cell hybridoma with a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene. Other examples of reporter cell lines with a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene include, but are not limited to, T cell and B cell clones or hybridomas, HEK cells or 3T3 cells.

[0076] Exome sequencing can be used to identify TSAs that are uniquely present in tumors. For example, TSAs can be identified by sequencing tumor DNA from a patient and comparing it with DNA from a healthy subject. TSAs can also be identified for a single patient by sequencing the tumor and normal DNA of each patient. Based on this analysis, multiple candidate peptides, each with tumor-derived mutations, can be used to generate MHC-peptide complexes. Thus, in one embodiment of the present invention, a library of peptides is used. These peptides can have tumor-derived mutations or can be native peptides.

[0077] Thus, in various embodiments, the invention relates to the method of any one of the preceding embodiments described herein, wherein said candidate peptides are tumor-specific peptides carrying individual tumor-derived mutations.

[0078] As used herein, the term "tumor-derived mutation" refers to a mutation in a nucleic acid sequence that is specific to a tumor and is not present in DNA from healthy tissue.

[0079] In certain embodiments, the tumor-derived mutation is a single nucleotide variation (SNV). SNVs include, but are not limited to, various mutations in p53, KRAS, and BRAF.

[0080] In another embodiment, the candidate peptide is an antigen that elicits an immune response. For example, the peptide can be derived from a pathogen.

[0081] In yet another embodiment, the candidate peptide is a compound undergoing immunogenicity testing.

[0082] In one embodiment, the present invention relates to a method of using a library of candidate peptides, said library comprising mutants of native peptides. For example, native peptides may be known not to be presented efficiently by a given MHC molecule. Thus, a library comprising mutant peptides may be used to screen for peptide mutants that are more efficiently presented by a given MHC molecule.

[0083] In another embodiment, the present invention relates to a method using a library of candidate peptides, said library comprising peptides generated by random shearing or digestion of cDNA or DNA from cells or pathogens of interest. Such libraries cover all peptides present in such cells. For example, the generation of MHC display libraries from cancer tissues or tissues under autoimmune attack will allow the definition of all tissue peptides that may be presented by a given MHC. Thus, in the future, one can look up in a table whether a particular peptide is efficiently presented by a particular MHC without having to carry out tests.

[0084] In one embodiment, the MHC-peptide complex library is generated using a recombinant expression vector. A recombinant expression vector is a replicable DNA construct that contains an assembly of agents that have a regulatory role in gene expression, such as a promoter, operator or enhancer, operably linked to (2) a nucleotide sequence that encodes a desired protein (e.g., an MHC-peptide complex) to be transcribed into mRNA and translated into a protein, and (3) appropriate transcriptional and translational initiation and termination sequences. The choice of promoter and other regulatory elements generally varies with the reporter cell line of interest. Expression vectors are often in the form of "plasmids" (which refer to circular double-stranded DNA loops that are not bound to the chromosome in their vector form). Suitable prokaryotic expression vectors include plasmids from E. coli, such as Col E1, pCR1, pBR322, pMB9 and their derivatives, broader host range plasmids, such as RP4, phage DNA, such as phage lambda and its various derivatives, M13, and the like. Further E. coli vectors are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). However, the present invention is intended to include such other forms of expression vectors that perform equivalent functions and become known in the art thereafter. Eukaryotic expression vectors that replicate episomally, such as pCEP4 or BKV, or other vectors derived from viruses, such as retroviruses, e.g., pMY, pMX, pSIR, adenoviruses, e.g., pAd, etc., can be used. In expression vectors, regulatory elements that control transcription or translation can generally be derived from mammalian, microbial, viral or insect genes. Replication capability, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can further be incorporated.Expression vectors containing regulatory elements derived from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the CMV promoter, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Ruth sarcoma virus promoter, the polyhedrin promoter, or other promoters that have been shown to be effective for expression in eukaryotic cells.

[0085] A "promoter" is defined as a set of nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter also includes distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the transcription start site, as needed. Promoters for use in eukaryotic host cells are known to those skilled in the art. Illustrative examples of such promoters include, but are not limited to, promoters from Simian Virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoters, such as the HIV long terminal repeat (LTR) promoter, Moloney virus promoter, ALV promoter, cytomegalovirus (CMV) promoters, such as the CMV immediate early promoter, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, and promoters from human genes, such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Yet another example of a suitable promoter is the CAG promoter (a hybrid promoter comprising a CMV enhancer, a chicken β-actin promoter and a rabbit β-globin splicing acceptor and a poly(A) sequence).

[0086] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter or a set of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0087] The MHC peptide library is introduced into a suitable reporter cell line by transducing the expression vector into the host cell using standard techniques known in the art. Suitable methods are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). To produce pseudoretroviruses for transduction, a packaging cell line (e.g., Phoenix cell line) that constantly expresses retroviral proteins GAG, POL and ENV is transiently transfected with a construct containing a viral genome composed of LTR, packaging signal and a gene of interest (in this case, a peptide with MHC chain). Alternatively, a suitable cell line, such as HEK, 3T3, is transiently transfected with a mixture of vectors separately encoding retroviral proteins GAG, POL and ENV and a viral genome composed of LTR, packaging signal and a gene of interest. These commonly used strategies ensure the production of defective pseudoretroviruses that can infect target cells and introduce a gene of interest into their genomic DNA. However, since pseudoretroviruses do not have the gag, pol and env genes in their genome, the infected target cells cannot produce retrovirus. Alternatively, MHC-peptide libraries can be introduced into suitable reporter cell lines by transfection with lipid, calcium phosphate, cationic polymer or DEAE-dextran based reagents, or by electroporation.

[0088] The terms "infection" and "transduction" of a reporter cell line are used interchangeably herein.

[0089] Methods for detecting cells that display cell surface-exposed MHC-peptide complexes are known in the art.Suitable methods include cell sorting techniques, such as flow cytometry, fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS).

[0090] Thus, in various embodiments, the invention relates to a method of any one of the foregoing embodiments described herein, wherein reporter cells efficiently expressing MHC on their surface are enriched by fluorescence activated cell sorting (FACS).

[0091] FACS refers to a method of separating a population of cells into one or more subpopulations based on the presence, absence or level of one or more specific polypeptides expressed by the cells. FACS relies on the optical properties (including fluorescence) of individual cells to classify cells into subpopulations. Cell sorters suitable for carrying out the methods described herein are well known in the art and are commercially available. Exemplary cell sorters include MoFlo sorter (DakoCytomation, Fori Collins, Colo.), FACSAria™, FACSArray™, FACS Vantage™, BD™ LSR II and FACSCaiibur™ (BD Biosciences, San Jose, Calif.), and other equivalent cell sorters produced by other commercial suppliers such as Sony, Bio-Rad and Beckman Coulter.

[0092] In another embodiment, the invention relates to a method of any one of the preceding embodiments described herein, wherein reporter cells efficiently expressing MHC on their surface are enriched by MACS-based cell sorting.

[0093] "MACS" refers to a method of separating a population of cells into one or more subpopulations based on the presence, absence or level of one or more MACS selective polypeptides expressed by the cells.MACS relies on the sensitivity properties of tagged individual cells to sort cells into subpopulations.For MACS, magnetic beads (e.g., available from Miltenyi Biotec Bergisch Gladbach, Germany; 130-048-402) can be used as labels.MACS cell sorters suitable for carrying out the methods described herein are well known in the art and are commercially available.Exemplary MACS cell sorters include autoMACS Pro Separator (Miltenyi Biotec).

[0094] In various embodiments, the cells may be contacted with an antibody specific for MHC-I or MHC-II, or with an antibody that detects a hybrid-associated protein. For example, if the MHC extracellular domain is fused to the TCR transmembrane region, an antibody that detects CD3γ, CD3δ, or CD3ε may be used. The antibody may be directly conjugated to a detectable label. Alternatively, a secondary antibody conjugated to a detectable label and specific for the primary antibody may be contacted with the cells. Detectable labels suitable for use include any compound that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the present invention include biotin, magnetic beads (e.g., Dynabeads™), fluorescent labels (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, dansyl, umbelliferone, PE, APC, CY5, Cy7, PerCP, Alexa dyes, etc.), radioactive labels (e.g., 3 H, 125 1. 35 S, 1 C or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, etc.) and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. A variety of suitable fluorescent labels are further described, for example, in The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition.

[0095] Sorting results in a population of non-fluorescent cells and at least one population of fluorescent cells, depending on how many fluorescent labels are used. The presence of at least one population of cells with fluorescent cells indicates that at least one candidate peptide is efficiently presented by APC. Thus, FACS allows sorting of a population of cells to produce a population of cells enriched for cells that contain surface-exposed MHC-I or MHC-II.

[0096] An embodiment of the present invention utilizes DNA isolation methods known to those skilled in the art. In general, the objective is to separate DNA present in the nucleus of a cell from other cellular components. DNA isolation usually begins with lysis or disintegration of the cell. This process is essential for the disruption of protein structures, allowing the release of nucleic acids from the nucleus. Lysis is performed in a salt solution containing a detergent to denature proteins or a protease (an enzyme that digests proteins), such as proteinase K, or in some cases both. It results in the disintegration of the cells and dissolution of the membranes. DNA isolation methods include, but are not limited to, phenol:chloroform extraction, high salt precipitation, alkaline denaturation, ion exchange column chromatography, resin binding and paramagnetic bead binding.

[0097] An embodiment of the present invention utilizes cDNA generation methods known to those skilled in the art. In general, the objective is to convert isolated RNA present in cells into DNA, referred to as copy DNA, for use as a template for polymerase chain reaction (PCR). RNA isolation usually begins with lysis or degradation of the cells. This process is essential for the destruction of protein structures, allowing the release of nucleic acids therefrom. Lysis is usually performed in a phenol-containing solution (e.g., TRIzol™), which results in the disintegration of the cells and the lysis of membranes, allowing the separation of RNA from other cellular components. The isolated RNA is then converted into cDNA by reverse transcriptase (e.g., Superscript™, Goscript™).

[0098] The sequences of candidate peptides within the MHC-peptide complexes displayed on the cell surface can then be amplified by PCR and sequenced by any method known in the art.

[0099] In various embodiments, the invention relates to the method of any one of the preceding embodiments described herein, wherein the sequence of the candidate peptide is determined by PCR and sequencing.

[0100] In one embodiment, the sequence of the candidate peptide is determined by digital PCR. Digital polymerase chain reaction (digital PCR, Digital PCR, dPCR or dePCR) is an improved version of the traditional polymerase chain reaction method that can be used to directly quantify and clonal amplify nucleic acids, including DNA, cDNA or RNA.

[0101] Sequencing can also be performed using microfluidics. Microfluidics involves microscale devices that handle small volumes of fluid. Because microfluidics can accurately and reproducibly control and dispense small volumes of fluid, especially volumes less than 1 μl, the application of microfluidics offers significant cost savings. The use of microfluidics technology reduces cycle times, shortens time to results, and increases throughput. Furthermore, the incorporation of microfluidics technology enhances the integration and automation of the system. Microfluidic reactions are generally performed in microdroplets.

[0102] In some embodiments, sequencing is performed using second generation (or next generation or Next-Gen), third generation (or Next-Next-Gen) or fourth generation (or N3-Gen) sequencing technologies, including but not limited to pyrosequencing, sequencing biligation, single molecule sequencing, sequencing by synthesis (SBS), massively parallel clonal, massively parallel single molecule SBS, massively parallel single molecule real-time, massively parallel single molecule real-time nanopore technologies. Morozova and Marra provide a review of such technologies in Genomics, 92:255 (2008).

[0103] In some embodiments, nucleic acid is amplified before, after or simultaneously with sequencing.Exemplary non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).Those skilled in the art will recognize that certain amplification techniques (e.g., PCR) require RNA to be reverse transcribed into DNA before amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

[0104] In one embodiment, the invention relates to the method of any one of the preceding embodiments described herein, wherein said candidate peptide is for use as a vaccine.

[0105] In certain embodiments, the vaccine is a tumor-specific antigen (TSA)-based cancer vaccine.

[0106] "Vaccination" or equivalent terms are well understood in the art. For example, the term vaccination can be understood as a process of increasing a subject's immune response to an antigen, thus increasing the ability to resist or overcome disease. "Vaccine" should be understood to mean a composition for generating immunity for the prevention and / or treatment of disease (e.g., cancer). Thus, a vaccine is a medicine that contains an antigen and is intended to be used in humans or animals to generate specific defense and protection by vaccination. The term "TSA-based cancer vaccine" is meant to refer to a vaccine that contains a pooled sample of tumor-specific antigens, such as at least one, at least two, at least three, at least four, at least five or more tumor-specific peptides. For a vaccine to be effective, short linear peptides containing the antigen or epitope must be presented on the surface of APCs by MHC. The set of expressed MHC variants varies greatly between different individuals.

[0107] Recurrent tumor-derived mutations can serve as public tumor-specific antigens, allowing the development of TSA-based cancer vaccines that can be applied to a broader patient cohort. Thus, the method of the present invention can be used to identify patients who contain specific MHC variants and efficiently present these common / public TSAs to the immune system. However, many tumor-derived mutations are likely to originate from patient-specific changes, and any of these changes may affect the binding of TSA peptides to MHC. Thus, the method of the present invention can also be used to identify patient-specific candidate peptides for personalized vaccines.

[0108] Moreover, in many cases, a patient's tumor contains many TSAs, each of which may only be contained in a subset of tumor cells. Thus, in some embodiments, a set of candidate TSAs may consist of TSAs from different sets of tumor cells. Thus, a key step in developing a cancer vaccine is to select candidates that have high affinity to MHC expressed in the patient from the TSA candidates contained in and preferably well expressed in the patient's tumor cells. For optimal immune response, the set of TSAs contained in the vaccine should further activate both CD4+ and CD8+ T cells that recognize MHCII or MHCI-peptide complexes, respectively. Thus, the method of the present invention can be used to identify TSAs that bind to specific MHCII and MHCI variants in a patient.

[0109] In one embodiment, the candidate peptide is for use in inducing immune tolerance to at least one of the epitopes it contains. As used herein, "immune tolerance" refers to a decrease in the immunological reactivity of the host to a particular antigen or antigens. The antigen comprises an immune determinant / epitope that in the absence of tolerance would cause an undesired immune response. Immune tolerance may be induced to prevent or ameliorate transplant rejection, autoimmunity, allergic reaction or another undesired immune response. Without being bound by theory, immune tolerance may be achieved by the generation of regulatory T cells that act as negative regulators of immune responses. The balance of different types of T cells may also depend on the interaction of the TCR with the MHC-peptide complex. Thus, the method of the present invention may be used to select an appropriate MHC-peptide complex to generate immune tolerance to the peptide. Other possible mechanisms for achieving immune tolerance include blocking the TCR of specific T cells and / or killing specific T cells.

[0110] Thus, in one embodiment, the candidate peptide is for use in blocking TCR in the context of MHC molecules. "TCR blocking" refers to any agent that comprises a peptide-MHC complex that blocks natural TCR-MHC interaction. To predict the TCR blocking efficacy of MHC-peptide complex, the stability of the complex is important.

[0111] In another embodiment, the candidate peptides are for use in MHC-mediated toxin delivery to cells, particularly T cells.

[0112] "MHC-mediated toxin delivery" refers to a method of covalently linking a toxic agent (such as a protein) to a peptide-MHC tetramer or other MHC multimer in order to deliver the toxin to a cell, particularly a T cell, causing cell death.

[0113] The methods disclosed herein allow one to measure the stability of specific MHC-peptide pairs and can therefore be used to develop MHC-peptide complexes for use in specific TCR blockade or specific T cell killing, particularly for in vivo applications.

[0114] In yet another embodiment, the candidate peptides are for use in redirecting T cells to other peptide-MHC specific T cells via MHC-CAR.

[0115] The redirection of T cells to other cells using CARs composed of antibodies linked to the intracellular chain of CD247 (CD3 zeta) is commonly used and is already in the clinic. T cells with CARs composed of peptide-MHC linked to the intracellular chain of CD247 (CD3 zeta), particularly cytotoxic T cells or T cells with MCRs, can use these receptors to recognize and kill T cells specific for these peptide-MHC complexes (Jyothi et al., Nat. Biotech, 20, 1215-1220 (2002)). The methods disclosed herein allow the stability of specific MHC-peptide pairs to be measured, and therefore can be used to develop MHC-peptide complexes for use in CARs or MCRs for redirected killing of specific T cells, particularly for in vivo applications.

[0116] Many therapies that modulate immune responses require the identification of TCRs involved in the immune response. There are various methods known in the art that allow these TCRs to be identified and rely on the interaction of TCRs with epitopes contained in MHC-peptide complexes. An important prerequisite is the information of how MHC-peptide complexes are presented on the surface of APCs. If an epitope is not presented by the MHC of the APC used in an experimental in vitro assay, the appropriate TCR that binds to that epitope cannot be identified. Furthermore, if one of the peptides is preferentially presented in the library or in the MHC-peptide complexes presented by the APC in such an assay, this may lead to the selection of a suboptimal TCR. Therefore, it is important to overcome the uncertainties associated with the presentation of MHC-peptide complexes for an optimized TCR identification and selection process. Thus, the method of the present invention provides quantitative information on the affinity of a particular peptide to a particular MHC and can be used to identify the TCR that recognizes the epitope or to select the appropriate complex for the in vitro assay used to enrich the T cells expressing such TCR.

[0117] In one embodiment, the candidate peptides are for use in T cell reactivity tests.

[0118] As used herein, the term "T cell reactivity" refers to the ability of a substance to induce T cell activation. More specifically, "T cell reactivity" refers to the ability of a peptide to induce T cell proliferation, differentiation and / or cytokine production. T cell reactivity assays are well known in the art and often involve co-culture of T cells with APCs presenting MHC-peptide complexes. These assays can be used to expand T cells for cell therapy or to identify TCRs that bind to specific epitopes, for example, for adaptive T cell therapy or chimeric antigen receptor (CAR) T cell therapy. T cell reactivity tests can further be used for immunogenicity testing.

[0119] In certain embodiments, the candidate peptides are for use in immunogenicity studies.

[0120] As used herein, the term "immunogenicity testing" refers to measuring potential immune responses to biotherapeutics. Biotherapeutics may induce immune responses that may affect their safety and efficacy. Immunogenicity testing is used to monitor and evaluate humoral (antibody) or cellular (T cell) responses during clinical and preclinical studies. Typically, testing the immunogenicity of a biotherapeutic involves measuring antibodies generated specifically against the biotherapeutic. Using the methods of the present invention, it is possible to identify peptides that are efficiently presented by MHC molecules, particularly individual-specific MHC, and thus may induce T cell-mediated immune responses in in vitro procedures. This may help provide a more complete and accurate picture of the overall immunogenicity profile of a compound, while at the same time reducing the burden on patients or clinical trial participants.

[0121] In a further embodiment, the invention provides an MHC-peptide complex comprising a candidate peptide covalently linked to the extracellular portion of an alpha or beta domain of an MHC molecule, the extracellular portion of an MHC beta domain, and at least one transmembrane domain of a heterologous molecule.

[0122] In one embodiment, the invention provides an MHC-peptide complex of the preceding embodiment, wherein the transmembrane domain is the transmembrane domain of a heterologous molecule, such as TCR α / β, TCR γ / δ, CD3 γ / δ / ε / ζ, CD4 or CD8 α / β.

[0123] In one embodiment, the MHC molecule is an MHC class II molecule comprising an extracellular MHC class II alpha chain, an extracellular MHC class II beta chain and at least two transmembrane domains.

[0124] In another embodiment, the MHC molecule is an MHC class I molecule comprising an extracellular MHC class I alpha chain, beta-2 macroglobulin and at least one transmembrane domain.

[0125] In yet another embodiment, the MHC molecule is an MHC hybrid molecule comprising a fusion of a peptide, β2-microglobulin and an extracellular MHC class I alpha chain and at least one transmembrane domain.

[0126] In certain embodiments, the MHC alpha chain of the MHC class I molecule has a Y84A mutation.

[0127] In various embodiments, the invention provides MHC-peptide complexes, where the candidate peptides are tumor-specific peptides carrying individual tumor-derived mutations.

[0128] In certain embodiments, the tumor-derived mutation is a single nucleotide variation (SNV).

[0129] In a further embodiment, the present invention provides a recombinant construct comprising a nucleotide sequence encoding an MHC-peptide complex as defined in any one of the preceding embodiments.

[0130] In yet a further embodiment, the present invention provides an expression cassette comprising a promoter sequence linked to the recombinant construct of the preceding embodiments.

[0131] In another embodiment, the present invention provides a vector comprising the expression cassette of the preceding embodiments.

[0132] In yet another embodiment, the invention provides a cell comprising an MHC-peptide complex of the invention.

[0133] In one embodiment, the invention relates to a cell of the previous embodiment comprising an MHC class II peptide complex, wherein the cell is a mammalian cell lacking MHC class II peptide loading machinery.

[0134] In another embodiment, the cell comprises an MHC class I peptide complex and the cell is a mammalian cell that lacks functional TAP1, TAP2 and / or beta-2-microglobulin genes.

[0135] In yet another embodiment, the cell comprises an MHC class I peptide complex and the cell is a T cell hybridoma having a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene.

[0136] Further included by the present invention is a method for determining the MHC binding affinity of a candidate peptide, comprising expressing in a reporter cell line a recombinant MHC-peptide complex comprising a covalently bound candidate peptide, and detecting reporter cells presenting said MHC-peptide complex on the surface of said reporter cell, including measuring the level of such presentation / expression. The reporter cell line may be a mammalian cell line. In one embodiment, the mammalian cell line used exhibits altered characteristics relative to the native cell line, for example, it has enzyme deletions, mutations and / or overexpression of enzymes and / or MHC molecules.

[0137] In one embodiment, the present invention relates to a method of the previous embodiment, (i) generating a library containing candidate peptides cloned upstream of a recombinant MHC alpha or beta domain connected to a transmembrane region; (ii) transducing such libraries together with the corresponding MHC alpha or beta domains into a suitable reporter cell line; (iii) detecting cells that display one or more MHC-peptide complexes on their cell surface; (iv) determining the level of such expression / presentation. The present invention relates to a method comprising the steps of:

[0138] The method of the present invention can also be applied to high-throughput screening. High-throughput screening (HTS) techniques are commonly used to define rapid processing of cells on a large scale. In certain embodiments, multiple screenings can be performed in parallel with different candidate peptide libraries. High-throughput screening systems are commercially available and typically automate the entire procedure, including all sample and reagent pipetting, liquid dispensing, timed incubation, and final reading of the microplate with a detector appropriate for the assay. These configurable systems offer high throughput and rapid start-up, as well as a high degree of flexibility and customization.

[0139] As used herein, the term "peptide" means at least two covalently attached amino acids. Generally, MHC class I peptides are 8 or 9 amino acids in length, but can vary from 7 to 10 amino acids in length. MHC class II peptides can be 15 to 24 amino acids in length. Optionally, they can vary from 10 amino acids to 30 amino acids or more in length.

[0140] As used herein, binding or "binding affinity" of a peptide to MHC or interaction of a peptide with MHC refers to a peptide-MHC interaction that occurs in the peptide binding groove or cleft. Such binding or interaction may also occur naturally.

[0141] As used herein, "covalent attachment" or "tethering" of a peptide to an MHC refers to a recombinant MHC-peptide complex in which the covalent attachment is produced by genetic engineering.

[0142] As used herein, an MHC-peptide complex refers to a complex in which a peptide is bound to the MHC. This binding can occur through the peptide binding groove or cleft and / or by covalent binding of the peptide to the MHC.

[0143] In a recombinant MHC-peptide complex, as used herein, the candidate peptide is always covalently bound to the MHC, but the peptide may or may not bind efficiently into the cleft of the MHC.

[0144] The stability of an MHC-peptide complex refers to the stability of the peptide-MHC interaction that occurs in the peptide binding groove that correlates with the surface expression of said MHC-peptide complex. It does not refer to the stability of the covalent peptide-MHC bond.

[0145] As used herein, the term "antigen" refers to all or a portion of a peptide or protein capable of eliciting an immune response against itself or a portion thereof. This immune response may involve antibody production, or activation of specific immunologically competent cells, or both.

[0146] As used herein, the term "peptide loading machinery" refers to all the necessary components required for loading peptides into MHC molecules.For example, the MHC class I peptide loading machinery exists in almost all cells, and includes TAP transporter, tapasin and calreticulin, among others.The MHC class II peptide loading machinery exists only in APCs, but it can be induced in other cells, such as T cells.

[0147] As used herein, the term "transmembrane domain" is defined as a predominantly hydrophobic amino acid sequence capable of spanning a cell membrane. The term "transmembrane region" is used herein as a synonym for "transmembrane domain."

[0148] As used herein, "library" or equivalent terms refer to a plurality of molecules. In the case of MHC-peptide complexes, the library provides a sufficiently structurally diverse population of peptides to provide a sufficient range of cellular responses probabilistically and provide one or more cells with a desired response. In a preferred embodiment, at least 10, preferably at least 50, more preferably at least 200, and most preferably at least 1000 peptides are analyzed simultaneously in the method of the present invention. The library can be designed to maximize the size and diversity of the library.

[0149] The term "recombinant", for example, when used in reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all. Recombinant nucleic acids are originally formed in vitro, typically by the manipulation of nucleic acids using, for example, polymerases and endonucleases, in a form not normally found in nature. In this way, operative linkage of different sequences is achieved. Thus, both isolated nucleic acids in linear form, or expression vectors formed in vitro by linking DNA molecules that are not normally connected, are considered recombinant for the purposes of the present invention. It is understood that once a recombinant nucleic acid has been made and reintroduced into a host cell or organism, it replicates non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulation; however, such a nucleic acid, once produced recombinantly, may subsequently be replicated non-recombinantly but is still considered to be recombinant for purposes of the present invention. Similarly, a recombinant protein, such as an MHC-peptide complex of the present invention, is a protein that has been made using recombinant techniques, i.e., through expression of a recombinant nucleic acid as described above.

[0150] The term "heterologous" when used in reference to a portion of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly to have two or more sequences, e.g., produced recombinantly from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein often refers to two or more subsequences (e.g., fusion proteins) that are not normally found in the same relationship to each other in nature.

[0151] As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0152] Moreover, in the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used to carry out or test the present invention, but suitable methods and materials are described below.In case of conflict, the present specification, including definitions, shall prevail.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0154] Specific embodiments of the present invention are further illustrated by the following examples. However, it should be understood that the present invention is not limited to the specific details of these examples. The following examples are included to demonstrate preferred embodiments of the present invention. It should be recognized by those skilled in the art that the techniques disclosed in the following examples correspond to techniques used in the present invention to function well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize, in light of the present disclosure, that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. [Brief description of the drawings]

[0155] [Figure 1] FIG. 1 shows the structure of the MHC-TCR fusion molecule (MCR2) and native MHC class II.

[0156] [Figure 2-1]Constructs were generated that encode MCRs with the alpha and beta chains of human MHC molecules (HLA-DRB1_4, DRB1_15 and DRB5_1) covalently linked to influenza matrix protein 1 peptides MP1103-120 (a), MP162-72 (b) or a library of influenza cDNA-derived peptides (c). After transduction of MHC class II-deficient cell lines, MCR surface expression was measured by FACS using anti-HLA-DR APC antibodies. The graph shows efficient surface expression of MCRs containing HLA-DRB1_4-MP1103-120 complexes, whereas MCRs containing DRB1_15-MP1103-120 were detectable at very low levels (a). We barely detected MCRs containing DRB5_1-MP1103-120 (a), but detected efficient expression of MCRs containing DRB5_1-MP162-72 (b). Importantly, when a library of influenza cDNA-derived peptides was tethered to HLA DBR1_4 or HLA DRB1_15, similar levels of MCR surface expression were observed (c). "UI" refers to uninfected cells. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0157] [Diagram 3] Figure 3 shows an example of an analysis of cells transduced with MCR containing various HLA beta chains with different peptides (pep1, pep2 and pep3) stained with anti-HLA-DR and anti-CD3e. The double positive staining shows that both antibodies are equally efficient in detecting MCR surface expression and the diagonal-shaped population (efficient association of MCR with CD3 chains).

[0158] [Figure 4] Figure 4 shows low surface expression of MHC and MCR molecules without covalently linked peptides. Cells transduced with HLA-DRB1.4 (left) or MCR2-DRB1.4 constructs (right) were stained with either αHLA-DR (top) or αCD3 (bottom).

[0159] [Diagram 5] FIG. 5 shows peptide binding predictions for MP1103-120 using the Immune Epitope Database (IEDB) analysis resource.

[0160] [Figure 6] Figure 6 shows FACS analysis of cells transduced with MCR containing various HLA-DRB chains with different peptides containing the core epitope MP1107-117. MCR surface expression was measured using anti-CD3e or anti-HLA-DR PE antibodies. The graph shows that the core MP1107-117 peptide can be efficiently presented only on HLA-DRB1_4, but not by HLA-DRB1_15 and HLA-DRB5_1, and that the adjacent peptide region does not play a major role. PCR and sequencing confirmed the presence of MCR in all samples (not shown).

[0161] [Figure 7] Figure 7. A library of random peptides tethered to the alpha chain of mouse MCRs was transduced into reporter cells, single cells expressing MCRs on their surface were sorted, and exemplary clones were sequenced. The graph represents FACS analysis of exemplary clones with MCRs bearing such peptides stained with anti-mouse MHC class II and the corresponding peptide sequences. EXAMPLES

[0162] Example 1 - Differential surface expression of MCRs containing influenza matrix protein 1 peptides We constructed MHC-TCR fusion proteins (MCRs) composed of a peptide linked to the native extracellular domain of MHC fused to the transmembrane unit of the TCR (Figure 1; see also WO 2016 / 097334). In particular, we covalently linked influenza matrix protein 1 peptide (MP1 103-120 ), MP1 62-72We generated constructs encoding MCRs with alpha and beta chains of human MHC molecules (HLA-DRB1_4, DRB1_15, and DRB5_1) along with the beta chain with a library of influenza cDNA-derived peptides. Retrovirus-containing supernatants were produced in ecotropic phoenix packaging cell lines according to standard protocols and used to infect reporter cell lines and sorted cells. After transduction of MHC class II-deficient cell lines (T-cell hybridomas), MCR surface expression was measured by FACS using anti-HLA-DR APC antibodies (Figure 2). MP1-derived peptides containing amino acids 103-120 tethered to HLA DRB1_4 result in efficient expression of MCRs on the surface of mammalian reporter cell lines. The same peptides tethered to HLA DRB1_15 result in very low MCR expression and are not expressed at all in the context of HLA DRB5_1 (Figure 2a). However, another MP1-derived peptide containing amino acids 62-72 results in efficient expression of the MCR containing HLA DRB5-1, apart from the other HLAs tested (Figure 2b). Importantly, when a library of influenza cDNA-derived peptides was tethered to HLA DRB1_4 or HLA DRB1_15, similar levels of surface expression were observed, indicating that both HLA1_4 and HLA1_15 can be efficiently expressed on the cell surface as part of the MCR (Figure 2c). From published studies (Schmid et al., Immunity 2007), it is known that MP1 peptides containing amino acids 103-117 and 107-121 are efficiently presented by HLA DRB1-4, suggesting that only if the covalently attached peptide efficiently binds to the MHC peptide-binding groove is the MHC complex stable enough for surface expression to be detected with antibody staining. Figure 6 further illustrates the core MP1 107-117 We show that peptides can be efficiently presented only on HLA-DRB1_4, regardless of the flanking peptide regions.

[0163] Example 2 - Detection of MCR surface expression with different antibodies Surface expression of MCRs containing various HLA beta chains with different peptides (pep1, pep2 and pep3) was detected by antibody staining using anti-HLA-DR and anti-CD3e and analyzed on a BD Fortessa flow cytometer (Figure 3). Double positive staining indicates that both antibodies are efficient in detecting MCR surface expression and that anti-CD3e staining can be universally used to detect MCRs with different HLA alleles. Furthermore, the diagonal linear shaped population indicates efficient association of MCRs with CD3 chains.

[0164] Example 3 -MP1 103-120 MHC binding affinity prediction for Using the Immune Epitope Database (IEDB) analysis resources, MP1 103-120 MHC binding affinity for MP1 has been predicted (Wang, P. et al., A Systematic Assessment of MHC Class II Peptide Binding Predictions and Evaluation of a Consensus Approach. PLoS Comput Biol 4, e1000048 (2008); Wang, P. et al., Peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics 11, 568 (2010)). 103-121 For HLA DRB 4_1, the method of the present invention showed that HLA DRB 4_1 binds best, resulting in high surface expression of HLA, HLA DRB 1_15 binds moderately, resulting in low surface expression, and HLA DRB 5_1 does not bind at all. However, using the IEDB analysis resource, peptide binding prediction analysis showed that HLA DRB 5_1 was the best MP1. 103-120 It was shown to be a binding product (Figure 5).

[0165] Example 4 - Detection of surface expression of MCRs containing peptides fused to the alpha chain. We constructed a library of mouse MCRs with random peptides tethered to the alpha chain. After transduction of MHC class II-deficient cell lines (T cell hybridomas), MCR surface expression was measured by FACS using anti-MHC antibodies (Figure 7). Efficient expression of MCRs with many different peptides was detected, indicating that peptides linked to the alpha chain of MCRs can also stabilize the MHC complex sufficiently for surface expression. The present invention provides, for example, the following items. (Item 1) 1. A method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), comprising expressing a recombinant MHC-peptide complex comprising a covalently linked candidate peptide in a reporter cell line, detecting reporter cells exhibiting surface expression of the MHC-peptide complex, and determining the sequence of the candidate peptide displayed on the cell surface. (Item 2) (i) generating a library comprising candidate peptides cloned in-frame upstream of recombinant MHC beta and / or alpha chains; (ii) transducing such libraries together with the corresponding MHC alpha and / or beta chains into a suitable reporter cell line; (iii) detecting and isolating cells that express one or more MHC-peptide complexes on the cell surface; (iv) isolating DNA from cells that display one or more MHC-peptide complexes on their cell surface; (v) determining the sequence of candidate peptides encoded by vectors incorporating said DNA isolated from cells that display MHC-peptide complexes on their cell surface. The method according to item 1, comprising: (Item 3) 1. A method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), comprising expressing a recombinant MHC-peptide complex comprising a covalently linked candidate peptide in a reporter cell line, detecting reporter cells exhibiting surface expression of the MHC-peptide complex, and determining the level of surface expression of the MHC-peptide complex and the sequence of the candidate peptide displayed on the cell surface. (Item 4) (i) generating a library comprising candidate peptides cloned in-frame upstream of recombinant MHC beta and / or alpha chains; (ii) transducing such libraries together with the corresponding MHC alpha and / or beta chains into a suitable reporter cell line; (iii) detecting and isolating cells that express one or more MHC-peptide complexes on the cell surface; (iv) determining the level of cell surface expression of said one or more MHC-peptide complexes. (v) isolating DNA from cells that display one or more MHC-peptide complexes on their cell surface; (vi) determining the sequence of candidate peptides encoded by vectors integrated into said DNA isolated from cells that display MHC-peptide complexes on their cell surface; The method according to item 3, comprising: (Item 5) 5. The method according to any one of items 1 to 4, wherein the MHC molecule is an MHC class II molecule comprising an extracellular MHC class II alpha chain and a transmembrane domain and an extracellular MHC class II beta chain and a transmembrane domain. (Item 6) 5. The method according to any one of items 1 to 4, wherein the MHC molecule is an MHC class I molecule comprising the extracellular MHC class I alpha chain and transmembrane domain and beta-2 microglobulin. (Item 7) 5. The method according to any one of items 1 to 4, wherein the MHC-peptide complex is a fusion protein comprising the candidate peptide, beta-2 macroglobulin, the extracellular MHC class I alpha chain and a transmembrane domain. (Item 8) 8. The method of claim 6 or 7, wherein the MHC alpha chain has a Y84A mutation. (Item 9) 9. The method according to any one of items 1 to 8, wherein each chain of the MHC molecule comprises a transmembrane domain. (Item 10) 10. The method according to any one of items 1 to 9, wherein the transmembrane domain is the native transmembrane domain of the MHC molecule. (Item 11) 10. The method according to any one of items 1 to 9, wherein the transmembrane domain is a transmembrane domain of a heterologous molecule, such as TCR α / β, TCR γ / δ, CD3 γ / δ / ε / ζ, CD4 or CD8 α / β. (Item 12) 12. The method according to any one of items 1 to 11, wherein the reporter cell line is a mammalian cell line. (Item 13) 13. The method of any one of items 1 to 12, wherein the reporter cell line is a cell line lacking the MHC class II peptide loading machinery. (Item 14) 14. The method of claim 13, wherein the reporter cell line is a T cell hybridoma. (Item 15) 15. The method of any one of items 1 to 14, wherein the reporter cell line is a cell line lacking a functional TAP1, TAP2 and / or beta-2-microglobulin gene. (Item 16) 16. The method of claim 15, wherein the reporter cell line is a T cell hybridoma having a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene. (Item 17) The method of any one of the preceding items, wherein the candidate peptides are tumor-specific peptides carrying individual tumor-derived mutations. (Item 18) 18. The method of claim 17, wherein the mutation is an SNV. (Item 19) 17. The method according to any one of items 1 to 16, wherein the candidate peptide is an antigen that induces an immune response. (Item 20) 17. The method according to any one of items 1 to 16, wherein the candidate peptide is a compound undergoing immunogenicity testing. (Item 21) The method according to any one of the preceding items, wherein reporter cells that efficiently express MHC on their surface are enriched by FACS- or MACS-based cell sorting. (Item 22) The method of any one of the preceding claims, wherein the sequence of the candidate peptide displayed on the cell surface is determined by PCR and sequencing. (Item 23) 23. The method according to any one of items 1 to 22, wherein the candidate peptide is for use as a vaccine. (Item 24) 24. The method of claim 23, wherein the vaccine is a tumor-specific antigen (TSA)-based cancer vaccine. (Item 25) 23. The method according to any one of items 1 to 22, wherein the candidate peptide is for use in inducing immune tolerance to at least one of the epitopes it contains. (Item 26) 26. The method of any one of items 1 to 22 or 25, wherein the candidate peptide is for use in blocking TCR in the context of an MHC molecule. (Item 27) 26. The method of any one of items 1 to 22 or 25, wherein the candidate peptide is for use in MHC-mediated toxin delivery to cells, in particular to T cells. (Item 28) 26. The method of any one of items 1 to 22 or 25, wherein the candidate peptide is for use in redirecting T cells using an MHC-CAR. (Item 29) 23. The method according to any one of items 1 to 22, wherein the candidate peptide is for use in an immunogenicity test. (Item 30) 30. The method according to any one of items 1 to 22 or 29, wherein the candidate peptide is for use in a T cell reactivity test. (Item 31) 1. A method for determining the MHC binding affinity of a candidate peptide, comprising expressing in a reporter cell line a recombinant MHC-peptide complex comprising a covalently bound candidate peptide, detecting reporter cells which present said MHC-peptide complex on the surface of said reporter cells, and determining the level of such presentation / expression. (Item 32) (i) generating a library comprising candidate peptides cloned upstream of a recombinant MHC alpha or beta domain connected to at least one transmembrane region; (ii) transducing such libraries together with the corresponding MHC alpha or beta domains into a suitable reporter cell line; (iii) detecting cells that display one or more MHC-peptide complexes on their cell surface; (iv) determining the level of such expression / presentation. 32. The method according to claim 31, comprising:

Claims

1. 1. A method for identifying candidate peptides that are efficiently presented by major histocompatibility complex (MHC), the method comprising: a) expressing in a reporter cell line a recombinant MHC-peptide complex comprising an extracellular MHC molecule covalently linked to a candidate peptide and a transmembrane domain of TCRα / β, said reporter cell line being a mammalian cell line; b) detecting and isolating reporter cells exhibiting surface expression of said MHC-peptide complexes, said detecting and isolating reporter cells comprising contacting said reporter cells with an antibody against said recombinant MHC-peptide complexes; c) determining the amino acid sequence of the candidate peptide displayed on the cell surface of the reporter cells isolated in step (b); and d) identifying said candidate peptides of step (c) that are presented by MHC wherein the recombinant MHC-peptide complex is composed of a peptide linked to the extracellular domain of MHC fused to the transmembrane domain of the TCR α / β.

2. (i) generating a library comprising candidate peptides cloned in frame upstream of recombinant MHC beta and / or alpha chains; and (ii) transducing said library together with the corresponding MHC alpha and / or beta chains into said reporter cell line. The method of claim 1 further comprising:

3. The method of claim 1 or 2, comprising the further step of determining the level of surface expression of the MHC-peptide complexes and / or the binding affinity of the peptide candidates for the MHC molecules.

4. The method of any one of claims 1 to 3, wherein the MHC molecule is an MHC class II molecule comprising an extracellular MHC class II alpha chain and a transmembrane domain and an extracellular MHC class II beta chain and a transmembrane domain.

5. The method of any one of claims 1 to 3, wherein the MHC molecule is an MHC class I molecule comprising an extracellular MHC class I alpha chain and a transmembrane domain and beta-2 microglobulin.

6. The method of any one of claims 1 to 3, wherein the MHC-peptide complex is a fusion protein comprising a candidate peptide, beta-2 microglobulin, an extracellular MHC class I alpha chain and a transmembrane domain.

7. The method of claim 5 or 6, wherein the extracellular MHC class I alpha chain has a Y84A mutation.

8. The method of any one of claims 1 to 7, wherein the reporter cell line is a cell line that lacks MHC class II peptide loading machinery.

9. The method of claim 8, wherein the reporter cell line is a T cell hybridoma.

10. The method of any one of claims 1 to 9, wherein the reporter cell line is a cell line lacking functional TAP1, TAP2 and / or beta-2-microglobulin genes.

11. The method of claim 10, wherein the reporter cell line is a T cell hybridoma that has a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene.

12. The method according to any one of claims 1 to 11, wherein the candidate peptides are tumor-specific peptides carrying individual tumor-derived mutations.

13. The method of claim 12, wherein the mutation is a SNV.

14. The method according to any one of claims 1 to 11, wherein the candidate peptide is an antigen that elicits an immune response.

15. The method of any one of claims 1 to 11, wherein the candidate peptide is a compound undergoing immunogenicity testing.

16. The method of any one of claims 1 to 15, wherein the candidate peptides are encoded in an MHC-peptide complex library.

17. 17. The method of claim 16, wherein the MHC-peptide complex library is obtained by cloning a plurality of polynucleotides encoding the candidate peptides in frame upstream of a recombinant MHC beta and / or alpha chain.

18. The plurality of polynucleotides encoding the candidate peptides a) by mutagenesis of one or more polynucleotides encoding the native peptide; or b) by random shearing or digestion of cDNA or DNA from the cell or pathogen of interest. The method of claim 17, wherein

19. The method of any one of claims 16 to 18, wherein the MHC-peptide complex library encodes at least 10, at least 50, at least 200, or at least 1000 peptide candidates.

20. The method of any one of claims 16 to 19, wherein the MHC-peptide complex library is encoded by a plurality of viral vectors.

21. The method of any one of claims 1 to 20, wherein the antibody is directed against MHC-I or MHC-II, or the antibody is directed against CD3γ, CD3δ or CD3ε.

22. The method according to any one of claims 1 to 21, wherein reporter cells efficiently expressing MHC on their surface are enriched by FACS- or MACS-based cell sorting.

23. The method of any one of claims 1 to 22, wherein the sequence of the candidate peptide displayed on the cell surface is determined by PCR and sequencing.

24. The method of any one of claims 1 to 23, wherein the candidate peptide is for use as a vaccine.

25. 25. The method of claim 24, wherein the vaccine is a tumor-specific antigen (TSA)-based cancer vaccine.

26. The method of any one of claims 1 to 23, wherein the candidate peptide is for use in inducing immune tolerance to at least one epitope that it contains.

27. 27. The method of any one of claims 1 to 23 or 26, wherein the candidate peptide is for use to block a TCR in the context of an MHC molecule.

28. 27. The method of any one of claims 1 to 23 or 26, wherein the candidate peptide is for use in MHC-mediated toxin delivery to a cell, such as to a T cell.

29. 27. The method of any one of claims 1 to 23 or 26, wherein the candidate peptide is for use in redirecting T cells using MHC-CAR.

30. The method of any one of claims 1 to 23, wherein the candidate peptide is for use in an immunogenicity test.

31. The method of any one of claims 1 to 23 or 30, wherein the candidate peptide is for use in a T cell reactivity test.

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