Mammal mhc peptide display as epitope selection tool for vaccine design

Abstract

The present invention relates to a method for identifying candidate peptides presented by the major histocompatibility complex (MHC) for vaccination, induction of immune tolerance, blocking of TCR, MHC-mediated toxin delivery and redirection of T cells with CAR, for immunogenic tests and other in vitro T cell reactivity tests. The invention further relates to a method for determining the MHC binding affinity of a candidate peptide.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980035785.7. The original application is the Chinese national phase application of the international application PCT / EP2019 / 059563 with the application date of April 12, 2019.

[0002] The present invention relates to a method for identifying candidate peptides presented by the major histocompatibility complex (MHC) for use in in vivo and / or in vitro interventions, including vaccination, induction of immune tolerance, blocking TCR and MHC-mediated toxin delivery, for immunogenicity testing and other in vitro T cell reactivity tests.

[0003] T cells play a central role in the immune system. The function of T cells depends primarily 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 effective in presentation. There are different T cell sets with different functions, in particular CD4+T cells or CD8+T cells that recognize MHCII-bound epitopes presented by professional APCs or MHC I-bound epitopes presented by most cell types, respectively. MHC molecules play an important role in presenting cell antigens or epitopes to T cells in the form of short linear peptides.

[0004] MHC is composed of α-chain and β-chain and peptides bound in the grooves formed by these chains. The stability of the complex is highly dependent on peptide binding, so empty MHC molecules are downregulated and degraded from the cell surface. However, empty MHC molecules are present on the cell surface and can bind extracellular peptides and present them to T cells. In addition, two different isomers of empty MHC have been described (Rabunowitz et al., Immunity, 9, 699–709 (1998)): less stable active isomers and more stable inactive isomers. The MHC repertoire of an individual is polygenic and the MHC genes are highly polymorphic. Therefore, the set of MHC that an individual can express is likely to be very different from the set of another random individual.

[0005] The MHC-bearing peptide interacts with the T cell receptor (TCR) on the surface of the T cell, leading to T cell activation. APCs, such as dendritic cells or macrophages, can internalize antigens by phagocytosis or receptor-mediated endocytosis.

[0006] In order to control the activity of T cells, it is important to determine their specific peptide ligands and control or regulate the interaction between MHC-peptide complexes and TCR. Therefore, it is important to determine the binding affinity of peptides to MHC. Suitable peptides or MHC-peptide complexes can be used for a variety of 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 block TCR, deliver toxins to T cells in vivo or redirect T cells with MHC-CAR containing chimeric antigen receptors (Jyothi et al., Nat. Biotech, 20, 1215–1220 (2002)) to induce immune tolerance to epitopes (otherwise the epitopes will activate T cells specific to the epitopes). Vaccines, especially the development of cancer vaccines based on tumor-specific antigens (TSAs) that activate T cells to eliminate cancer cells is one of the main goals of modern medicine. Tumor-specific peptides, so-called neoantigens, are only present in tumor cells and are completely absent in normal tissues. Such tumor-specific peptides are produced by tumor-specific DNA changes that lead to the formation of new protein sequences. Due to the genetic heterogeneity of cells within and between different tumors, the set of neoantigens present in a patient's tumor is largely specific to that 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. Once tumor-specific peptides are presented on the cell surface, they can be recognized and destroyed by the immune system. Vaccines containing tumor-specific peptides can therefore stimulate the immune system to detect and destroy cancer cells that present these molecules on their surface.

[0007] Recently, it has been demonstrated that TSAs caused by somatic mutations accumulated in tumors are often 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 tissue can reveal such somatic mutations that are specific to the patient's tumor cells. However, many mutations are usually detected (Vormehr et al., Curr Opin Immunol 39, 14–22 (2016)), but due to technical problems and in order to minimize the risk of autoimmunity, only a small amount of TSA (i.e., tumor-specific peptides) should be mixed in a vaccine. The selection of these TSAs is crucial because antigen presenting cells (APCs) do not present all possible peptides on their MHC molecules, but present a subset of specific MHC alleles that are suitable for patients. For many applications involving T cells, it is crucial to select peptides that can be effectively presented by APCs, especially tumor-specific peptides, and it is still a bottleneck for tumor vaccine design. To address this problem, candidate peptides are often ranked according to their MHC binding affinities, which are predicted with the aid of bioinformatics analysis (Sahin et al., Nature 1–19 (2017); Ott et al., Nature 547, 217–221 (2017)). However, recent data indicate that bioinformatics predictions for peptide presentation are quite poor, especially for MHC class II epitopes (Sofron et al., Eur J Immunol 46, 319–328 (2015)). A “wet lab” method for measuring the binding of T cells to MHC-peptide complexes (via their TCR) using an antigen presenting reporter cell line has been disclosed; WO2016097334A1. The reporter cell line expresses a chimeric antigen receptor comprising an MHC-peptide complex that activates a reporter gene in the reporter cell line after binding to the TCR of the T cell. Indirectly, the stability of the MHC-peptide complex is also measured with this method, since only sufficiently stable MHC-peptide complexes will appear on the surface of the reporter cell line, interact with the T cells and ultimately activate the reporter. However, this method does not allow specific measurement of the stability of the MHC-peptide complex, since the activation of the reporter also depends on the affinity of the TCR for the peptide epitope and other immunomodulatory factors.

[0008] Therefore, there is a need to provide devices and methods for reliably determining peptides, particularly TSA, that are efficiently presented by APCs.

[0009] The present invention now satisfies this need in that it provides such an apparatus and method as is more particularly defined in the claims and the following detailed description of the invention.

[0010] In its main aspects, the present invention relates to:

[0011] 1. A method for identifying candidate peptides presented by the major histocompatibility complex (MHC), the method comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting reporter cells showing surface expression of the MHC-peptide complex, and determining the sequence of the candidate peptide presented on the cell surface.

[0012] 2. The method according to aspect 1, the method comprising

[0013] (i) generating a library comprising candidate peptides, the candidate peptides being cloned upstream and in-frame with a recombinant MHC β-chain and / or α-chain;

[0014] (ii) transducing such a library together with the corresponding MHC α-chain and / or β-chain into a suitable reporter cell line;

[0015] (iii) detecting and isolating cells expressing one or more MHC-peptide complexes on the cell surface;

[0016] (iv) isolating DNA from cells presenting one or more MHC-peptide complexes on their cell surface;

[0017] (v) determining the sequence of the candidate peptide encoded by the vector integrated in the DNA isolated from cells presenting MHC-peptide complexes on the cell surface.

[0018] 3. A method for identifying candidate peptides presented by the major histocompatibility complex (MHC), the method comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting reporter cells showing surface expression of the MHC-peptide complex, and determining the level of surface expression of the MHC-peptide complex, as well as the sequence of the candidate peptide presented on the cell surface.

[0019] 4. The method according to aspect 3, the method comprising

[0020] (i) generating a library comprising candidate peptides, the candidate peptides being cloned upstream and in-frame with a recombinant MHC β-chain and / or α-chain;

[0021] (ii) transducing such a library together with the corresponding MHC α-chain and / or β-chain into a suitable reporter cell line;

[0022] (iii) detecting and isolating cells expressing one or more MHC-peptide complexes on the cell surface;

[0023] (iv) determining the level of cell surface expression of the one or more MHC-peptide complexes;

[0024] (v) isolating DNA from cells that present one or more MHC-peptide complexes on their cell surface;

[0025] (vi) determining the sequence of the candidate peptide encoded by the vector integrated into said DNA isolated from a cell presenting the MHC-peptide complex on the cell surface.

[0026] 5. The method according to any one of aspects 1 to 4, wherein the MHC molecule is an MHC class II molecule comprising an extracellular MHC class II α chain and a transmembrane domain, and an extracellular MHC class II β chain and a transmembrane domain.

[0027] 6. The method according to any one of aspects 1 to 4, wherein the MHC molecule is an MHC class I molecule comprising an extracellular MHC class I α chain and a transmembrane domain and β-2 microglobulin.

[0028] 7. The method according to any one of aspects 1 to 4, wherein 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.

[0029] 8. The method according to aspect 6 or 7, wherein the MHC α chain carries a Y84A mutation.

[0030] 9. The method according to any one of aspects 1 to 8, wherein each chain of the MHC molecule comprises a transmembrane domain.

[0031] 10. The method according to any one of aspects 1 to 9, wherein the transmembrane domain is a native transmembrane domain of an MHC molecule.

[0032] 11. A method according to any one of aspects 1 to 9, wherein the transmembrane domain is a transmembrane domain of a heterologous molecule such as TCRα / β, TCRγ / δ, CD3γ / δ / ε / ζ, CD4 or CD8α / β.

[0033] 12. The method according to any one of aspects 1 to 11, wherein the reporter cell line is a mammalian cell line.

[0034] 13. The method according to any one of aspects 1 to 12, wherein the reporter cell line is a cell line lacking the MHC class II peptide loading machinery.

[0035] 14. A method according to aspect 13, wherein the reporter cell line is a T cell hybridoma.

[0036] 15. The method according to any one of aspects 1 to 14, wherein the reporter cell line is a cell line lacking a functional TAP1, TAP2 and / or beta-2-microglobulin gene.

[0037] 16. The method according to aspect 15, wherein the reporter cell line is a T cell hybridoma having a defective or deleted TAP1, TAP2 and / or β-2-microglobulin gene.

[0038] 17. The method according to any of the preceding aspects, wherein the candidate peptide is a tumor-specific peptide carrying a mutation derived from an individual tumor.

[0039] 18. The method according to aspect 17, wherein the mutation is a SNV.

[0040] 19. The method according to aspects 1 to 16, wherein the candidate peptide is an antigen that evokes an immune response.

[0041] 20. The method according to aspects 1 to 16, wherein the candidate peptide is a compound that is tested for immunogenicity.

[0042] 21. The method according to any one of the preceding aspects, wherein reporter cells that efficiently express MHC on their surface are enriched by FACS-based or MACS-based cell sorting.

[0043] 22. The method according to any one of the preceding aspects, wherein the sequence of the candidate peptide presented on the cell surface is determined by PCR and sequencing.

[0044] 23. The method according to any one of aspects 1 to 22, wherein the candidate peptide is used as a vaccine.

[0045] 24. The method according to aspect 23, wherein the vaccine is a tumor specific antigen (TSA) based cancer vaccine.

[0046] 25. The method according to any one of aspects 1 to 22, wherein the candidate peptide is used to induce immune tolerance against at least one epitope it comprises.

[0047] 26. The method according to any one of aspects 1 to 22 or 25, wherein the candidate peptide is used to block TCR in the context of an MHC molecule.

[0048] 27. A method according to any one of aspects 1 to 22 or 25, wherein the candidate peptide is used for MHC-mediated toxin delivery to cells, particularly T cells.

[0049] 28. A method according to any one of aspects 1 to 22 or 25, wherein the candidate peptide is used to redirect T cells with MHC-CAR.

[0050] 29. The method according to any one of aspects 1 to 22, wherein the candidate peptide is used for immunogenicity testing.

[0051] 30. The method according to any one of aspects 1 to 22 or 29, wherein the candidate peptide is used in a T cell reactivity test.

[0052] 31. A method for determining the MHC binding affinity of a candidate peptide, the method comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting reporter cells presenting the MHC-peptide complex on the surface of the reporter cells, and determining the level of such presentation / expression.

[0053] 32. The method according to aspect 31, comprising:

[0054] (i) generating a library comprising candidate peptides cloned upstream of a recombinant MHC α domain or β domain linked to at least one transmembrane region;

[0055] (ii) transducing this library together with the corresponding MHC α domain or β domain into a suitable reporter cell line;

[0056] (iii) detecting cells presenting one or more MHC-peptide complexes on their cell surface;

[0057] (iv) determining the level of such presentation / expression.

[0058] The present invention provides a method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), the method comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting the reporter cells showing surface expression of the MHC-peptide complex, and determining the sequence of the candidate peptide presented on the cell surface.

[0059] The present invention is based at least in part on the unexpected discovery that the surface expression level of recombinant peptide-MHC complexes in mammalian cell lines can be directly and quantitatively measured. Further surprisingly, this direct measurement of peptide-MHC complexes is feasible by standard laboratory techniques based on antibody staining and flow cytometry, and without complex co-culture systems involving T cells and / or complex reporter systems. Although those skilled in the art may know that predictions based on bioinformatics methods are not always accurate, it is still surprising that the bioinformatics methods of the prior art are completely unable to predict the binding of peptides determined by the methods disclosed herein to specific MHC variants. Further surprisingly, regardless of the specific peptide and / or MHC variant to be analyzed, the MHC contained in the recombinant MCR (peptide-MHC-TCR chimera disclosed in WO2016097334 A1) can be detected with CD3e antibodies. Without being bound by theory, the methods disclosed herein allow the biological variability of peptide presentation caused by MHC to be measured primarily without the need to introduce technical variability that may be present when different antibodies are used to detect different MHC variants.

[0060] Peptides can be covalently bound to MHC molecules to overcome the need for peptide processing / loading mechanisms. The method has been used 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 produce stable MHC-tetramers (Crawford et al., Immunity 8, 675–682 (1998)), to produce mice expressing single peptides on their APCs (Ignatowicz et al., Cell 84, 521–529 (1996)) or designed in CAR to redirect T cells to other T cells (Jyothi et al., Nat Biotechnol 20, 1215–1220 (2002)). Typically, MHC molecules must be mutated in order to be expressed on the surface of yeast or insect cells (Wen et al., Protein Eng Des Sel 24, 701-709 (2011)), and in some studies some measures are taken to stabilize the peptide in the groove of the MHC by incorporating additional disulfide bridges (Truscott et al., J. Immunol. 178, 6280-6289 (2007)).

[0061] However, it was shown in the context of the present invention that in cells that are unable to load MHC peptides, recombinant MHC molecules without bound peptides are either undetectable or unstable (detectable at low levels) on the cell surface even when overexpressed ( Figure 4 ).

[0062] Thus, even if present, the relatively stable isoform of empty MHC described by Rabunowitz (Rabunowitz et al., Immunity, 9, 699–709 (1998)) does not persist for long on the surface of non-professional antigen presenting cells. Importantly, it was shown in the context of the present invention that not all peptides tethered to recombinant MHC lead to efficient cell surface expression of the MHC complex. In fact, some prevent expression on the surface ( Figure 2a and Figure 2b ). Thus, high expression of recombinant MHC molecules with bound candidate peptides on the surface of a suitable reporter cell line compared to uninfected reporter cells and / or MHC-transduced reporter cells without covalently bound peptides or unstable MHC-peptide complexes can be used as an indicator of effective peptide presentation by MHC. In other words, reporter cells showing surface expression of recombinant MHC-peptide complexes can be detected by comparing the surface expression of said MHC-peptide complexes to the background signal of uninfected reporter cells or MHC-transduced reporter cells without covalently bound peptides or unstable MHC-peptide complexes. In addition, the level of such presentation / expression is measured ( Figure 2a and Figure 7 ) allows the peptides to be ranked according to their MHC binding capacity. The detection and / or ranking of peptides presented by MHC may, if necessary, include one or more normalization steps, such as normalization of the background signal of the corresponding MHC variants of the candidate peptide or random peptide that do not have covalent binding or exert unstable interactions with the candidate peptide or random peptide. If necessary, the binding affinity of the candidate peptide to MHC can be determined based on the measured surface expression of the recombinant MHC-peptide complex using normalization and / or standardization methods well known in the art.

[0063] In certain embodiments, the ranking of candidate peptides and / or the determination of the binding affinity of candidate peptides to MHC can be directly correlated with the surface expression of recombinant MHC-peptide complexes, and the surface expression of recombinant MHC-peptide complexes is determined by CD3 staining using anti-CD3e, anti-CD3δ or anti-CD3γ antibodies. Preferably, for said correlation with said CD3 staining, the MHC is contained in an MCR.

[0064] Here we demonstrate 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.

[0065] Furthermore, it was found that within the scope of the present invention, when using an MHC-TCR fusion protein (MCR) consisting of a peptide attached to the native extracellular domain of the MHC fused to the transmembrane unit of the TCR, not all MHC-II class peptide combinations were effectively expressed on the cell surface. For example, when testing different MHC-peptide combinations, surface differential expression of influenza matrix protein 1 (MP1)-derived peptides was observed. Importantly, predictive analysis of peptide binding did not indicate a similar pattern of MHC peptide binding affinity.

[0066] In various embodiments, the present invention relates to a method for identifying a candidate peptide according to the aforementioned embodiments as described herein, wherein the method comprises the following steps:

[0067] (i) generating a library comprising candidate peptides, in particular a library comprising candidate peptides cloned upstream and in frame with a recombinant MHC β chain and / or α chain;

[0068] (ii) transducing this library together with the corresponding MHC α chain and / or β chain into a suitable reporter cell line;

[0069] (iii) detecting cells presenting one or more MHC-peptide complexes on their cell surface;

[0070] (iv) isolating DNA from cells that present one or more MHC-peptide complexes on their cell surface;

[0071] (v) Determining the sequence of the candidate peptide within the MHC-peptide complex presented on the cell surface.

[0072] As used herein, the term "major histocompatibility complex (MHC)" refers to MHC class I and MHC class II genes and the proteins encoded therefrom. The terms MHC-I, MHC-II, MHC-1, and MHC-2 are used differently herein to indicate these classes of molecules.

[0073] In certain embodiments, the present invention relates to a method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), the method comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, detecting reporter cells that display surface expression of the MHC-peptide complex, and determining the level of surface expression of the MHC-peptide complex, as well as the sequence of the candidate peptide presented on the cell surface.

[0074] In various embodiments, the present invention relates to a method for identifying a candidate peptide according to the aforementioned embodiments as described herein, wherein the method comprises the following steps:

[0075] (i) generating a library comprising candidate peptides cloned upstream and in frame with recombinant MHC β chain and / or α chain;

[0076] (ii) transducing this library together with the corresponding MHC α chain and / or β chain into a suitable reporter cell line;

[0077] (iii) detecting and isolating cells that express one or more MHC-peptide complexes on their cell surface;

[0078] (iv) determining the level of cell surface expression of said one or more MHC-peptide complexes;

[0079] (v) isolating DNA from cells that present one or more MHC-peptide complexes on their cell surface;

[0080] (vi) determining the sequence of the candidate peptide encoded by the vector integrated into said DNA isolated from a cell presenting the MHC-peptide complex on the cell surface.

[0081] In mammals, there are two types of MHC molecules called class I and class II. MHC class I molecules are present in almost all nucleated cells in the body and are recognized by CD8+ cells. MHC class II molecules are mainly expressed on professional APCs of the immune system and are recognized by CD4+ cells. The molecules are further subdivided by antigen subtypes. Human MHC class I molecules, also known as human leukocyte antigens (HLA), are divided into HLA-A, -B and -C. Human MHC class II molecules are divided into HLA-DR, -DQ and -DP.

[0082] MHC class I molecules are found in almost every cell of the human body. MHC class I molecules are heterodimers composed of a monomorphic (in humans) non-MHC-encoded light (β) chain protein of approximately 12 kDa (called β 2 Microglobulin (β 2 The heavy α chain is a polymorphic transmembrane glycoprotein of approximately 45 kDa, consisting of three extracellular domains, each of which contains approximately 90 amino acids (the α at the N-terminus). 1 , α 2 and α 3 ), a transmembrane region of about 40 amino acids and a cytoplasmic tail of about 30 amino acids. 1 and α 2 The membrane distal domain forms a peptide binding groove or cleft that is large enough to bind peptides of 8-10 amino acids, while the α 3 The domain is close to the plasma membrane. The presented peptide is bound by the peptide binding groove in the α 1 and α 2 The central region of the domain is fixed.

[0083] MHC class II molecules are found only in a few specialized cell types, especially antigen presenting cells (APCs), such as macrophages, dendritic cells, and B cells. MHC class II molecules contain two different polypeptide chains, a 33kDa α chain and a 28kDa β chain, which are conjugated by non-covalent interactions. Similar to MHC class I molecules, MHC class II molecules are membrane-bound glycoproteins that contain an extracellular domain, a transmembrane segment, and a cytoplasmic tail. Each chain in these non-covalent heterodimeric complexes contains two extracellular domains, a transmembrane domain, and a cytoplasmic tail. The extracellular domain is an α chain for the α chain and a β chain for the β chain, respectively. 1 and α 2 Domain and β 1 and β 2 Domain. The membrane distal domain of class II molecules consists of α 1 and β 1 The domains form a peptide-binding groove or cleft of sufficient size to bind peptides, which are usually 13-18 amino acids in length, or 10-18 amino acids or longer. The membrane-proximal pair of class II, α 2 and β 2 , with structural similarity to the Ig constant (C) domain. There are three pairs of class II α and β chain genes in humans, called HLA-DR, HLA-DP and HLA-DQ. The highest level of polymorphism has been recorded for HLA-DR.

[0084] As used herein, a candidate peptide refers to a peptide having 8-18 amino acids or up to 50 amino acids in length. 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 contained in an MHC-peptide complex encoded by a recombinant nucleic acid.

[0085] In various embodiments, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein the MHC molecule is an MHC class II molecule comprising an extracellular MHC class II α chain and a transmembrane domain and an extracellular MHC class II β chain and a transmembrane domain.

[0086] In other embodiments, 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.

[0087] In 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.

[0088] For MHC class I molecules, it has been shown that the Y84A mutation in the α chain opens the peptide binding pocket, allowing for better accommodation of attached peptides (Mitaksov, V. et al. Chem Biol 14, 909-922 (2007)).

[0089] Thus, in a specific embodiment, the MHC alpha chain of the MHC class I molecule carries a Y84A mutation. For example, the Y84A mutation can be introduced by site-directed mutagenesis.

[0090] In some embodiments, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein the MHC molecule comprises one or two transmembrane domains.

[0091] In various embodiments, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein the above-mentioned transmembrane domain is a native transmembrane domain of an MHC molecule.

[0092] 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.

[0093] In a specific embodiment, the transmembrane domain is a transmembrane domain of a heterologous molecule, including but not limited to TCRα / β, TCRγ / δ, CD3γ / δ / ε / ζ, CD4 or CD8α / β.

[0094] The reporter cell line for implementing the method of the present invention can be any cell line or derivative of the cell line that can be used for biological applications. Suitable cell lines or derivatives thereof are, for example, but not limited to, humans, mice, rats, monkeys, hamsters, fish, frogs or insect sources or yeast, for example, but not limited to HEK, CHO, 3T3, hybridoma, HeLa, Sf9, Schneider 2 cells, Jurkat, HUVEC, HL-60, MCF-7, Saos-2 cells, IMR-90, Raw264.7, PC3, Vero, Cos, GH3, PC12, dog MDCK, African clawed frog A6 or zebrafish AB9.

[0095] In a specific embodiment, the reporter cell line is a mammalian cell line. 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.

[0096] In particular, reporter cell lines suitable for the present invention, preferably mammalian cell lines, are cell lines lacking the ability to display endogenous protein-derived peptides initially present in cells. Therefore, cells should only present MHC peptide complexes carrying candidate peptides derived from a peptide library of interest. The processing of peptides and their loading on MHC molecules for display is a multi-step process involving a variety of molecules constituting processing and presentation mechanisms. MHC class I molecules are loaded with peptides via a transporter called TAP (transporter associated with antigen processing), which transports 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 and loaded with peptides before they are transferred to the cell surface. The peptide processing / loading mechanism for MHC class II molecules is present only in APCs.

[0097] According to the above, in one embodiment, a reporter cell line suitable for practicing the method of the invention is a cell line lacking the MHC class II peptide loading machinery. Preferably, the reporter cell line lacking the MHC class II peptide loading machinery is a mammalian cell line.

[0098] In a specific embodiment, the reporter cell line is a T cell hybridoma.

[0099] In another embodiment, a reporter cell line suitable for practicing the methods of the invention is a cell line lacking a functional TAP1, TAP2 and / or beta-2-microglobulin gene.

[0100] In a specific embodiment, 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 defective or deleted TAP1, TAP2 and / or beta-2-microglobulin genes include, but are not limited to, T cell and B cell clones or hybridomas, HEK cells or 3T3 cells.

[0101] Exome sequencing can be used to identify TSAs that are uniquely present in tumors. For example, TSAs can be identified by sequencing tumor DNA derived from the patient and comparing it to DNA derived from healthy subjects. TSAs can also be identified for individual patients by sequencing tumor and normal DNA from each patient. Based on this analysis, multiple candidate peptides, each carrying a tumor-derived mutation, can be used to generate MHC-peptide complexes. Therefore, in one embodiment of the present invention, a peptide library is used. These peptides can carry tumor-derived mutations or be natural peptides.

[0102] Therefore, in various embodiments, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein said candidate peptide is a tumor-specific peptide carrying an individual tumor-derived mutation.

[0103] 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.

[0104] In a specific embodiment, the tumor-derived mutation is a single nucleotide variation (SNV). SNVs include, but are not limited to, various mutations of p53, KRAS, and BRAF.

[0105] In another embodiment, the candidate peptide is an antigen that elicits an immune response. For example, the peptide can be derived from a pathogen.

[0106] In another embodiment, the candidate peptide is a compound that is tested for immunogenicity.

[0107] In one embodiment, the present invention relates to a method of using a candidate peptide library, wherein the library contains mutant forms of natural peptides. For example, the natural peptide may be known not to be effectively presented by a given MHC molecule. Therefore, in order to screen for peptide mutants that are more effectively presented by a given MHC molecule, a library containing mutant peptides can be used.

[0108] In another embodiment, the present invention relates to a method for using a candidate peptide library, wherein the library comprises peptides generated by random diversion or digestion of cDNA or DNA derived from cells or pathogens of interest. Such a library will cover all peptides present in such cells. For example, generating an MHC display library from cancer tissue or tissue subjected to autoimmune attack will allow the definition of all tissue peptides that may be presented by a given MHC. Therefore, in the future, testing will not be required and a table can be queried to see whether a specific peptide is effectively presented by a given MHC.

[0109] In one embodiment, a library of MHC-peptide complexes is produced using a recombinant expression vector. A recombinant expression vector is a replicable DNA construct comprising the following components: (1) one or more agents that have a regulatory role in gene expression, such as a promoter, an operator, or an enhancer, operably linked to (2) a nucleotide sequence encoding a desired protein (e.g., an MHC-peptide complex) that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation start and stop sequences. The choice of promoter and other regulatory elements typically varies depending on the intended reporter cell line. An expression vector is typically in the form of a "plasmid," which refers to a circular double-stranded DNA loop that is not bound to a chromosome in its vector form. Suitable prokaryotic expression vectors include plasmids from Escherichia coli, such as Col E1, pCR1, pBR322, pMB9, and derivatives thereof, broader host range plasmids, such as RP4, phage DNA, such as phage λ and its various derivatives, M13, and the like. Other 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 other forms of expression vectors that have equivalent functions and will be 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 such as pMY, pMX, pSIR, adenoviruses such as pAd, etc., can also be used. In expression vectors, regulatory elements that control transcription or translation can generally be derived from mammalian, microbial, viral or insect genes. Replication ability generally conferred by a replication origin and selection genes that help identify transformants can be additionally incorporated. Expression vectors containing regulatory elements from eukaryotic viruses are generally used for eukaryotic expression vectors, such as SV40 vectors, papillomavirus 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, SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0110] "Promoter" is defined as an array of nucleic acid control sequences that direct nucleic acid transcription. As used herein, a promoter includes an essential nucleic acid sequence near the transcription start site, such as in the case of a polymerase II type promoter, i.e., a TATA element. The promoter also optionally includes a distal enhancer or repressor element, which may be located up to several thousand base pairs from the transcription start site. Promoters for 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) promoters, human immunodeficiency virus (HIV) promoters, such as HIV long terminal repeats (LTR) promoters, Moloney virus promoters, ALV promoters, cytomegalovirus (CMV) promoters, such as CMV immediate early promoters, Epstein Barr virus (EBV) promoters, Rous sarcoma virus (RSV) promoters, and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Other examples of suitable promoters include the CAG promoter (a hybrid promoter comprising a CMV enhancer, a chicken β-actin promoter and a rabbit β-globin splice acceptor and a poly(A) sequence).

[0111] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, an array of transcription factor binding sites or a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0112] By using standard techniques known in the art, the expression vector is transduced into the host cell, so that the MHC-peptide library is introduced into a suitable reporter cell line. Suitable methods are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). In order to produce a pseudoretrovirus for transduction, a packaging cell line (such as Phoenix cell line) containing a construct (in this case, a peptide carrying an MHC chain) of a viral genome consisting of LTR, packaging signals and a gene of interest is transiently transfected to continuously express retroviral proteins GAG, POL and ENV. Alternatively, a suitable cell line, such as HEK, 3T3 or other, is transiently transfected with a vector mixture of a viral genome consisting of LTR, packaging signals and a gene of interest, respectively. These common strategies ensure that defective pseudoretroviruses are produced, and the pseudoretroviruses can infect target cells and introduce the gene of interest into their genomic DNA. However, the infected target cells cannot produce retroviruses, since pseudoretroviruses do not carry the gag, pol, and env genes in their genome. Alternatively, the MHC-peptide library can be introduced into a suitable reporter cell line by transfection with lipid-, calcium phosphate-, cationic polymer-, or DEAE-dextran-based reagents or by electroporation.

[0113] The terms "infection" and "transduction" of reporter cell lines are used interchangeably herein.

[0114] Methods for detecting cells presenting MHC-peptide complexes exposed on the cell surface 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).

[0115] Thus, in various embodiments, the invention relates to a method according to any of the preceding embodiments as described herein, wherein reporter cells that efficiently express MHC on their surface are enriched by fluorescence activated cell sorting (FACS).

[0116] FACS refers to a method for separating a cell population into one or more subpopulations based on the presence, absence, or level of presence of one or more specific polypeptides expressed by the cells. FACS relies on the optical properties of individual cells, including fluorescence, to classify cells into subpopulations. Cell sorters suitable for practicing the methods described herein are well known in the art and are commercially available. Exemplary cell sorters include the MoFlo sorter (DakoCytomation, Fori Collins, Colo.), the FACSAria TM 、FACSArrayTM 、FACS Vantage TM BD TM LSR II and FACSCaiibur TM (BD Biosciences, San Jose, Calif.) and other equivalent cell sorters produced by other commercial suppliers such as Sony, BioRad, and Beckman Coulter.

[0117] In another embodiment, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein reporter cells that efficiently express MHC on their surface are enriched by MACS-based cell sorting.

[0118] "MACS" refers to a method for dividing a cell population into one or more subpopulations based on the presence, absence or level of presence of one or more MACS selectable polypeptides expressed by the cells. MACS relies on the magnetic susceptibility properties of labeled individual cells in order to classify cells into subpopulations. For MACS, magnetic beads (e.g., magnetic beads available from Miltenyi Biotec Bergisch Gladbach, Germany; 130-048-402) can be used as markers. MACS cell sorters suitable for implementing the methods described herein are well known in the art and are commercially available. Exemplary MACS cell sorters include autoMACS Pro Separator (Miltenyi Biotec).

[0119] In various embodiments, the cells may be contacted with antibodies specific for MHC-I or MHC-II or with antibodies for detecting proteins associated with the hybrid. For example, if the MHC extracellular domain is fused to the TCR transmembrane region, antibodies that detect CD3γ, CD3δ, or CD3ε may be used. The antibody may be directly conjugated to a detectable label. Alternatively, a second antibody conjugated to a detectable label and specific for the first antibody may be contacted with the cells. Detectable labels suitable for use include any compound detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels in the present invention include biotin, magnetic beads (e.g., Dynabeads TM ), fluorescent labels (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, dansyl chloride, 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. Various suitable fluorescent labels are further described in, for example, The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies (11th Edition).

[0120] Depending on how many fluorescent markers are used, the sorting produces a non-fluorescent cell population and at least one fluorescent cell population. The presence of at least one cell population carrying fluorescent cells indicates that at least one candidate peptide is effectively presented by the APC. Thus, FACS is able to sort cell populations to produce cell populations enriched for cells containing surface-exposed MHC-I or MHC-II.

[0121] Embodiments of the present invention adopt DNA separation methods known to those skilled in the art. Generally, the purpose is to separate the DNA present in the nucleus from other cellular components. The separation of DNA usually starts from the lysis or decomposition of the cell. This process is essential for destroying the protein structure and allows the nucleic acid to be released from the nucleus. The lysis is carried out in a saline solution, which contains a detergent to denature proteins or proteases (enzymes that digest proteins) such as proteinase K, or both in some cases. This causes cell rupture and membrane dissolution. The method of DNA separation includes but is not limited to phenol: chloroform extraction, high salt precipitation, alkaline denaturation, ion exchange column chromatography, resin binding and paramagnetic bead binding.

[0122] Embodiments of the present invention employ cDNA production methods known to those skilled in the art. Typically, the goal is to convert isolated RNA present in a cell into DNA, called copy DNA, so that it can be used as a template for a polymerase chain reaction (PCR). Isolation of RNA typically begins with the lysis or decomposition of the cell. This process is essential for destroying protein structures and allows the release of nucleic acids therefrom. The lysis is typically performed in a phenol-containing solution (e.g., TRIzol TM This causes the cells to disintegrate and the membranes to dissolve, allowing the RNA to be separated from other cellular components. The separated RNA is then passed through a reverse transcriptase (e.g., Superscript TM , Goscript TM ) was converted into cDNA.

[0123] The sequence of the candidate peptide within the MHC-peptide complex presented on the cell surface is then amplified by PCR and can be sequenced by any method known in the art.

[0124] In various embodiments, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein the sequence of the candidate peptide is determined by PCR and sequencing.

[0125] In one embodiment, the sequence of the candidate peptide is determined by digital PCR. Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR or dePCR) is an improvement of the traditional polymerase chain reaction method that can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA or RNA).

[0126] Sequencing can also be performed using microfluidics. Microfluidics involves miniature devices that handle small amounts of fluid. Since microfluidics can accurately and reproducibly control and dispense small fluid volumes, especially fluids less than 1 μl, the application of microfluidics provides significant cost savings. The use of microfluidics reduces cycle time, shortens the time to obtain results and increases throughput. In addition, the introduction of microfluidics enhances system integration and automation. Microfluidic reactions are usually carried out in microdroplets.

[0127] In some embodiments, sequencing is performed using second generation sequencing (or next generation or Next-Gen), third generation (or next next generation) or fourth generation (or N3-Gen) sequencing technology, including but not limited to pyrosequencing, ligation sequencing, single molecule sequencing, sequence-by-synthesis (SBS), massively parallel cloning, massively parallel single molecule SBS, real-time massively parallel single molecule, real-time massively parallel single molecule nanopore technology. Morozova and Marra in Genomics, 92:255 (2008) review some of these technologies.

[0128] In some embodiments, nucleic acids are amplified before, after, or simultaneously with sequencing. Illustrative 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). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

[0129] In one embodiment, the invention relates to a method according to any one of the preceding embodiments as described herein, wherein said candidate peptide is used as a vaccine.

[0130] In a specific embodiment, the vaccine is a tumor specific antigen (TSA) based cancer vaccine.

[0131] The term "vaccination" or equivalents are well known in the art. For example, the term vaccination can be understood as a process of increasing a subject's immune response to an antigen and thereby increasing the ability to resist or overcome a disease. "Vaccine" should be understood to refer to a composition used to generate immunity for the prevention and / or treatment of a disease (e.g., cancer). Therefore, a vaccine is a drug comprising an antigen and is intended to be used in humans or animals to produce specific defenses and protective substances through vaccination. The term "TSA-based cancer vaccine" refers to a vaccine comprising a pooled sample of tumor-specific antigens, for example, comprising at least one, at least two, at least three, at least four, at least five or more tumor-specific peptides. In order for the vaccine to be effective, short linear peptides comprising antigens or epitopes must be presented by MHC on the surface of APCs. The set of MHC variants expressed between different individuals varies greatly.

[0132] Recurrent tumor-derived mutations can serve as public tumor-specific antigens, enabling the development of TSA-based cancer vaccines applicable to a wider patient population. Therefore, the methods of the present invention can be used to identify patients containing certain MHC variants and effectively present these common / public TSAs to the immune system. However, many tumor-derived mutations appear to be derived from patient-specific changes and any of those changes may affect the binding of TSA peptides to MHC. Therefore, the methods of the present invention can also be used to identify patient-specific candidate peptides for personalized vaccines.

[0133] In addition, a patient's tumor typically contains many TSAs, each of which may only be contained in a subset of tumor cells. Therefore, in some embodiments, the collection of candidate TSAs may be composed of TSAs derived from different tumor cell collections. Therefore, an important step in developing cancer vaccines is to select those with high affinity to the MHC expressed in the patient from among the TSA candidates contained in the patient's tumor cells and preferably well expressed in the patient's tumor cells. For optimal immune response, a group of TSAs contained in the vaccine should further activate both CD4+ and CD8+ T cells that recognize MHCII or MHCI-peptide complexes, respectively. Therefore, the method of the present invention can be used to identify TSAs that bind to specific MHCII and MHCI variants of a patient.

[0134] In one embodiment, the candidate peptide is used to induce immune tolerance to at least one epitope contained therein. As used herein, "immune tolerance" refers to a reduction in the host's immunoreactivity to one or more specific antigens. Antigens contain immune determinants / epitopes that, in the absence of tolerance, can cause unwanted immune responses. Immune tolerance can be induced to prevent or improve transplant rejection, autoimmunity, allergic reactions, or other adverse immune responses. Without being bound by theory, immune tolerance can be achieved by producing 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 TCR and MHC peptide complexes. Therefore, the method of the present invention can be used to select an MHC-peptide complex suitable for producing 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.

[0135] Thus, in one embodiment, the candidate peptide is used to block TCR in the context of an MHC molecule. "Blocking of TCR" refers to any agent comprising a peptide-MHC complex that blocks the natural TCR-MHC interaction. In order to predict the TCR blocking efficacy of an MHC peptide complex, the stability of the complex is important.

[0136] In another embodiment, the candidate peptides are used for MHC-mediated toxin delivery to cells, particularly T cells.

[0137] "MHC-mediated toxin delivery" refers to the covalent attachment of a toxic agent (protein or other) to a peptide-MHC tetramer or other MHC polymer to deliver the toxin to a cell, particularly a T cell, leading to cell death. The methods disclosed herein allow for the measurement of the stability of a specific MHC-peptide pair and can therefore be used to develop MHC-peptide complexes for blocking specific TCRs or killing specific T cells, particularly for in vivo application.

[0138] In yet another embodiment, the candidate peptide is used to redirect T cells to other peptide-MHC specific T cells via MHC-CAR.

[0139] Redirecting T cells to other cells with a CAR consisting of antibodies connected to the intracellular chain of CD247 (CD3zeta) has been widely used and has been used in the clinic. T cells, particularly cytotoxic T cells or T cells equipped with MCR, which are composed of peptide-MHC connected to the intracellular chain of CD247 (CD3zeta), can use these receptors to identify T cells specific to these peptide-MHC complexes and kill them (Jyothi et al., Nat.Biotech, 20, 1215–1220 (2002)). The method disclosed herein allows the stability of specific MHC-peptide pairs (MHC-peptide pair) to be measured, and therefore can be used to develop MHC-peptide complexes used in CAR or MCR to redirect killing of specific T cells, particularly for in vivo application of MHC-peptide complexes.

[0140] For many therapies that regulate immune responses, it is necessary to identify the TCR involved in the immune response. There are different ways well known in the art that allow identification of those TCRs and rely on the interaction of TCRs with epitopes contained in MHC-peptide complexes. A key prerequisite is information about the presentation of MHC-peptide complexes on the surface of APCs. If the epitope is not presented by the MHC of the APC used in the in vitro experimental assay, it may not be possible to identify the appropriate TCR that binds to the epitope. In addition, if in such an assay, in the MHC-peptide complex or library presented by APC, one of the peptides is preferentially presented, which may lead to the selection of suboptimal TCRs. Therefore, for optimized TCR recognition and selection processes, it is important to overcome the uncertainty associated with the presentation of MHC-peptide complexes. Therefore, the method of the present invention can be used to provide quantitative information about the affinity of a specific peptide to a specific MHC and select suitable complexes for in vitro assays for identifying TCRs that recognize epitopes or for enriching T cells expressing such TCRs.

[0141] In one embodiment, candidate peptides are tested for T cell reactivity.

[0142] The term "T cell reactivity" as used herein refers to the ability of a substance to cause 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 generally include co-culture of APCs and T cells presenting MHC-peptide complexes. Those assays can be used to expand T cells for cell therapy or to identify TCRs bound to specific epitopes, such as for adaptive T cell therapy or chimeric antigen receptor (CAR) T cell therapy. T cell reactivity tests can be further used for immunogenicity testing.

[0143] In certain embodiments, candidate peptides are used for immunogenicity testing. As used herein, the term "immunogenicity testing" refers to measuring potential immune responses to biotherapeutics. Biotherapeutics can cause immune responses that may affect their safety and effectiveness. 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 biotherapeutics involves measuring antibodies specifically produced against biotherapeutics. With the method of the present invention, peptides effectively presented by MHC molecules, particularly MHC specific to an individual, can be identified, and therefore may cause T cell-mediated immune responses in in vitro procedures. This can help provide a more complete and accurate description of the overall immunogenicity characteristics of a compound, and at the same time can reduce the burden on patients or clinical trial participants.

[0144] In another embodiment, the present invention provides an MHC-peptide complex comprising a candidate peptide covalently bound to the extracellular portion of the α domain or β domain of an MHC molecule, the extracellular portion of the MHC β domain, and at least one transmembrane domain of a heterologous molecule.

[0145] In one embodiment, the present invention provides an MHC-peptide complex according to the aforementioned embodiment, wherein the transmembrane domain is a transmembrane domain of a heterologous molecule such as TCRα / β, TCRγ / δ, CD3γ / δ / ε / ζ, CD4 or CD8α / β.

[0146] In one embodiment, the MHC molecule is an MHC class II molecule comprising an extracellular MHC class II α chain, an extracellular MHC class II β chain, and at least two transmembrane domains.

[0147] 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.

[0148] In yet another embodiment, the MHC molecule is an MHC hybrid molecule comprising a fusion of a peptide, beta-2-microglobulin, and an extracellular MHC class I alpha chain and at least one transmembrane domain.

[0149] In a specific embodiment, the MHC alpha chain of the MHC class I molecule carries a Y84A mutation.

[0150] In various embodiments, the present invention provides MHC-peptide complexes, wherein the candidate peptide is a tumor-specific peptide carrying a mutation derived from an individual tumor.

[0151] In a specific embodiment, the tumor-derived mutation is a single nucleotide variation (SNV).

[0152] In another 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.

[0153] In yet another embodiment, the present invention provides an expression cassette comprising a promoter sequence linked to the recombinant construct of the preceding embodiment.

[0154] In another embodiment, the present invention provides a vector comprising the expression cassette of the preceding embodiment.

[0155] In another embodiment, the present invention provides a cell comprising the MHC-peptide complex of the present invention.

[0156] In one embodiment, the invention relates to a cell according to the preceding embodiments comprising an MHC class II peptide complex, wherein the cell is a mammalian cell lacking MHC class II peptide loading machinery.

[0157] In another embodiment, the cell comprises an MHC class I peptide complex, wherein the cell is a mammalian cell lacking a functional TAP1, TAP2 and / or beta-2-microglobulin gene.

[0158] In yet another embodiment, the cell comprises an MHC class I peptide complex, wherein the cell is a T cell hybridoma having a defective or deleted TAP1, TAP2 and / or beta-2-microglobulin gene.

[0159] The present invention also includes a method for determining the MHC binding affinity of a candidate peptide, the method comprising expressing a recombinant MHC-peptide complex comprising a covalently bound candidate peptide in a reporter cell line, and detecting reporter cells presenting the MHC-peptide complex on the surface of the reporter cells, including measurement of such presentation / expression levels. The reporter cell line can be a mammalian cell line. In one embodiment, the mammalian cell line used exhibits altered properties relative to the native cell line, for example, it carries an enzyme deficiency, a mutation and / or overexpression of an enzyme and / or MHC molecule.

[0160] In one embodiment, the invention relates to a method according to the preceding embodiment, comprising:

[0161] (i) generating a library comprising candidate peptides cloned upstream of a recombinant MHC α domain or β domain linked to a transmembrane region;

[0162] (ii) transducing this library together with the corresponding MHC α domain or β domain into a suitable reporter cell line;

[0163] (iii) detecting cells presenting one or more MHC-peptide complexes on their cell surface;

[0164] (iv) determining the level of such presentation / expression.

[0165] The method of the present invention can also be applied to high-throughput screening. High-throughput screening (HTS) technology is generally used to define large-scale rapid cell processing. In certain embodiments, multiple screenings can be run in parallel with different candidate peptide libraries. High-throughput screening systems are commercially available and generally automate the entire procedure, including all sample and reagent pipetting, liquid distribution, timed incubations, and the final reading of the microplate in the detector suitable for the assay. These configurable systems provide high throughput and rapid startup as well as a high degree of flexibility and customization.

[0166] As used herein, the term "peptide" refers to at least two covalently linked amino acids. Typically, 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 vary from 15 to 24 amino acids in length. Optionally, they can vary from 10 amino acids to 30 amino acids or more in length.

[0167] As used herein, peptide binding to MHC or "binding affinity" or peptide interaction 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.

[0168] As used herein, "covalent binding" or "tethering" of a peptide to MHC refers to a recombinant MHC-peptide complex wherein the covalent bond is produced by genetic engineering.

[0169] As used herein, an MHC-peptide complex refers to a complex in which a peptide is bound to an MHC. This binding may occur via a peptide binding groove or cleft and / or by covalent binding of the peptide to the MHC.

[0170] 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 to the MHC cleft effectively.

[0171] The stability of the MHC-peptide complex refers to the stability of the peptide-MHC interaction occurring in the peptide binding groove, which is associated with the surface expression of the MHC-peptide complex. It does not refer to the stability of the covalent peptide-MHC bond.

[0172] As used herein, the term "antigen" refers to all or part of a peptide or protein that is capable of eliciting an immune response to itself or a portion thereof. Such an immune response may involve the production of antibodies, or the activation of specific immunologically active cells, or both.

[0173] As used herein, the term "peptide loading machinery" refers to all necessary components required to load MHC molecules with peptides. For example, the MHC class I peptide loading machinery is present in almost all cells and includes the TAP transporter, trypsin, and calreticulin. The MHC class II peptide loading machinery persists only in APCs, but may be induced in other cells (such as T cells).

[0174] As used herein, the term "transmembrane domain" is defined as a predominantly hydrophobic amino acid sequence capable of crossing a cell membrane. The term "transmembrane region" is used herein as a synonym for "transmembrane domain".

[0175] As used herein, the term "library" or equivalent refers to a plurality of molecules. In the case of MHC-peptide complexes, the library provides a sufficiently diverse population of peptides to achieve a sufficiently probabilistic range of cellular responses to provide one or more cells that exhibit the 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.

[0176] When it comes to, for example, a cell or nucleic acid, protein or vector, the term "recombinant" means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or a change in a natural nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene not found in the natural (non-recombinant) form of the cell or expresses a natural gene that would otherwise be expressed in an abnormal manner, insufficiently expressed or not expressed at all. Typically, recombinant nucleic acids are initially formed in vitro by manipulating nucleic acids in a form not usually found in nature, for example using polymerases and endonucleases. In this way, an operable connection of different sequences is achieved. Therefore, for the purposes of the present invention, an isolated nucleic acid in linear form or an expression vector formed in vitro by connecting DNA molecules that are not usually connected are considered to be recombinant. It should be understood that once a recombinant nucleic acid is prepared and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulation; however, once such a nucleic acid is produced by recombination, it is still considered to be recombinant for the purposes of the present invention, although it is subsequently replicated non-recombinantly. Similarly, a recombinant protein, such as an MHC-peptide complex of the invention, is a protein produced using recombinant technology (ie, by expressing a recombinant nucleic acid as described above).

[0177] The term "heterologous" when used with reference to portions of a nucleic acid means that the nucleic acid comprises two or more subsequences that are not typically found in the same relationship to each other in nature. For example, the nucleic acid is typically recombinantly produced, having, for example, two or more sequences from unrelated genes that are arranged to make a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein typically refers to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0178] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the blood and lymphatic systems. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0179] Furthermore, in the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article ("a", "an") and the definite article ("the") include plural referents unless the context clearly indicates otherwise.

[0180] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, the present specification including definitions shall prevail. In addition, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0181] The 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 used to illustrate the preferred embodiments of the present invention. It should be understood by those skilled in the art that the technology disclosed in the following examples represents the technology used to work well in the practice of the present invention, and therefore can be considered to constitute the preferred mode of its implementation. However, according to this disclosure, it should be understood by those skilled in the art that many changes can be made in the disclosed specific embodiments, and similar or similar results can still be obtained without departing from the spirit and scope of the present invention.

[0182] Figure 1 Shown are the structures of an MHC-TCR fusion molecule (MCR2) and native MHC class II.

[0183] FIG2 Preparation of constructs encoding MCRs carrying the α and β chains of human MHC molecules (HLA-DRB1_4, DRB1_15 and DRB5_1) with covalently linked influenza matrix protein 1 peptide MP1 103-120 (a) MP1 62-72 (b) or influenza cDNA derived peptide library (c). After transduction of MHC class II deficient cell lines, MCR surface expression was measured by FACS using anti-HLA-DR APC antibody. The graph shows the presence of HLA-DRB1_4-MP1 103-120 Efficient surface expression of the MCR complex containing DRB1_15-MP1 103-120 (a) The MCR was detected only at very low levels. We detected almost no MCR containing DRB5_1-MP1 103-120 (a) MCR, but did detect DRB5_1-MP1 62-72 (b) Efficient expression of MCR. Importantly, similar levels of MCR surface expression were observed when influenza cDNA-derived peptide libraries were tethered to HLA DBR 1_4 or HLA DRB 1_15 (c). "UI" refers to uninfected cells.

[0184] Figure 3 An example analysis of cells transduced with MCR containing various HLAβ chains carrying different peptides (pep1, pep2 and pep3) stained with anti-HLA-DR and anti-CD3e is shown. Double positive staining indicates that both antibodies are equally effective in detecting MCR surface expression, and the diagonal shape of the population indicates efficient conjugation of MCR to CD3 chains.

[0185] Figure 4 Showing 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 αHLA-DR (top) or αCD3 (bottom).

[0186] Figure 5 Shows MP1 using the Immune Epitope Database (IEDB) analysis resource 103-120 Peptide binding prediction.

[0187] Figure 6 Shown is FACS analysis of cells transduced with MCR containing various HLA-DRB chains carrying the core epitope MP1 107-117 Anti-CD3e or anti-HLA-DR PE antibodies were used to measure MCR surface expression. The graph shows core MP1 107-117The peptide was only efficiently presented on HLA-DRB1_4, but not by HLA-DRB1_15 and HLA-DRB5_1, and the flanking peptide regions did not play a major role. The presence of MCR in all samples was confirmed by PCR and sequencing (not shown).

[0188] Figure 7 A library of random peptides tethered to the alpha chain of mouse MCR was transduced into reporter cells, single cells expressing MCR on the surface were sorted, and the sequence of an exemplary clone was determined. The figure represents a FACS analysis of an exemplary clone of MCR carrying such a peptide, stained with anti-mouse MHC class II and the corresponding peptide sequence.

[0189] Example 1 - Surface Differential Expression of MCRs Containing Influenza Matrix Protein 1 Peptides

[0190] The inventors constructed an MHC-TCR fusion protein (MCR) consisting of a peptide linked to the native extracellular domain of MHC fused to the transmembrane unit of TCR ( Figure 1 ; see also WO 2016 / 097334A1). Specifically, constructs encoding MCRs carrying the α and β chains of human MHC molecules (HLA-DRB1_4, DRB1_15 and DRB5_1) have been generated, wherein the β chain carries a covalently linked influenza matrix protein 1 peptide (MP1 103-120 )、MP1 62-72 or 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), anti-HLA-DR APC antibodies were used to measure MCR surface expression by FACS (Figure 2). MP1 derived peptides containing amino acids 103-120 tethered to HLA DRB1_4 resulted in efficient expression of MCR on the surface of mammalian reporter cell lines. The same peptide tethered to HLA DRB 1_15 resulted in very low MCR expression and no expression at all in the case of HLA DRB 5_1 ( Figure 2a ). However, another MP1-derived peptide comprising amino acids 62-72 resulted in efficient expression of MCRs containing HLA DRB 5-1, but not the other HLAs tested ( Figure 2b Importantly, similar surface expression levels were observed when the influenza cDNA-derived peptide library was tethered to either HLA DBR1_4 or HLA DRB 1_15, indicating that both HLA 1_4 and HLA 1_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 the MP1 peptide comprising amino acids 103-117 and 107-121 is efficiently presented by HLA DRB 1-4, suggesting that only the covalently linked peptide binds efficiently to the MHC peptide binding groove and the MHC complex is sufficiently stable for surface expression to be detected by antibody staining. Figure 6 It was further shown that regardless of the flanking peptide regions, the core MP1 107-117 The peptide can only be efficiently presented on HLA-DRB1_4.

[0191] Example 2 - Detection of MCR surface expression using different antibodies

[0192] Antibody staining with anti-HLA-DR and anti-CD3e was performed to detect the surface expression of MCR containing various HLAβ chains carrying different peptides (pep1, pep2 and pep3) and analyzed on a BD Fortessa flow cytometer ( Figure 3 ). Double positive staining indicates that both antibodies can effectively detect MCR surface expression and anti-CD3e staining can be universally used to detect MCR carrying different HLA alleles. The diagonal shape of the population further indicates efficient conjugation of MCR to CD3 chains.

[0193] Example 3-MP1 103-120 MHC binding affinity prediction

[0194] The Immune Epitope Database (IEDB) was used to analyze the resource to predict the expression of MP1 103-120 MHC binding affinity (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 , the methods of the present invention showed that HLA DRB 4_1 bound best and resulted in high surface expression of HLA, HLA DRB 1_15 bound moderately and resulted in low surface expression, and HLA DRB 5_1 did 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 Binder( Figure 5 ).

[0195] Example 4 - Detection of surface expression of MCR containing peptide fused to α chain

[0196] The inventors constructed a library of mouse MCRs carrying random peptides tethered to the α chain. After transduction of an MHC class II-deficient cell line (T cell hybridoma), anti-MHC antibodies were used to measure MCR surface expression by FACS ( Figure 7 ). Efficient expression of MCR carrying many different peptides was detected, suggesting that peptides linked to the α chain of MCR can also stabilize the MHC complex sufficiently for surface expression.

Claims

1. A method for identifying a candidate peptide presented by a major histocompatibility complex (MHC), the method comprising the steps of: a) expressing a recombinant MHC-peptide complex in a reporter cell line, wherein the recombinant MHC-peptide complex is a peptide-MHC-TCR chimera in which the candidate peptide is covalently bound to the MHC, and wherein the reporter cell line is a mammalian cell line, b) staining reporter cells showing surface expression of MHC-peptide complexes, and c) isolating the reporter cells stained in step (b).

2. The method according to claim 1, further comprising the steps of: d) determining the amino acid sequence of the candidate peptide presented on the cell surface of the reporter cells isolated in step c), and e) identifying the candidate peptide of step d) presented by MHC.

3. The method according to claim 1 or 2, further comprising the steps of: (i) generating a library comprising candidate peptides cloned upstream and in frame with recombinant MHC β chain and / or α chain; and (ii) transducing the library together with the corresponding MHC α chain and / or β chain into the reporter cell line.

4. The method according to claim 1 or 2, further comprising the step of determining the level of surface expression of the MHC-peptide complex and / or the binding affinity of the candidate peptide to MHC.

5. The method according to claim 1 or 2, wherein the MHC is a class II MHC molecule comprising an extracellular MHC class II α chain and a transmembrane domain, and an extracellular MHC class II β chain and a transmembrane domain; or The MHC is a class I MHC molecule comprising an extracellular MHC class I alpha chain and a transmembrane domain and beta-2 microglobulin.

6. The method according to claim 1 or 2, wherein the MHC-peptide complex is a fusion protein comprising a candidate peptide, beta-2 macroglobulin, an extracellular MHC class I α chain, and a transmembrane domain.

7. The method of claim 6, wherein the MHC class I α chain carries a Y84A mutation.

8. The method of claim 1 or 2, wherein each chain of the MHC comprises a transmembrane domain.

9. The method of claim 5, wherein the transmembrane domain is a native MHC transmembrane domain; or The transmembrane domain is a transmembrane domain of a heterologous molecule.

10. The method of claim 9, wherein the heterologous molecule is TCRα / β, TCRγ / δ, CD3γ / δ / ε / ζ, CD4 or CD8α / β.

11. The method of claim 1 or 2, wherein the reporter cell line is a cell line lacking MHC class II peptide loading machinery, or wherein the reporter cell line is a cell line lacking functional TAP1, TAP2 and / or β-2-microglobulin genes.

12. The method of claim 11, wherein the reporter cell line is a T cell hybridoma.

13. The method of claim 1 or 2, wherein the candidate peptide is a tumor-specific peptide carrying a mutation derived from an individual tumor; or wherein the candidate peptide is an antigen that elicits an immune response; or The candidate peptides are compounds that are tested for immunogenicity.

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

15. The method of claim 1, wherein the candidate peptides are encoded in a library of MHC-peptide complexes.

16. The method of claim 15, wherein the library of MHC-peptide complexes comprises candidate peptides cloned upstream and in frame with recombinant MHC β chain and / or α chain.

17. The method of claim 16, wherein the library of MHC-peptide complexes comprises: a) a mutant form of a native peptide; or b) Peptides generated by random transformation or digestion of cDNA or DNA derived from cells or pathogens of interest.

18. The method according to any one of claims 15 to 17, wherein the library of MHC-peptide complexes encodes at least 10 candidate peptides.

19. The method of claim 1 or 2, wherein the staining of the reporter cells is achieved using antibodies.

20. The method of claim 19, wherein the antibody is directed against MHC-I or MHC-II, or wherein the antibody is directed against CD3γ, CD3δ or CD3ε.

21. The method of claim 1 or 2, wherein reporter cells expressing MHC on their surface are enriched by FACS-based or MACS-based cell sorting.

22. The method of claim 2, wherein the sequence of the candidate peptide presented on the cell surface is determined by PCR and sequencing.

23. The method of claim 1 or 2, wherein the candidate peptide is used as a vaccine; or wherein the candidate peptide is used to induce immune tolerance to at least one epitope contained therein; or wherein the candidate peptide is used to block TCR in the context of an MHC molecule; or wherein the candidate peptide is used for MHC-mediated toxin delivery to T cells; or wherein the candidate peptide is used to redirect T cells with MHC-CAR; or wherein the candidate peptide is used for immunogenicity testing; or The candidate peptides are used in T cell reactivity testing.

24. The method of claim 23, wherein the vaccine is a tumor specific antigen (TSA) based cancer vaccine.

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