NOVEL PEPTIDE MIMETICS AND USES THEREOF - Patent application

Abstract

The present invention relates to mimetics of post-translationally modified naturally occurring peptides, which bind to the peptide-binding groove of human leukocyte antigen (HLA) molecules to the same extent as the naturally occurring post-translationally modified peptides, which are recognized by T cells to the same extent as the naturally occurring post-translationally modified peptides, and which have substantially the same three-dimensional structure as the post-translationally modified naturally occurring peptides. Such peptidomimetics can be used alone, conjugated to a carrier, and are particularly useful in methods for the treatment, alleviation and prevention of autoimmune diseases, as well as components of tolerogenic vaccines.

Description

[Technical field]

[0001] The present disclosure relates to the field of medicine, and more specifically to immunotherapy, in particular to novel synthetic mimetics of naturally occurring post-translationally modified peptides, MHC class II peptide complexes including vaccines, and other compositions comprising said mimetics, and their use in therapeutic and prophylactic methods, e.g., for the induction of tolerance to a particular antigen in a subject. [Background technology]

[0002] Much effort has been expended on the development of immunotherapies for the treatment and prevention of autoimmune diseases, the ultimate goal of which is the induction of antigen-specific tolerance. In recent years, this goal has become more achievable due to the increased recognition and knowledge of antigens recognized by potentially pathogenic T and B cells. Ideally, such knowledge may enable the development of antigen-specific therapeutic approaches to eliminate or re-regulate such pathogenic immunity.

[0003] Of particular interest in this development is detailed knowledge of peptides that bind to specific allele genotypes of major histocompatibility complex (MHC) class II molecules, where these peptides are recognized by potentially pathogenic T cells from the patient. One tolerization procedure currently being developed by several academic and pharmaceutical / biotech groups is based on the preparation of constructs containing MHC class II peptide complexes. These complexes can then be administered to the patient together with a suitable carrier to induce antigen-specific tolerance.

[0004] In 2012, International Application No. 2012138294 presented novel peptides derived from human alpha-enolase, collagen type II and vimentin that are capable of binding to different types of MHC class II molecules.

[0005] Application AU2013204094A1, published in 2013 and entitled "Citrullinated peptides for diagnosis and prognosis of rheumatoid arthritis," presented a mimetic of a post-translationally modified naturally occurring nine-residue peptide within the vimentin polypeptide, replacing arginine residues with glutamine to mimic citrullination.

[0006] Harauz and Musse et al. (Harauz, G. and Musse AA et al., 2006) investigated post-translational modifications of myelin basic protein (MBP) and found, among other things, that the degree of deamination (or citrullination) of MBP correlated with the severity of MS. No information is available regarding potential HLA binding or T cell recognition.

[0007] In the 2020 issue of Lancet Rheumatology, Nel et al. provide a review of novel therapeutic approaches, discussing early-phase clinical trial results that suggest immunotherapy may be able to extend remission periods or prevent disease progression, suggesting that modulating tolerance in rheumatoid arthritis may represent a promising therapeutic opportunity. Summary of the Invention

[0008] While the above approaches may be promising for many autoimmune diseases, the inventors noticed that the only peptides so far described as being associated with rheumatoid arthritis (RA) and binding to the appropriate MHC molecule are post-translationally modified, i.e., citrulline is required as an amino acid involved in binding to the MHC molecule or in the recognition of the peptide-MHC complex by the RA-derived T cell receptor (TCR).

[0009] A major problem in producing such therapeutic MHC class II peptide complexes, or other entities that require the synthesis of post-translationally modified amino acids, is that the procedures for producing these entities require a post-translational modification step after the initial synthesis. An example is the synthesis of a bound MHC molecule and a peptide that binds to the peptide-binding groove of this molecule. The production of such complexes is not possible if key amino acids in the peptide are post-translationally modified, a feature that represents a major obstacle to the development of potential therapeutic MHC class II peptide-containing constructs.

[0010] Although in a different technical context, similar issues are relevant for the possibility of using mRNA-based vaccines for tolerization. As shown in a recent paper from BioNTech (Krienke et al., 2021), mRNA vaccines encoding peptides relevant for tolerization in an experimental allergic encephalomyelitis model, said mRNA vaccines (which do not contain tags that activate the immune system, as used for example in COVID vaccines) can also induce antigen-specific tolerance. In this case, it is not possible to generate an mRNA vaccine for the treatment of RA, since it is not possible to produce post-translationally modified amino acids from the mRNA code.

[0011] However, based on the knowledge of the crystal structures of certain relevant allelic variants of MHC class II (HLA-DR0401 and DR0404), related citrullinated peptides, and where these citrullinated peptides are recognized by T cells of RA patients, the inventors have succeeded in synthesizing novel synthetic and alternative non-citrullinated peptidomimetics that bind to the peptide binding groove of HLA-DR0401 and 0404 and are recognized by T cell receptors from activated T cells in RA patients.

[0012] The inventors developed a system to identify peptides that bind to RA-associated allele genotypes of MHC class II molecules (HLA-DR) and used T cell clones arising from RA patients that recognize the appropriate MHC class II-citrullinated peptide complexes to test whether novel peptidomimetics (peptides that do not contain citrulline) are able to bind to the appropriate MHC class II molecules and be recognized by T cell clones derived from RA patients.

[0013] Accordingly, a first aspect of the present disclosure relates to synthetic mimetics of post-translationally modified naturally occurring peptides, in which citrulline has been replaced with another amino acid to form a peptidomimetic, which binds to the peptide-binding groove of human leukocyte antigen (HLA) molecules to the same extent as the naturally occurring post-translationally modified peptide.

[0014] Preferably, the synthetic peptidomimetic is also recognized by T cells to the same extent as the post-translationally modified naturally occurring peptide.

[0015] According to one embodiment, the peptidomimetic has a crystal structure, e.g., determined by X-ray diffraction crystallography, that is substantially identical to the crystal structure of a naturally occurring peptide determined using the same method. Preferably, the peptidomimetic also binds to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring post-translationally modified peptide, and more preferably, it is also recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0016] The crystal structure of a molecule can be determined using methods and equipment available to those skilled in the art, most commonly X-ray diffraction crystallography. Since X-ray diffraction crystallography has been practiced for several decades, those skilled in the art are familiar with the methods and equipment available to perform X-ray diffraction crystallography. For example, the double helix structure of DNA, discovered by James Watson and Francis Crick, has already been revealed by X-ray crystallography. Similarly, molecular binding can be studied and quantified using binding assays and associated equipment. Competitive binding assays typically measure the binding of a labeled ligand to a target protein in the presence of a competing, but unlabeled, second ligand. Such assays can be used to assess qualitative binding information as well as the relative affinity of two or more molecules for one target.

[0017] In the above, it is preferably an amino acid that binds to a pocket in the peptide-binding groove of an HLA molecule that has been substituted, for example, in which citrulline has been replaced by glutamine, which is referred to herein either by its full name, the three-letter code (gln) or the one-letter code (Q).

[0018] According to an embodiment of the first aspect of the present invention, the synthetic peptide is a mimetic of a peptide selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated cartilage intermediate lamina protein (CILP), citrullinated tenascin C, and citrullinated alpha-enolase.

[0019] According to a particular embodiment of the first aspect of the invention, the peptide is fibrinogen and the citrulline at position 74 is replaced by glutamine. The relevant sequence of the fibrinogen β-chain (amino acids 69-81) is shown as SEQ ID NO:1 and the first mimetic is shown by SEQ ID NO:2.

[0020] An alternative, in which the tyrosine at position 71 is replaced with a phenylalanine, is shown as SEQ ID NO:3.

[0021] According to an alternative embodiment of the first aspect of the invention, the peptide is fibrinogen, in which, in addition to the substitution of citrulline at position 74 by glutamine, the tyrosine at position 71 is substituted by phenylalanine, as shown by SEQ ID NO:4.

[0022] According to another embodiment of the first aspect of the invention, the peptide is vimentin, the relevant portion, the T cell epitope of the vimentin peptide, amino acids 66-78, is shown in SEQ ID NO: 5. Three synthetic peptidomimetics have been produced according to the invention:

[0023] According to one embodiment, the peptide is vimentin, as shown in SEQ ID NO:6, and the citrulline at position 71 is substituted with glutamine.

[0024] Alternatively, the peptide is vimentin, and valine at position 68 is replaced with phenylalanine, as shown in SEQ ID NO: 7. According to yet another embodiment of the first aspect of the present invention, the peptide is vimentin, and citrulline at position 71 is replaced with glutamine, and valine at position 68 is replaced with phenylalanine, as shown in SEQ ID NO: 8.

[0025] Tenascin-C is an oligomeric, multidomain matrix glycoprotein consisting of six monomers. The size of these tenascin-C monomers varies between 180 and 250-300 kDa as a result of alternative splicing of fibronectin repeats at the pre-mRNA level. Tenascin-C has recently been implicated as an antibody target in rheumatoid arthritis. In 2021, five novel citrullinated tenascin CT cell epitopes were identified by Song et al. Two epitopes, amino acids 871-885 (SEQ ID NO: 9) and amino acids 2067-2081 (SEQ ID NO: 10), are presented herein.

[0026] According to a specific embodiment of the first aspect of the invention, the peptide is tenascin C, as shown in SEQ ID NO:11, and the citrulline at position 877 is substituted with glutamine.

[0027] According to another particular embodiment of the first aspect of the invention, the peptide is tenascin C, as shown in SEQ ID NO: 12, and the citrulline at position 2073 is substituted with glutamine.

[0028] According to an embodiment of the first aspect and freely combinable with any of its embodiments, the synthetic peptide binds to the P4 pocket (binding groove) of a human leukocyte antigen (HLA) molecule with substantially the same affinity as a naturally occurring peptide.

[0029] This binding can be confirmed by methods known in the art, for example, by fluorescence polarization-based competitive assays by examining the bound peptide-HLA complexes developed in a DELFIA® time-resolved fluorescence assay using europium-labeled streptavidin (PerkinElmer). For a description of this method, see Pieper et al., J Autoimmunity, 2018 (incorporated herein by reference).

[0030] Furthermore, the ability of the peptides to be recognized by T cells is confirmed by functional T cell readout, i.e., T cells that respond to the original peptide also respond to the synthetic mimetic peptide. For a description of the method, see the Examples section of this patent application and the scientific literature, e.g., Boddul et al., J Trans Autoimm, 2021 (incorporated herein by reference).

[0031] The novel synthetic peptidomimetics presented above are expected to be useful in methods for the induction of tolerance in a subject, preferably in methods in which the induction of tolerance is a step in the treatment, mitigation, or prevention of an autoimmune disease (e.g., but not limited to, rheumatoid arthritis).

[0032] A second aspect of the present disclosure relates to a conjugate of a carrier and a peptide, wherein said peptide is a synthetic mimetic of a post-translationally modified naturally occurring peptide, wherein in said peptidomimetic, citrulline is replaced with other amino acids to form the peptidomimetic compared to the naturally occurring peptide, and wherein said peptidomimetic binds to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as the naturally occurring post-translationally modified peptide.

[0033] Preferably, the synthetic peptidomimetic is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0034] The carrier is selected from nanoparticles, proteins, blood cells, and MHC class II molecules. The peptidomimetic peptide / peptide mimic can be bound to the carrier either alone or in complex with other molecules, preferably MHC class II molecules or complexes containing MHC class II molecules. MHC class II molecules can bind and display peptides derived from intracellular proteins on the cell surface, forming MHC class II peptide complexes. The structure and function of MHC class II peptide complexes have been extensively studied (see, for example, Dessen et al., 1997).

[0035] In the above MHC class II-peptide complex, preferably, citrulline is replaced with glutamine (Q).

[0036] According to an embodiment of said second aspect, the peptidomimetic has a crystal structure, e.g., determined by X-ray diffraction crystallography, which is substantially identical to a crystal structure of a naturally occurring peptide determined using the same method; wherein said peptidomimetic binds to the peptide binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring post-translationally modified peptide, and is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0037] The crystal structure of a molecule can be determined by methods and equipment available to those skilled in the art, most commonly X-ray diffraction crystallography. Similarly, molecular binding can be tested and quantified using binding assays and related equipment. Competitive binding assays usually measure the binding of a labeled ligand to a target protein in the presence of a competing but unlabeled second ligand. Such assays can be used to evaluate qualitative binding information as well as the relative affinity of two or more molecules to one target.

[0038] According to an embodiment of the second aspect of the invention, the synthetic peptide is a mimetic of a peptide selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated tenascin C, citrullinated collagen type II, cartilage intermediate lamina protein (CILP), and citrullinated alpha-enolase.

[0039] According to a particular embodiment of the second aspect of the invention, the peptide is fibrinogen and the citrulline at position 74 is replaced by glutamine. The relevant sequence of the fibrinogen β-chain (amino acids 69-81) is shown as SEQ ID NO:1 and the first mimetic is shown by SEQ ID NO:2.

[0040] An alternative, in which the tyrosine at position 71 is replaced with a phenylalanine, is shown as SEQ ID NO:3.

[0041] According to an alternative embodiment of the second aspect of the invention, the peptide is fibrinogen, in which, in addition to the substitution of citrulline at position 74 by glutamine, the tyrosine at position 71 is substituted by phenylalanine. This is represented by SEQ ID NO:4.

[0042] According to another embodiment of the second aspect of the invention, the peptide is vimentin, the relevant portion, the T cell epitope of the vimentin peptide, amino acids 66-78, is shown in SEQ ID NO: 5. Three synthetic peptidomimetics have been produced according to the invention:

[0043] According to one embodiment, the peptide is vimentin, as shown in SEQ ID NO:6, and the citrulline at position 71 is substituted with glutamine.

[0044] Alternatively, the peptide is vimentin, and valine at position 68 is replaced with phenylalanine, as shown in SEQ ID NO: 7. According to yet another embodiment of the first aspect of the present invention, the peptide is vimentin, and citrulline at position 71 is replaced with glutamine, and valine at position 68 is replaced with phenylalanine, as shown in SEQ ID NO: 8.

[0045] According to a specific embodiment of the second aspect of the invention, the peptide is tenascin C, as shown in SEQ ID NO:11, and the citrulline at position 877 is substituted with glutamine.

[0046] According to another particular embodiment of the second aspect of the invention, the peptide is tenascin C, as shown in SEQ ID NO: 12, and the citrulline at position 2073 is substituted with glutamine.

[0047] According to an embodiment of the second aspect and freely combinable with any of its embodiments, the synthetic peptide binds to the P4 pocket (binding groove) of a human leukocyte antigen (HLA) molecule with substantially the same affinity as a naturally occurring peptide.

[0048] This binding can be confirmed by methods known in the art, for example, by fluorescence polarization-based competitive assays by examining the bound peptide-HLA complexes developed in a DELFIA® time-resolved fluorescence assay using europium-labeled streptavidin (PerkinElmer). For a description of this method, see Pieper et al., J Autoimmunity, 2018 (incorporated herein by reference).

[0049] Furthermore, the ability of the peptides to be recognized by T cells is confirmed by functional T cell readout, i.e., T cells that respond to the original peptide also respond to the synthetic mimetic peptide. For a description of the method, see the Examples section of this patent application and the scientific literature, e.g., Boddul et al., J Trans Autoimm, 2021 (supra).

[0050] A third aspect of the present invention relates to a method of inducing tolerance to a particular antigen in a subject, said method comprising administering to said subject a construct comprising a carrier-peptide complex, wherein the peptide incorporated in said carrier-peptide complex is a synthetic peptidomimetic as defined in the first aspect and embodiments thereof set out above and in the appended claims.

[0051] A parallel aspect is a method of inducing tolerance to a particular antigen in a subject, said method comprising administering to said subject a construct comprising an MHC class II-peptide complex as defined in the second aspect and embodiments thereof set out above and in the accompanying claims.

[0052] Preferably, this induction of tolerance is a step in the treatment, alleviation, or prevention of autoimmune diseases. Regimens have been developed recently for inducing tolerance, particularly self-tolerance, i.e., the immune system's ability to recognize (and therefore not react to) self-produced antigens. Several systems for tolerance induction have been described in the scientific literature, so far, mainly in mouse models, which are now being applied to human diseases. See, for example, Yang Y et al., Adv. Drug Deliv. Rev. 2021; Yang Y et al., Curr Opin Biotechnol, 2022 and Neef T et al., Cells, 2021 (all incorporated herein by reference). However, for RA, some of these approaches are not feasible, since there is no way to produce the exact peptide when it contains one or more citrulline residues.

[0053] According to a preferred embodiment, the autoimmune disease is rheumatoid arthritis (RA) or an autoimmune condition that confers an increased risk of future development of RA, the antigen is a peptide antigen, and the non-post-translationally modified peptidomimetic binds to the peptide binding groove of HLA-DRB1*04:01 and 04:04 and is recognized by a T cell receptor derived from an RA patient, the T cell being derived from an activated T cell in the RA patient.

[0054] According to one embodiment, the antigen is selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated type II collagen, citrullinated tenascin C, citrullinated CILP and citrullinated alpha-enolase.

[0055] A fourth aspect of the present invention relates to a tolerogenic mRNA vaccine for inducing tolerance to a specific antigen in a subject, the tolerogenic mRNA vaccine comprising a modified non-inflammatory mRNA encoding a non-post-translationally modified mimic of said antigen, which may be used to treat, alleviate, or prevent the onset of an autoimmune disease, such as, but not limited to, rheumatoid arthritis (RA).

[0056] A general introduction to mRNA vaccines has been described in "mRNA vaccines manufacturing: challenges and bottlenecks" by Rosa et al., Vaccine 39 (2021) 2190-2200, incorporated herein by reference. These techniques for mRNA vaccination for tolerance are not feasible for RA with the current knowledge of T cells involved in the pathogenesis of RA, since mRNA cannot code for citrullinated residues. Herein, the present invention provides important new findings that make mRNA vaccination for tolerance in RA feasible for the first time.

[0057] According to an embodiment of the fourth aspect, the non-post-translationally modified mimetic of the antigen is a non-citrullinated peptide that binds to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as the naturally occurring post-translationally modified peptide.

[0058] Preferably, the synthetic peptidomimetic is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0059] Preferably, the vaccine is administered for the treatment, alleviation or prevention of rheumatoid arthritis.

[0060] According to one embodiment, the peptidomimetic binds to the peptide binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring post-translationally modified peptide, e.g., the peptidomimetic has a crystal structure, e.g., determined by x-ray diffraction crystallography; preferably, the synthetic peptidomimetic is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0061] According to a further embodiment, which can be freely combined with the above, citrulline is replaced by another amino acid that maintains binding to the peptide-binding groove of human leukocyte antigen (HLA) molecules to the same extent as the naturally occurring post-translationally modified peptide.Preferably, citrulline is replaced by glutamine (Q).

[0062] According to a preferred embodiment, the synthetic antigen binds to the P4 pocket (binding groove) of human leukocyte antigen (HLA) class II molecules.

[0063] According to a particular embodiment of the fourth aspect, the synthetic peptide is a mimic of an antigen selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated tenascin C, citrullinated CILP and citrullinated alpha-enolase.

[0064] According to a particular embodiment of the fourth aspect of the invention, the peptide is fibrinogen, in which citrulline at position 74 is replaced by glutamine. The relevant sequence of the fibrinogen β-chain (amino acids 69-81) is shown as SEQ ID NO:1 and the first mimetic is shown by SEQ ID NO:2.

[0065] An alternative, in which the tyrosine at position 71 is replaced with a phenylalanine, is shown as SEQ ID NO:3.

[0066] According to an alternative embodiment of the fourth aspect of the invention, the peptide is fibrinogen, in which citrulline at position 74 is replaced by glutamine and tyrosine at position 71 is replaced by phenylalanine, as represented by SEQ ID NO:4.

[0067] According to another embodiment of the fourth aspect of the invention, the peptide is vimentin, the relevant portion, the T cell epitope of the vimentin peptide, amino acids 66-78, is shown in SEQ ID NO: 5. Three synthetic peptidomimetics have been produced according to the invention:

[0068] According to one embodiment, the peptide is vimentin and the citrulline at position 71 is replaced with glutamine, as shown in SEQ ID NO:6.

[0069] Alternatively, the peptide is vimentin, and valine at position 68 is replaced with phenylalanine, as shown in SEQ ID NO: 7. According to yet another embodiment of the first aspect of the present invention, the peptide is vimentin, and citrulline at position 71 is replaced with glutamine, and valine at position 68 is replaced with phenylalanine, as shown in SEQ ID NO: 8.

[0070] According to a particular embodiment of the fourth aspect of the invention, the peptide is tenascin C, as shown in SEQ ID NO:11, and the citrulline at position 877 is substituted with glutamine.

[0071] According to another particular embodiment of the fourth aspect of the invention, the peptide is tenascin C, as shown in SEQ ID NO: 12, and the citrulline at position 2073 is substituted with glutamine.

[0072] The vaccines are useful in the treatment, amelioration and / or prevention of autoimmune diseases, such as, but not limited to, rheumatoid arthritis.

[0073] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the following drawings, detailed description, and examples. [Brief description of the drawings]

[0074] The invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0075] [Figure 1] Figure 1 shows a conceptual image of how a citrullinated peptide docks into the peptide-binding groove of HLA DRB1*04:01, the most common MHC class II molecule associated with RA. Note that citrulline docks into the P4 pocket of the MHC groove and is therefore not exposed towards the specific T cell receptor (TCR). Original image published in Malmstrom et al., Nat Rev Immunol 2017. [Diagram 2] Figure 2 is a graph showing the results of a peptide binding assay, called a competition assay, performed according to the method outlined in Example 1. It is clear that the fibrinogen mimetic peptides tested have the same ability to compete with an already bound reference peptide, here an influenza (HA) peptide, as the original citrullinated peptide. [Diagram 3] FIG. 3 is a graph showing activation of artificial T cell lines expressing TCRs specific for citrullinated fibrinogen peptides. Shown from left to right are T cell receptor (TCR)-dependent nuclear factor of activated T cells (NFAT)-mediated activated T cells by optical fluorescence imaging (OFI) of FibF71Q74 (SEQ ID NO: 4), FibF71X74 (SEQ ID NO: 3), FibQ74 (SEQ ID NO: 2), FibX74 (SEQ ID NO: 1), and VimX71 (SEQ ID NO: 5), demonstrating that the mimetic peptides are capable of inducing T cell activation similar to the original citrullinated peptides. [Figure 4]FIG. 4 is a graph showing activation of artificial T cell lines expressing TCRs specific for citrullinated fibrinogen peptides, showing T cell receptor (TCR)-dependent programmed cell death protein 1 (PD1) expression for FibF71Q74 (SEQ ID NO: 4), FibF71X74 (SEQ ID NO: 3), FibQ74 (SEQ ID NO: 2), FibX74 (SEQ ID NO: 1), and VimX71 (SEQ ID NO: 5) from left to right, demonstrating that the mimetic peptides are capable of inducing T cell activation similar to the original citrullinated peptides. [Diagram 5] 5 is a graph showing the results of a peptide binding assay, also called a competition assay, of citrullinated vimentin peptides compared to non-post-translationally modified mimetic peptides. It is clear that the tested vimentin mimetic peptides have the same ability to compete with the already bound reference peptide (here, influenza (HA) peptide) as the original citrullinated peptide. [Figure 6] Figure 6 shows flow cytometric staining of polyclonal CD4+ T cells with HLA class II tetramers that capture antigen-specific T cells by binding to their TCR. Here, quadrant 2 (bold) shows T cells that respond to both the citrulline and glutamine tetramers of vimentin, suggesting that there are T cells in this culture that cannot distinguish between the original and mimetic peptides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Before describing the present invention, it is to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and their equivalents.

[0077] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0078] When referring to peptide sequences, the amino acids are numbered, the number indicating the position of the amino acid residue in the polypeptide chain as counted from the amino terminus, thus, for example, Glu74 means that the 74th amino acid residue in the chain is glutamine.

[0079] The term "peptidomimetic" refers to a molecule, such as a peptide, modified peptide, or any other molecule that biologically mimics the action or activity of some other peptide. A "peptidomimetic" is sometimes also called a "peptide mimetic."

[0080] The term "synthetic" is used to distinguish modified non-naturally occurring molecules, such as peptidomimetics, from naturally occurring molecules.

[0081] The terms "post-translational modification" and "post-translationally modified" refer to reversible or irreversible chemical changes that peptides and proteins can undergo after translation. In other words, post-translational modification is the chemical modification of a polypeptide chain that occurs after DNA is transcribed into RNA and translated into peptides and proteins. These chemical changes range from the enzymatic cleavage of peptide bonds to the covalent addition of specific chemical groups, lipids, carbohydrates, and even entire proteins to amino acid side chains (Uversky VN, Posttranslational Modification, in Brenner's Encyclopaedia of Genetics (Second Edition) Elsevier Inc. 2013, pages 425-430, incorporated herein by reference).

[0082] "Substantially identical", e.g., a phrase such as "a synthetic non-post-translationally modified peptidomimetic having substantially the same three-dimensional structure as a corresponding post-translationally modified naturally occurring peptide", means that the peptidomimetic has a functionally identical three-dimensional structure, as indicated by the fact that the peptidomimetic binds to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as the corresponding naturally occurring post-translationally modified peptide.

[0083] This disclosure frequently refers to the "peptide binding groove" of HLA class II molecules. The peptide binding groove of class I and class II HLA molecules is known to be formed by a β-sheet floor consisting of eight antiparallel β-sheets packed against two antiparallel α-helices forming a channel. In class I molecules (HLA-A, -B, and -C), the binding groove is divided into six pockets (AFs), which are defined by specific polymorphic amino acid residues that determine their topography and function. These class I HLA molecules typically bind peptides of 8-11 amino acids in length. Compared to class I, class II HLA-DRB1 molecules bind longer peptides of variable length, e.g., 12-15 amino acids. The most polymorphic HLA-DRB1 element is the structural pocket that accommodates peptide positions 1 (P1), P4, P6, P7, and P9.

[0084] Figure 1 shows a conceptual image of how citrullinated peptides dock into the peptide-binding groove of HLA DRB1*04:01, the most common MHC class II molecule implicated in RA in Caucasians. A similar groove is also present in Asian populations and is involved in the DRB1*04:05 MHC class II variant. Original image published in Malmstrom et al., Nat Rev Immunol 2017.

[0085] Note that citrulline docks into the P4 pocket of the MHC groove and is therefore not exposed towards a specific T cell receptor (TCR). Thus, as defined herein, in the examples and in the claims, functionally identical peptidomimetics can bind to the peptide binding groove of human leukocyte antigen (HLA) molecules to the same extent as the corresponding naturally occurring post-translationally modified peptides.

[0086] - Synthetic peptide mimetics -

[0087] Thus, a first aspect of the present disclosure relates to synthetic mimetics of post-translationally modified naturally occurring peptides in which citrulline has been replaced with another amino acid forming a peptidomimetic that binds to the peptide-binding groove of human leukocyte antigen (HLA) molecules to the same extent as the naturally occurring post-translationally modified peptide.

[0088] Preferably, the synthetic peptidomimetic is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0089] According to one embodiment, the peptidomimetic has a crystal structure, e.g., determined by X-ray diffraction crystallography, that is substantially identical to the crystal structure of a naturally occurring peptide determined using the same method. Preferably, the peptidomimetic also binds to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring, post-translationally modified peptide, and most preferably, the synthetic peptidomimetic is also recognized by T cells to the same extent as a post-translationally modified, naturally occurring peptide.

[0090] According to an embodiment of the first aspect of the present invention, the synthetic peptide is a mimetic of a peptide selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated tenascin C, citrullinated cartilage intermediate lamina protein (CILP), and citrullinated alpha-enolase.

[0091] In the above, preferably, citrulline is replaced with glutamine (Q).

[0092] Fibrinogen in its citrullinated form is a classical candidate autoantigen in RA, and its presence has been demonstrated in the joints of RA patients by mass spectrometry (Hermansson et al., 2010). It has also been suggested that citrullinated forms of fibrinogen may form immune complexes with ACPA autoantibodies, leading to cell activation such as macrophages.

[0093] A T cell epitope from the beta chain of citrullinated fibrinogen has been identified (amino acids 69-81) and has been widely used to detect, enumerate and phenotype autoreactive T cells in healthy donors and RA patients (e.g., James et al. Arthritis Rheum 2014, Gerstner et al. BMC Immunol 2020). The crystal structure of a peptide presented by the HLA-DRB1*04:01 molecule has been solved (Lim et al. Sci Immunol 2021, incorporated herein by reference), demonstrating that citrulline is located within the P4 pocket as originally predicted.

[0094] The original sequence of fibrinogen beta chain, amino acids 69-81, is shown as SEQ ID NO:1, where X represents citrulline, and the P1 (71) and P4 (74) positions are underlined: GG Y RA X PAKAAAT (SEQ ID NO:1).

[0095] We have synthesized three modified versions, shown below as SEQ ID NOs: 2, 3 and 4: GG Y RA Q PAKAAAT - (SEQ ID NO: 2) glutamine at position 74. GG F RA XPAKAAAT - (SEQ ID NO: 3) phenylalanine at position 71 and citrulline at position 74. GG F RA Q PAKAAAT - (SEQ ID NO: 4) phenylalanine at position 71 and glutamine at position 74.

[0096] Thus, according to a particular embodiment of the first aspect of the invention, the peptide is fibrinogen and the citrulline at position 74 is replaced by glutamine.

[0097] According to an alternative embodiment of the first aspect of the invention, the peptide is fibrinogen, in which the citrulline at position 74 is replaced by a glutamine and the tyrosine at position 71 is replaced by a phenylalanine.

[0098] Citrullinated forms of vimentin are classical candidate autoantigens in rheumatoid arthritis, and their presence has been demonstrated by mass spectrometry in both the joints and lungs of rheumatoid arthritis patients (Ytterberg et al., Ann Rheum Dis. 2015 Sep;74(9):1772-7). Citrullinated vimentin has been suggested to appear on the cell surface of cells differentiating into bone-resorbing osteoclasts (Harre et al., Nat Commun. 2015 Mar 31;6:6651).

[0099] Furthermore, some ACPA autoantibodies have been shown to have the ability to enhance osteoclast differentiation and increase their bone resorption capacity (Steen et al., Arthritis Rheumatol. 2019 Feb;71(2):196-209; Krishnamurthy A et al., Citrullination Controls Dendritic Cell Transdifferentiation into Osteoclasts, in .J Immunol. 2019 Jun 1;202(11):3143-3150; and Krishnamurthy A et al., Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss, Ann Rheum Dis. 2016 Apr;75(4):721-9. doi: 10.1136,).

[0100] A T cell epitope derived from citrullinated vimentin has been identified (amino acid positions 66-78) and has been widely used to detect, enumerate, and phenotype autoreactive T cells in healthy donors and RA patients (see, e.g., Snir et al., Arthritis Rheum 2011, James et al., Arthritis Rheum 2014, and Gerstner et al., BMC Immunol 2020). The crystal structure of a peptide presented by HLA-DRB1*04:01 molecules has been solved (Scally et al., J Exp Med 2013), demonstrating that citrulline is located in the P4 pocket as originally predicted.

[0101] As a result, we also investigated vimentin, and the original sequence of amino acids 66-78 of vimentin is shown as SEQ ID NO:5, where X represents citrulline and the P1 and P4 positions are underlined: SA V R.L. X SSVPGVR - (SEQ ID NO:5)

[0102] Three modified versions were synthesized and are shown as SEQ ID NOs: 6, 7 and 8: SA V R.L. Q SSVPGVR - (SEQ ID NO: 6) glutamine at position 71 SA F R.L. X SSVPGVR - (SEQ ID NO: 7) phenylalanine at position 68 and citrulline at position 71. SA F R.L. Q SSVPGVR - (SEQ ID NO: 8) Phenylalanine at position 68 and Glutamine at position 71.

[0103] Thus, according to another embodiment of the first aspect of the invention, the peptide is vimentin and the citrulline at position 71 is replaced by glutamine.

[0104] According to an alternative embodiment, the peptide is vimentin, in which the citrulline at position 71 is replaced by a glutamine and the valine at position 68 is replaced by a phenylalanine.

[0105] Citrullinated forms of tenascin-C are candidate autoantigens in RA, and their presence in the joints of RA patients has been demonstrated by mass spectrometry (Tutturen et al., 2014). It has also been suggested that citrullinated forms of tenascin-C may form immune complexes with ACPA autoantibodies, leading to cell activation such as macrophages.

[0106] Several T cell epitopes derived from citrullinated tenascin-C have been identified and widely used to detect, enumerate, and phenotype autoreactive T cells in healthy donors and RA patients (e.g., Song et al., JCI Insights 2021 and Sharma et al., Sci. Rep 2021). For two peptides, citrulline is modeled to be located in the P4 pocket (Song et al., JCI Insights 2021).

[0107] The original sequences of tenascin C, amino acids 871-885 and amino acids 2067-2081, are shown as SEQ ID NO:9 and SEQ ID NO:10, where X represents citrulline and the P1 (71) and P4 (74) positions are underlined: VSLISR X GDMSSNPA (SEQ ID NO: 9) Citrulline at position 877. QGQYEL X VDLRDHGE (SEQ ID NO: 10) Citrulline at position 2073.

[0108] Two modified versions were synthesized and shown as SEQ ID NOs: 11 and 12: VSLISR Q GDMSSNPA - (SEQ ID NO: 11) Currently, glutamine is located at position 877. QGQYEL Q VDLRDHGE - (SEQ ID NO: 12) Currently, glutamine is located at position 2073.

[0109] According to an embodiment of the first aspect, and freely combinable with any of its embodiments, the synthetic peptides of the present invention bind to the P4 pocket (binding groove) of human leukocyte antigen (HLA) molecules with substantially the same affinity as the corresponding naturally occurring peptides.

[0110] This binding can be confirmed by methods known in the art, for example, by fluorescence polarization-based competitive assays by examining the bound peptide-HLA complexes developed in a DELFIA® time-resolved fluorescence assay using europium-labeled streptavidin (PerkinElmer). For a description of this method, see Pieper et al., J Autoimmunity, 2018 (incorporated herein by reference).

[0111] Furthermore, the ability of the peptides to be recognized by T cells is confirmed by functional T cell readout, i.e., T cells that respond to the original peptide also respond to the synthetic mimetic peptide. For a description of the methods, see the Examples section of this patent application and the scientific literature incorporated herein by reference, e.g., Boddul et al., 2021 (supra).

[0112] The peptide mimetics defined above are useful in methods for the induction of tolerance in a subject, preferably in methods in which the induction of tolerance is a step in the treatment, alleviation or prevention of an autoimmune disease, such as, but not limited to, the treatment, alleviation or prevention of RA.

[0113] - Peptide + carrier complex -

[0114] A second aspect of the present disclosure is a conjugate of a carrier and a peptide, wherein the peptide is a synthetic mimetic of a post-translationally modified naturally occurring peptide, in which citrulline is replaced with other amino acids compared to the corresponding naturally occurring peptide to form the peptidomimetic, and wherein the peptidomimetic is capable of binding to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as the naturally occurring post-translationally modified peptide.

[0115] According to an embodiment of the second aspect, and freely combinable with any embodiment thereof, the synthetic peptide binds to the P4 pocket (binding groove) of a human leukocyte antigen (HLA) molecule with substantially the same affinity as a naturally occurring peptide.

[0116] This binding can be confirmed by methods known in the art, for example, by fluorescence polarization-based competitive assays by examining the bound peptide-HLA complexes developed in a DELFIA® time-resolved fluorescence assay using europium-labeled streptavidin (PerkinElmer). For a description of this method, see Pieper et al., J Autoimmunity, 2018 (incorporated herein by reference).

[0117] Furthermore, the ability of the peptides to be recognized by T cells is confirmed by functional T cell readout, i.e., T cells that respond to the original peptide also respond to the synthetic mimetic peptide. For a description of the methods, see the Examples section of this patent application and the scientific literature incorporated herein by reference, e.g., Boddul et al., 2021 (supra).

[0118] Preferably, the synthetic peptidomimetic is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide.

[0119] According to an embodiment of the second aspect, the carrier is selected from nanoparticles, proteins, blood cells, and MHC class II molecules. Examples of nanoparticles include, but are not limited to, iron oxide nanoparticles, latex nanoparticles, gold nanoparticles, silica nanoparticles, carbon nanotubes, etc.

[0120] Carriers can be constructed to bind either peptidomimetics alone or to molecular constructs that contain peptides, such as complexes that contain peptides that bind to MHC class II molecules. If an MHC class II peptide complex is involved, it should be able to bind peptides derived from intracellular proteins and display them on the cell surface to form MHC class II peptide complexes. The structure and function of MHC class II peptide complexes have been extensively studied (see, for example, Dessenet et al., 1997).

[0121] According to an embodiment of the second aspect, the peptidomimetic has a crystal structure, e.g., determined by X-ray diffraction crystallography, that is substantially identical to a crystal structure of a naturally occurring peptide determined using the same method; and the peptidomimetic binds to the peptide binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring, post-translationally modified peptide; and the synthetic peptidomimetic is recognized by T cells to the same extent as a naturally occurring, post-translationally modified peptide.

[0122] For example, the crystal structure of citrullinated fibrinogen bound / presented by HLA class II molecules has been published (see Lim et al., Sci Immunol 2021 (supra)).

[0123] In the above MHC class II-peptide complex, citrulline is preferably replaced with glutamine (Q).

[0124] According to an embodiment of the second aspect of the present invention, the synthetic peptide is a mimetic of a peptide selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated tenascin C, citrullinated CILP, and citrullinated alpha-enolase.

[0125] According to an embodiment of the second aspect of the invention, the peptide is fibrinogen and the citrulline in position 74 is replaced by glutamine.

[0126] According to an alternative embodiment of the second aspect of the invention, the peptide is fibrinogen and the citrulline at position 74 is replaced by glutamine and the tyrosine at position 71 is replaced by phenylalanine.

[0127] According to another embodiment of the second aspect of the invention, the peptide is vimentin and the citrulline at position 71 is replaced by glutamine.

[0128] Alternatively, the peptide is vimentin and the citrulline at position 71 is replaced by glutamine and the valine at position 68 is replaced by phenylalanine.

[0129] According to another embodiment of the second aspect of the invention, the peptide is tenascin C and the citrulline at position 877 is replaced by glutamine.

[0130] According to another embodiment of the second aspect of the invention, the peptide is tenascin C and the citrulline at position 2073 is replaced by glutamine.

[0131] The conjugates defined herein are useful in methods for the induction of tolerance in a subject, preferably in methods in which the induction of tolerance is a step in the treatment, alleviation, or prevention of an autoimmune disease, such as, but not limited to, the treatment, alleviation, or prevention of RA.

[0132] - Induction of tolerance -

[0133] A third aspect of the present invention relates to a method of inducing tolerance to a specific antigen in a subject, said method comprising the step of administering to said subject a construct comprising a carrier-peptide complex as disclosed above, said peptide being a synthetic peptidomimetic as defined in the first aspect and embodiments thereof set out above and in the appended claims.

[0134] A parallel aspect is a method of inducing tolerance to a particular antigen in a subject, said method comprising administering to said subject a construct comprising at least one MHC class II-peptide complex as defined in the second aspect and embodiments thereof set out above and in the accompanying claims.

[0135] Preferably, this induction of tolerance is a step in the treatment, alleviation or prevention of an autoimmune disease.

[0136] According to a preferred embodiment, the autoimmune disease is rheumatoid arthritis (RA), the antigen is a peptide antigen, and the non-post-translationally modified peptidomimetic binds to the peptide-binding groove of HLA-DR0401 and 0404 and is recognized by a T cell receptor from a RA patient, which is derived from T cells activated in the RA patient.

[0137] According to one embodiment, the antigen is selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated tenascin C, citrullinated CILP and citrullinated alpha-enolase.

[0138] - Tolerogenic vaccine -

[0139] A fourth aspect of the present invention relates to a tolerogenic mRNA vaccine for inducing tolerance to a specific antigen in a subject, comprising a modified, non-inflammatory mRNA encoding a non-post-translationally modified mimic of said antigen.

[0140] According to an embodiment of the fourth aspect, said non-post-translationally modified mimetic of an antigen is a non-citrullinated peptide that is capable of binding to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring post-translationally modified peptide.

[0141] Preferably, the synthetic peptidomimetic is also recognized by T cells to the same extent as the post-translationally modified naturally occurring peptide.

[0142] According to one embodiment, the peptidomimetic has a crystal structure determined, for example, by X-ray diffraction crystallography, which is substantially identical to the crystal structure of a naturally occurring peptide determined using the same method. Preferably, the peptidomimetic binds to the peptide-binding groove of a human leukocyte antigen (HLA) molecule to the same extent as a naturally occurring post-translationally modified peptide, and preferably, the synthetic peptidomimetic is recognized by T cells to the same extent as a post-translationally modified naturally occurring peptide. An example of a method for determining the crystal structure of a peptide-HLA molecule is given in Lim et al., Sci Immunol 2021, incorporated herein by reference.

[0143] According to a further embodiment, freely combinable with the above, citrulline is replaced with another amino acid that maintains binding to the peptide-binding groove of human leukocyte antigen (HLA) molecules to the same extent as in naturally occurring post-translationally modified peptides.

[0144] According to a preferred embodiment, the synthetic antigen binds to the P4 pocket (binding groove) of a human leukocyte antigen (HLA) molecule.

[0145] According to an embodiment of the fourth aspect, citrulline in the synthetic antigen is replaced by another amino acid that maintains binding to the peptide-binding groove of human leukocyte antigen (HLA) molecules to the same extent as in the naturally occurring post-translationally modified peptide.Preferably, citrulline is replaced by glutamine (Q).

[0146] According to certain embodiments of the fourth aspect, the synthetic peptide is a mimic of an antigen selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated tenascin C, CILP and citrullinated enolase.

[0147] According to an embodiment of the fourth aspect, which can be freely combined with all other embodiments of the fourth aspect, the modified non-inflammatory mRNA is 1-methylpseudouridine modified mRNA formulated in nanoparticles.

[0148] The invention disclosed herein allows the design of novel mRNA vaccines. For example, mRNA vaccines can be designed to mediate the production of peptidomimetic peptides / peptidomimetics in a non-immunogenic and tolerogenic manner in subjects with a pre-existing immune response when the mRNA is administered to a subject in need thereof. The response can be measured using known methods, such as T-cell or B-cell assays, or both, for the citrullinated peptides for which the peptidomimetics are surrogates. As has been shown in other experiments, such as the mIG-induced EAE model (Krienke C. et al., Science 2021, incorporated herein by reference), mRNA vaccines encoding the above-mentioned peptidomimetics can also exert a suppressive function against other specific immune responses targeting the same organs and / or acting on the same disease as the immune responses against the citrullinated peptides on which the peptidomimetic peptides were generated, due to a "bystander effect (bystander suppression)".

[0149] The inventors have checked the sequences of the modified peptides presented herein to determine whether they occur in other human proteins, and so far such investigations have shown that they do not. The inventors have made available previously unknown, non-naturally occurring, post-translational peptides that mimic the function and structure of citrullinated peptides involved in the pathogenesis of RA. This opens new possibilities for the development of new immunotherapeutic methods for the treatment and prevention of autoimmune diseases, particularly RA.

[0150] In the following examples, the inventors present experimental evidence supporting aspects and embodiments of the present invention. EXAMPLES

[0151] Example 1. Non-post-translationally modified fibrinogen mimetics

[0152] The inventors performed binding assays to verify that the modified peptides can indeed functionally bind both the relevant HLA molecules and the peptides of the present application to the T cell receptor (TCR). HLA binding was demonstrated in a competition assay, which showed that the mimetic peptides have the same ability as the original citrullinated peptides to compete with the already bound reference peptide (in this case, influenza (HA) peptide) (see Figure 2). The results can also be presented as curves or Kd values, demonstrating that the amino acid exchanges at positions P1 and P4 do not change the potential to be presented to T cells.

[0153] We have previously generated data on TCR re-expression into a TCR-deficient T cell line (58- / -) to study antigen specificity and T cell activation (Boddul et al., 2021, supra), and now we have re-expressed a cit-fib-specific TCR into the same line.

[0154] We now demonstrate that TCRs specific for citrullinated fibrinogen peptides cannot discriminate between cognate and mimetic peptides, as artificial T cell lines respond equally to both nuclear factor of activated cells (NFAT) signals (see Figure 3). Similarly, we compared peptide mimetics with respect to programmed death-1 (PD-1) expression (see Figure 4).

[0155] material and method

[0156] 58- / - cell line expressing a cit-fib-specific TCR in an ametrine expression vector and coexpressing human CD4 and GFP as a reporter for NFAT expression.

[0157] HLA-DRB1*04:01 monomer protein (500 μg / mL) was incubated with different versions of fib69-81 peptide (test) and VimX71 peptide (control) in sodium phosphate buffer (1X) containing n-octyl β-D-glucopyranoside (Sigma-Aldrich, USA) and Pefabloc SC (Sigma-Aldrich, USA) for 72 h at 37 °C and then stored at 4 °C until use. The loaded HLA monomers were then coated (0.03-2 μg / well) in 100 μl of PBS onto 48-well plates for 4 h at 37 °C. Subsequently, the HLA / peptide solution was flicked off the plate and specific T cells (5x104) and anti-CD28 (1 μg / well) were added to the monomer-coated wells and incubated for 48 h at 37 °C before cells were harvested.

[0158] As positive controls, anti-mouse anti-CD3 (BioLegend #100314) and anti-CD28 (BioLegend #101112) were used.

[0159] PD1 expression on 58-- / - cells was assessed using anti-mouse PD-1 PE-Cy7 antibody, while NFAT activation was studied using assessment of GFP expression after cell stimulation. Human CD4+ ametrine+ viable singlets were used as a population to confirm NFAT and PD1 expression.

[0160] A competitive binding assay was used to demonstrate the ability of the mimetic peptides to bind to HLA-DRB1*04:01, as compared to the original citrullinated peptide. The instrument used was a PerkinElmer 1420 Multilabel Counter VICTOR3™ V, using the settings shown in the table below: [Table 1]

[0161] The lines in Figure 2 represent the fitting of the experimental data to a one-site competition model using SigmaPlot software v.13 (Systat Software, Inc., San Jose, California, USA).

[0162] Increasing concentrations of peptides were incubated overnight at 37° C. in a humidified incubator in 384-well polypropylene plates in the presence of 30 nM HLA-DRB1*04:01 and 5 nM biotin-labeled HA306-318 peptide. The reaction mixture was transferred to a polystyrene plate coated with anti-HLA-DR monoclonal antibody L243 and incubated overnight at +4° C. Bound peptide-HLA complexes were developed with a DELFIA® time-resolved fluorescence assay using europium-labeled streptavidin (PerkinElmer).

[0163] Results show that T cell lines are unable to distinguish peptide presentation of citrulline-containing peptides from glutamine-containing (artificial) peptides when presented on HLA-DRB1*04:01. These cells responded equally well with NFAT signaling and PD1 upregulation.

[0164] To further increase the stability of the peptide-HLA complex, we replaced amino acids in the P1 pocket (not exposed to the TCR) and found that these peptides could induce T cells, whereas unrelated peptides presented by the same HLA-DRB1*04:01 molecule did not activate the cells.

[0165] Results show that T cell lines are unable to distinguish peptide presentation of citrulline-containing peptides from glutamine-containing and P1-optimized peptides when presented on HLA-DRB1*04:01. These cells responded equally well with upregulation of NFAT signaling and PD1.

[0166] Example 2. Non-post-translationally modified vimentin mimetics

[0167] A T cell epitope derived from citrullinated vimentin has been previously identified (amino acid positions 66–78) and has been widely used to detect, enumerate, and phenotype autoreactive T cells in healthy donors and RA patients (e.g., Snir et al., Arthritis Rheum 2011, James et al., Arthritis Rheum 2014, and Gerstner et al., BMC Immunol 2020). The crystal structure of a peptide presented by HLA-DRB1*04:01 molecules has been solved (Scally et al., J Exp Med 2013), demonstrating that citrulline is located in the P4 pocket as originally predicted.

[0168] Materials and Methods

[0169] Peptide competition is carried out similarly for fibrinogen peptides, see Materials and Methods of Example 1, mutatis mutandis.

[0170] Primary cells from RA patients were cultured in vitro for 14 days with the original citrullinated vimentin peptide. From day 5, the cell medium was supplemented with human serum and 50 U of recombinant IL-2. The cells were incubated in a 37 °C incubator with 5% CO2.

[0171] Upon harvesting, the cells were centrifuged, the pellet resuspended in PBS, and stained with HLA class II tetramers, reagents that can only interact with T cells that have a TCR that can interact with the peptide-HLA complex. Two sets of tetramers (different colors) were used for staining.

[0172] To prove that the modified peptides can indeed bind and thereby present the peptides to the TCR, we performed binding assays, which are competition assays and show the ability of the test peptide to compete with an already bound peptide (in this case, an influenza (HA) peptide). The results can be expressed as a curve or Kd values, both of which are shown in the accompanying figures, showing that exchanging one amino acid at position P4 reduces the Kd value, whereas exchanging two gives a better (lower) Kd value. (See FIG. 5). Note that these numbers only reflect the ability of the peptide to bind to HLA, and not to interact with the TCR.

[0173] Furthermore, short-term T cell lines derived from primary cells of RA patients were generated and we demonstrated that many of the cit-specific T cells identified by peptide-HLA tetramers also bound to tetramers loaded with the glutamine version of the peptide. Figure 6 shows the cit-peptide-reactive T cells on the x-axis and the glutamine-reactive T cells on the y-axis. This result clearly shows that there are T cells that cannot distinguish between the original and mimic peptides.

[0174] Furthermore, we sequenced the TCR from these cells, generated T cell lines, and began testing fibrinogen peptides as previously described.

[0175] In summary, the new findings presented herein can be used to develop methods for generating antigen-specific tolerance, where the peptides of the present invention are administered alone, in complex with the relevant MHC class II molecule, bound to other molecules or cellular complexes, and optionally bound to suitable carriers such as nanoparticles, proteins, or blood cells, to name a few. Knowledge of these peptides can also be used to design tolerizing mRNA vaccines. Such vaccines can be produced in a similar manner as described for example for mRNA vaccines encoding MOG peptides for tolerizing therapy against experimental allergic encephalomyelitis (EAE) (Krienke C. et al., Science 2021, incorporated herein by reference).

[0176] This principle of defining and producing non-post-translationally modified peptidomimetics will significantly improve the production of tolerogenic molecular constructs and appropriate mRNA molecules, and will provide a major breakthrough in the development of tolerogenic treatments for autoimmune diseases, especially RA.

[0177] The concept of creating non-naturally occurring non-post-translationally modified peptide mimetics instead of naturally occurring post-translationally modified peptides and using them for therapeutic purposes may open up new therapeutic principles, in which the design of therapeutic peptides will be significantly improved for the treatment or prevention of immune-mediated diseases driven by immunity against post-translationally modified proteins and peptides.

[0178] Furthermore, so-called "tolerogenic particles" designed to be used as biopharmaceuticals and administered to patients can now be produced in ways that make these medicines more stable and easier to manufacture, including producing them at higher quality, and in new and convenient ways to ensure this increased quality. Our contribution is an unexpected major advancement for the production of tolerogenic medicines for autoimmune diseases (e.g., but not limited to, RA).

[0179] Without further detailed description, it is believed that those skilled in the art can utilize the present invention to its fullest extent using the present description, including the examples. Also, while the present invention has been described herein with reference to preferred embodiments thereof which constitute the best mode currently known to the inventors, it should be understood that various changes and modifications, as would be obvious to one of ordinary skill in the art, may be made therein without departing from the scope of the invention as set forth in the claims appended hereto.

[0180] Thus, while various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

[0181] Sequence Listing

[0182] GGYRAXPAKAAAT - (SEQ ID NO: 1) GGYRAQPAKAAAT - (SEQ ID NO: 2) GGFRAXPAKAAAT - (SEQ ID NO: 3) GGFRAQPAKAAAT - (SEQ ID NO: 4) SAVRLXSSVPGVR - (SEQ ID NO: 5) SAVRLQSSVPGVR - (SEQ ID NO: 6) SAFRLXSSVPGVR - (SEQ ID NO: 7) SAFRLQSSVPGVR - (SEQ ID NO: 8) VSLISRXGDMSSNPA - (SEQ ID NO: 9) QGQYELXVDLRDHGE - (SEQ ID NO: 10) VSLISRQGDMSSNPA - (SEQ ID NO: 11) QGQYELQVDLRDHGE - (SEQ ID NO: 12) (amino acid sequence, one letter code)

[0183] References

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Claims

1. An analog of a naturally occurring post-translationally modified peptide for use in a method for inducing tolerance in a subject, preferably wherein said induction of tolerance is a step in the treatment, alleviation or prevention of an autoimmune disease, wherein compared to said naturally occurring peptide, citrulline is replaced by another amino acid to form a peptide mimic, and said peptide mimic binds to the peptide binding groove of a human leukocyte antigen (HLA) molecule to the same extent as said corresponding naturally occurring post-translationally modified peptide, and said peptide mimic is recognized by T cells to the same extent as said corresponding naturally occurring post-translationally modified peptide, an analog of said naturally occurring post-translationally modified peptide.

2. The peptide mimic has a crystal structure determined, for example, by X-ray diffraction crystallography, and said crystal structure is substantially identical to the crystal structure of said corresponding naturally occurring peptide determined using the same method, a peptide mimic for use according to claim 1.

3. The synthetic peptide is an analog of a peptide selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated type II collagen, citrullinated cartilage intermediate layer protein (CILP), citrullinated tenascin C and citrullinated α-enolase, a peptide mimic for use according to claim 1.

4. The peptide is fibrinogen, and citrulline at position 74 is replaced by glutamine, a peptide mimic for use according to claim 3.

5. The peptide is fibrinogen, citrulline at position 74 is replaced by glutamine, and tyrosine at position 71 is replaced by phenylalanine, a peptide mimic for use according to claim 3.

6. The peptide is vimentin, citrulline at position 71 is replaced by glutamine, and valine at position 68 is replaced by phenylalanine, a peptide mimic for use according to claim 3.

7. The peptide is tenascin C, and citrulline at position 877 is replaced by glutamine, a peptide mimic for use according to claim 3.

8. The peptide is tenascin C, and citrulline at position 2073 is replaced by glutamine, a peptide mimic for use according to claim 3.

9. The peptidomimetic according to claim 1 for use, wherein the peptidomimetic binds to the P4 pocket (binding groove) of a human leukocyte antigen (HLA) molecule with substantially the same affinity as the corresponding naturally occurring peptide.

10. A complex of a carrier and a peptide, wherein the peptide is a mimetic of a naturally occurring post-translationally modified peptide, and in the peptidomimetic, citrulline is replaced with another amino acid as compared to the naturally occurring peptide to form the peptidomimetic, and the peptidomimetic binds to the peptide binding groove of a human leukocyte antigen (HLA) molecule to the same extent as the corresponding naturally occurring post-translationally modified peptide, and the peptidomimetic is recognized by T cells to the same extent as the corresponding naturally occurring post-translationally modified peptide, and the carrier is selected from nanoparticles, blood cells, and MHC class II molecules, said complex.

11. The complex according to claim 10, wherein the peptidomimetic has a crystal structure determined, for example, by X-ray diffraction crystallography, and the crystal structure is substantially identical to the crystal structure of the corresponding naturally occurring peptide determined using the same method.

12. The complex according to claim 10, wherein the peptide is a mimetic of a peptide selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated type II collagen, citrullinated tenascin C, citrullinated cartilage intermediate layer protein (CILP), and citrullinated α-enolase.

13. The complex according to claim 10, wherein as shown in SEQ ID NO: 2, the peptide is fibrinogen, and citrulline at position 74 is replaced with glutamine.

14. The complex according to claim 10, wherein as shown in SEQ ID NO: 4, the peptide is fibrinogen, citrulline at position 74 is replaced with glutamine, and tyrosine at position 71 is replaced with phenylalanine.

15. The complex according to claim 10, wherein as shown in SEQ ID NO: 8, the peptide is vimentin, citrulline at position 71 is replaced with glutamine, and valine at position 68 is replaced with phenylalanine.

16. The complex according to claim 10, wherein as shown in SEQ ID NO: 11, the peptide is tenascin C, and citrulline at position 877 is replaced with glutamine.

17. The complex according to claim 10, wherein as shown by SEQ ID NO: 12, the peptide is tenascin C and the citrulline at position 2073 is replaced by glutamine.

18. The complex according to claim 10 for use in a method for inducing tolerance in a subject, preferably a method in which the induction of tolerance is a step in the treatment, alleviation or prevention of an autoimmune disease.

19. An immunogenic mRNA vaccine for inducing tolerance to a specific antigen in a subject, comprising a modified non-inflammatory mRNA encoding a mimic of the post-translational modification of said antigen, wherein in said peptide, citrulline is replaced with another amino acid while maintaining binding to the peptide-binding groove of the human leukocyte antigen (HLA) molecule to the same extent as the naturally occurring post-translationally modified peptide, and the mimic of the post-translational modification of said antigen is a peptide mimic that binds to the peptide-binding groove of the human leukocyte antigen (HLA) molecule to the same extent as the naturally occurring post-translationally modified peptide.

20. The vaccine according to claim 19, wherein said peptide has a crystal structure determined, for example, by X-ray diffraction crystallography, and said crystal structure is substantially identical to the crystal structure of said naturally occurring peptide determined using the same method; and said peptide binds to the peptide-binding groove of the human leukocyte antigen (HLA) molecule to the same extent as the naturally occurring post-translationally modified peptide.

21. The vaccine according to claim 19, wherein citrulline is replaced by glutamine.

22. The vaccine according to claim 19, wherein said peptide is a mimic of an antigen selected from citrullinated fibrinogen, citrullinated vimentin, citrullinated collagen type II, citrullinated tenascin C, citrullinated cartilage intermediate layer protein (CILP), and citrullinated enolase.

23. The vaccine according to claim 19, wherein said modified non-inflammatory mRNA is a nanoparticle-formed 1-methylpseudouridine-modified mRNA.

24. The vaccine according to claim 19 for use in the treatment, alleviation or prevention of an autoimmune disease.

25. The vaccine for use according to claim 24, wherein said autoimmune disease is rheumatoid arthritis.

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