Generation of MHC II / CII peptide complexes

Abstract

The present invention relates to MHC II / CII peptide complexes which are glycosylated in situ, i.e. which are naturally glycosylated during recombinant protein expression in a host cell. The present invention further relates to a method for producing glycosylated MHC II / CII peptide complexes in mammalian cells. Furthermore, the present invention relates to the use of such post-translationally modified, preferably glycosylated, MHC / CII complexes for the treatment of rheumatoid arthritis, preferably in humans.

Description

TECHNICAL FIELD

[0001] The present invention relates to in situ glycosylated MHC II / CII peptide complexes, i.e. complexes that are naturally glycosylated during recombinant protein expression in host cells. The present invention further relates to a method for producing glycosylated MHC II / CII peptide complexes in mammalian cells. Furthermore, the present invention relates to the use of such post-translationally modified, preferably glycosylated, MHC II / CII peptide complexes for the treatment of arthritis. TECHNICAL BACKGROUND

[0002] Rheumatoid arthritis (RA) is a common and severe disease, being a major health problem affecting 4-7 million people in Europe. It is caused by an abnormal autoimmune joint inflammation associated with pain, progressive cartilage and bone destruction, which, if not adequately treated, leads to disability and eventually to immobility / bed confinement. Today's pharmacological treatment is initiated immediately after clinical diagnosis is established and is effective in 60-70% of cases, but does not cure the disease. The pharmacological treatment is mainly directed against common effector pathways of inflammation, thus causing a broad immunosuppressive effect associated with an increased risk of infections.

[0003] Immunogenetics of RA indicate that abnormal pathways of T cell activation play a key role in the onset and / or persistence of the disease. During T cell activation, CD4+ T cells are bound by antigenic peptide fragments complexed with human leukocyte antigen (HLA) class II molecules (human major histocompatibility complex (MHC) class II), leading to their activation in the context of costimulatory signals provided by professional antigen-presenting cells. The strongest evidence supporting a role for CD4+ T cells in the pathogenesis of the disease is the genetic association between RA and certain alleles of the HLA-DRB1 locus, which encodes a common amino acid motif Q / R R / K RAA (amino acid positions 70-74, so-called "shared epitope") on the beta chain of the peptide-binding pocket of the MHC class II molecule HLA-DR (Gregersen PK et al., Arthritis Rheum. 1987; 30: 1205-1213). Convincing evidence for a pathogenic role of T cells in RA comes from their frequent detection in inflamed synovial infiltrates of moderate to severe disease, suggesting that they cooperate with B cells in the local immune response to promote maturation of specific autoantibody responses. Moreover, impaired CD4+ CD25(hi) regulatory T cell (Treg) function has been shown to be involved in the pathogenesis of RA. Thus, the chronically activated T cell compartment in RA represents a key target for therapeutic immunomodulatory interventions.

[0004] Today, it is believed that RA starts many years before clinical onset. RA as a polygenic disease, with the aforementioned shared epitope-encoding alleles at the HLA-DRB1 locus as the strongest risk factor, develops in susceptible individuals, respectively. However, it is not clear environmental and / or lifestyle factors (smoking) also participate in triggering the autoimmune response associated with IgG (rheumatoid factor) and citrullinated protein (ACPA) antibody production, which can persist for up to twenty years in preclinical stage in individuals with arthritic predisposition but still healthy. Immune responses to type II collagen (CII) and citrullinated CII can be detected before and after clinical onset (Burkhardt H et al., Eur J Immunol. 2005; 35: 1643-52). CII is the major protein component in the articular cartilage. RA patients carrying the DRB1*0401 allele (50% of Caucasian RA patients) have been shown to harbor T cells in their repertoire that specifically respond to the major CII epitope corresponding to the amino acid sequence 259-273 of the triple-helical CII region. However, the T cell determinant crucial for T cell receptor (TCR) activation depends on the physiologically galactosylated hydroxylysine residue at position 264 (Baecklund J. et al., Proc Natl Acad Sci U S A. 2002; 99: 9960-5).

[0005] The most commonly used animal model for RA is collagen-induced arthritis (CIA) in mice. Experimental arthritis depends on MHC class II, is associated with murine class II allele Aq, and depends on T cell recognition of the galactosylated 259-273 CII epitope (Holmdahl R. et al. Ageing Res Rev. 2002; 1 : 135-47). CIA is used as a standard model to test the therapeutic efficiency of new compounds with anti-arthritic potential in drug development. Various protocols have been developed to induce antigen-specific tolerance, and one of the candidate antigens to prevent and treat arthritis by vaccination is CII. The most efficient protocol in adult mice (with no side effects observed to date) is to induce tolerance by intravenous injection of a recombinant protein complex consisting of the extracellular domain of MHC class II molecule Aq with the major antigen CII peptide in the binding pocket, i.e. the galactosylated CII 259-273 peptide or Aq / galCII complex (Dzhambazov B et al. J Immunol 2006; 176: 1525-1533). Injection of the Aq / galCII complex after immunization with CII but before the onset of arthritis leads to almost complete prevention of the development of arthritis, and treatment of mice with chronic recurrent arthritis leads to downregulation of inflammatory activity. The Aq / galCII effect is dominant for tolerogenicity because of its anti-arthritic potential that can be transferred from treated mice to na'ive recipients with T cells.

[0006] The Aq complex containing the CII peptide without galactosylation in position 264 remained ineffective. The reason for this remarkable selective modulation can be related to the fact that galactosylated CII is expressed only in cartilage (Baecklund J. et al., Proc Natl Acad Sci U S A. 2002; 99:9960-5), whereas non-glycosylated CII is also expressed in the thymus (Chin R.K., et al., J Immunol. 2006; 177:290-7). Thus, the T cell response to non-glycosylated CII is regulated by central tolerance, whereas the T cell response to galactosylated antigens is regulated by peripheral tolerance mechanisms. It has thus been proposed that a disturbance of physiological peripheral self-tolerance, in particular interference with diarthrodial joint structural components, is a major driving force in the pathogenesis of RA, and that its reconstitution is a fundamental principle for the development of tolerance therapy strategies. This approach includes the parenteral administration of DR4 / galCII complexes to human RA patients selected by biomarkers, which are induced by prior genotyping to induce immunoregulatory T cells and identified as carriers of the DRB1*0401 allele, which down-regulate arthritogenic T cell responses by bystander suppression. Its mechanism of action includes selective immunomodulation of arthritogenic adaptive immune responses, while leaving protective immunity unaffected, compared to traditional treatment approaches. It is a personalized or HLA-restricted treatment approach, limited to patients with specific HLA alleles, such as DRB1*0401 positive patients. In addition, preclinical data on CIA treatment suggest that DR4 / galCII complexes have the potential to achieve therapeutic effects in established RA and prophylactic effects in individuals at risk of developing RA, i.e. before disease manifestation. Thus, the mode of action is fundamentally different from established therapies in RA.

[0007] WO 2007 / 058587 A1 relates to "Compounds comprising a self-antigen peptide and a carrier with an MHC binding motif" and discloses compounds comprising (a) a peptide and (b) a carrier, wherein the peptide has at least the motif X-X-X-X-X-X-X, and wherein at least one amino acid residue X is glycosylated. Furthermore, the peptide is linked to a peptide binding protein, and the carrier comprises at least an MHC binding motif, wherein the linkage can be a covalent linkage. However, the peptide is not expressed together with the MHC II protein by the same host cell, or linked to the MHC II protein by a linker peptide. The MHC II protein is initially expressed in SL2 cells, with a surrogate peptide in the binding groove, and then loaded with the peptide in vitro.

[0008] Production of MHC II proteins in HEK cells has been described before (Sareila et al., Antioxidants & Redox Signaling, 2017, 27(18): 1473-1490), however, the synthetic glycosylated peptides are no longer expressed with the MHC II together with the protein, but are loaded onto the MHC II protein after production, thus not connected to the MHC II protein with a linker peptide. Synthesis of galactosylated CII peptides is both time and money consuming. Furthermore, loading of synthetic peptides onto recombinant MHC class II molecules is not trivial and difficult to scale up, especially because of the need for excess addition of peptides. Therefore, there is a need for a simpler production method that can be scaled up for production of relevant amounts for therapeutic use. SUMMARY

[0010] The present invention relates to a composition comprising a recombinant MHC II / CII peptide complex, said complex comprising: (a) an extracellular region of an MHC class II alpha chain comprising at least one alpha 1 domain; (b) an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain; and (c) a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the N-terminus of the MHC class II beta chain, via a linker peptide; wherein the CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG and AGFKXEQGPXG, and wherein the MHC II / CII peptide complex comprises a post-translationally modified CII peptide, preferably wherein the first lysine residue of the CII peptide is a hydroxylysine (Hyl) or an O-glycosylated Hyl. In one embodiment, the first lysine residue is a galactosyl-hydroxylysine.

[0011] In certain embodiments, the CII peptide comprises the amino acid sequence of AGFKGEQGPKG, AGFKGEQGPX1G, AGFKGEX2GPKG, AGFKGX3QGPKG, AGFKX4EQGPKG, AGFKGEX2GPX1G, AGFKGX3QGPX1G, and AGFKX4EQGPX1G, wherein X1 is any protein amino acid except K, preferably R, A, G, or Q, more preferably R; X2 is any protein amino acid except Q, preferably A, R, H, or G; X3 is any protein amino acid except E, preferably A, D, Q, or G; and X4 is any protein amino acid except G, more preferably A, S, V, or L. Preferably, X2, X3, or X4 is not K, more preferably X1, X2, X3, or X4 is not K. In certain embodiments, the CII peptide comprises the amino acid sequence of AGFKGEQGPKG or AGFKGEQGPX1G, preferably AGFKGEQGPKGEP or AGFKGEQGPX1GEP, more preferably GIAGFKGEQGPKGEP or GIAGFKGEQGPX1GEP.

[0012] Preferably, the MHC class II is HLA-DR, and at least the alpha 1 domain is DRA*0101, and at least the beta 1 domain is selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303, preferably DRB1*0401. In certain embodiments, the CII peptide comprises only the first lysine residue, and any additional K is mutated, preferably to R, A, G, or Q, more preferably to R.

[0013] The present application also relates to a method for producing a post-translationally modified (e.g., O-glycosylated) CII peptide, comprising: (a) transfecting a mammalian cell with: (i) a polynucleotide encoding an extracellular region of an MHC class II alpha chain comprising at least one alpha 1 domain; (ii) a polynucleotide encoding an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain; and (iii) a polynucleotide encoding a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain via a linker peptide, preferably to the MHC class II beta chain, wherein the CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPX1G, AGFKGEX2GPKG, AGFKGX3QGPKG, AGFKX4EQGPKG, AGFKGEX2GPX1G, AGFKGX3QGPX1G, and AGFKX4EQGPX1G; (b) culturing the mammalian cell under conditions suitable for producing an MHC II / CII peptide complex, and (c) harvesting the cell supernatant and optionally the cells comprising the MHC II / CII peptide complex, the MHC II / CII peptide complex comprising a post-translationally modified CII peptide, preferably wherein the first lysine residue of the CII peptide is a hydroxylysine (Hyl) or an O-glycosylated Hyl; optionally further comprising a step of analyzing the post-translational modification (preferably the glycosylation profile) of the CII peptide of the MHC II / CII peptide complex. In one embodiment, the first lysine residue is galactosyl-hydroxylysine.

[0014] In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG, AGFKGEQGPX1G, AGFKGEX2GPKG, AGFKGX3QGPKG, AGFKX4EQGPKG, AGFKGEX2GPX1G, AGFKGX3QGPX1G, and AGFKX4EQGPX1G, wherein X1 is any protein amino acid except K, preferably R, A, G, or Q, more preferably R; X2 is any protein amino acid except Q, preferably A, R, H, or G; X3 is any protein amino acid except E, preferably A, D, Q, or G; and X4 is any protein amino acid except G, more preferably A, S, V, or L. Preferably, X2, X3, or X4 is not K, more preferably X1, X2, X3, or X4 is not K. In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG or AGFKGEQGPX1G, preferably AGFKGEQGPKGEP or AGFKGEQGPX1GEP, more preferably GIAGFKGEQGPKGEP or GIAGFKGEQGPX1GEP.

[0015] In certain embodiments, the CII peptide comprises only the first lysine residue and any additional K is mutated, preferably to R, A, G or Q, more preferably to R. Suitable mammalian cells comprise enzymes that post-translationally modify lysine residues in collagen, including: hydroxylation of lysine to hydroxy lysine (Hyl) and galactosylation of Hyl to galactosyl hydroxy lysine (Gal-Hyl), such as lysyl hydroxylases (e.g. lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2)) and collagen galactosyltransferases (e.g. collagen galactosyltransferases GLT25D1 and / or GLT25D2). In one embodiment, the mammalian cell is a kidney cell, a fibroblast cell or an osteoblast cell, preferably a kidney cell, more preferably a HEK293 cell line. In another embodiment, the mammalian cell is a genetically engineered cell recombinantly expressing lysyl hydroxylases and collagen galactosyltransferases, preferably lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2) and collagen galactosyltransferases GLT25D1 and / or GLT25D2. Preferably, the mammalian cell lacks galactosyl hydroxy lysyl glucosyltransferase activity.

[0016] Also provided is a recombinant MHC II / CII peptide complex comprising a post- translationally modified CII peptide, obtained by the method of the application. In one embodiment, the first lysine residue of the CII peptide is hydroxy lysine (Hyl) or O-glycosylated Hyl. In another aspect, the present application relates to a composition containing a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide, obtained by the method of the application. Preferably, the first lysine residue of the CII peptide is hydroxy lysine (Hyl) or O-glycosylated Hyl.

[0017] Also provided is the use of a composition according to the application or a recombinant MHC II / CII peptide complex according to the application for the treatment of a chronic inflammatory disease, wherein the chronic inflammatory joint disease is preferably selected from the group of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, non-radiographic axial spondyloarthritis, ankylosing spondylitis, juvenile idiopathic arthritis, relapsing polychondritis, systemic lupus erythematosus, Lyme disease, Meniere diseases, autoimmune inner ear disease (AIED) or Still’s disease.

[0018] In yet another aspect, the present application relates to a MHC II / CII peptide complex tetramer comprising: a recombinant MHC II / CII peptide complex of a composition according to the application, or a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide according to the application. Preferably, the tetramer comprises a multimerization molecule, such as streptavidin.

[0019] In yet another aspect, the present application relates to a method for preparing a MHC II / CII peptide complex tetramer comprising the following steps: (a) providing a composition according to the present application or a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide according to the present application, wherein the MHC II / CII peptide complex comprises at least one N-terminal biotinylation; (b) contacting the composition with a multimerization molecule (preferably streptavidin) and, optionally, isolating the tetramer comprising four MHC II / CII peptide complexes bound to streptavidin.

[0020] In yet another aspect, the present application provides an in vitro method for detecting and / or quantifying T cells specific for a given antigen, wherein the method comprises the following steps: providing a MHC II / CII peptide complex tetramer of the present application; contacting the MHC II / CII peptide complex tetramer with a sample of a subject (preferably a sample containing peripheral blood cells of said subject); and detecting the label of the MHC II / CII peptide complex tetramer bound to T cells. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : Schematic representation of the MHC II / CII peptide complex. The MHC II molecule has a covalently bound CII 259-273 peptide. BirA: biotinylation site, HIS: poly(6x)histidine tag, JUN / FOS: complementary domains of a leucine zipper (heterodimerization domain), TEV: Tobacco Etch Virus (TEV) cysteine protease cleavage site, linker: Gly-Ser linker peptide, Thrombin cleavage site, Strep-tag, CII peptide 259-273.

[0022] Figure 2 : IL-2 (FU) secretion of Aq / rCII (259-273) complex producing Aq-restricted T cell hybridoma clones in response to HEK293 cells (upper panel), S2 insect cells (middle panel) and anti-CD3 antibody stimulation (lower panel) (recombinant, in situ glycosylated Aq / rCII). Mouse T cell hybridoma clones used have the following specificities: HCQ3 (CII, Gal-HK264), HCQ.4 (CII, unmodified and HK264), HCQ.11 (Glc-Gal-HK264), HM1R.2 (CII, Gal-HK264 and Gal-HK264+270), HP3 (Aq-restricted pepsin peptide), where K is the abbreviation for lysine and HK is the abbreviation for hydroxylysine.

[0023] Figure 3Therapeutic vaccination using in situ glycosylated Aq / rCII produced in HEK293 cells in a mouse CIA model. A) Dose-response curve: Naive mice were immunized with CII to induce arthritis and received a booster at day 35. Mice were treated with different doses of MHC II / CII peptide complex: 10, 50 or 100 μg (n=9). The number of arthritic mice was significantly reduced in the 100 μg treatment group compared to the control group (p<0.05, Chi square). B) To administer the MHC II / CII peptide complex, osmotic pumps were implanted 7 days after the booster at day 35 to ensure continuous vaccine administration (e.g. 100 μg: 15 μg / 24h for 7 days).

[0024] Figure 4 Activation of glycosylation-restricted human T cell hybridomas. Human T cell hybridomas were activated upon stimulation with human MHC II / CII peptide complex (DR4 / hCII) in an antigen-specific manner. Recognition of human T cell hybridoma mDR1.1 and 3H8 was dependent on the glycosylation signature of the CII peptide. A) T cell hybridoma clone mDR1.1 was activated by HLA-DR4 presented galactosylated K264; B) whereas T cell hybridoma clone 3H8 was activated by HLA-DR4 presented unmodified CII epitope. Reactivity of these two different T cell hybridoma clones was compared by using human MHC II / CII peptide complex loaded with synthetic galactosylated or unmodified CII peptide (DR4 / galCII and DR4 / nCII, respectively) and naturally glycosylated MHC II / CII peptide complex (DR4 / hCII). IL-2 secretion was measured by ELISA.

[0025] Figure 5 Detection of antigen-specific T cells in peripheral blood of HLA-DRB1*0401 rheumatoid arthritis patients. A) Biotinylated DR4 / galCII peptide complex was incubated with streptavidin conjugated with a fluorescent dye (PE, APC). These tetramers were used to detect T cells specific for CII259-273 peptide galactosylated at K264. Antigen-specific (CII259-273, K264gal) T cells were detected in PBMC of RA patients and healthy donors using flow cytometry. B) Frequency comparison of antigen-specific T cells was detected using DR4 / galCII peptide tetramers, DR4 / nCII peptide tetramers or DR4 / hCII peptide tetramers. Frequency of tetramer-positive T cells in CD4+ T cell population was measured by flow cytometry.

[0026] Figure 6Human T cell activation. Detection of antigen-specific T cells in the peripheral blood of HLA-DRB1*0401 RA patients. T cells were activated by stimulation with galCII and to a lesser extent by unmodified CII peptides. Upregulation of CD154 was measured by flow cytometry (significance: p-value = 0.0332, Mann-Whitney test).

[0027] Figure 7 Legendplex of cytokines released by in vitro stimulated PBMC of HLA-DRB1*0401 positive RA patients (n=20) TM Analysis. Specific induction of IL-2, IL-17f, IFN-γ, IL-10, IL-17a and TNF-α release by in vitro stimulation with DR4 / nCII peptide complex or DR4 / galCII peptide complex compared to stimulation with standard TR1 cell differentiation conditions (TR1) and negative control (CO) is shown.

[0028] Figure 8 Comparison of complexes with and without His-tag. (A) ELISA comparing the coating efficiency of equimolar solutions of DR4 / nCII versus DR4 / nCII Tev-cleaved complex to microtiter wells using a DR4-specific antibody and a peroxidase-coupled secondary antibody. Shown is the absorbance at 405 nm [μg / ml] of the DR4 / nCII solution used to coat the microtiter plate at the indicated protein concentration. (B) Activation of 3H8 hybridoma cells by DR4 / nCII versus DR4 / nCII Tev-cleaved complex pre-coated into microtiter wells at the indicated concentrations. Shown is the IL-2 concentration [μg / ml] in the supernatant after activation at the indicated protein concentration of the DR4 / nCII solution used to coat the microtiter plate.

[0029] Figure 9 Effect of His-tag in DR4 / hCII peptide complex and its interaction with A) Chondroitin sulfate (CS), B) Hyaluronic acid, C) Heparan sulfate (HS) on T cell activation: IL-2 response induced by DR4 / hCII versus DR4 / hCII ΔHis in 3H8 hybridoma and also DR4 / hCII DED at the indicated concentrations in the solute phase in (A) microtiter wells were blocked or pre-coated with chondroitin sulfate. Shown is the IL-2 concentration in the supernatant after activation.

[0030] Figure 10: Activation of 3H8 hybridoma cells by DR4 / hCII versus DR4 / hCII Delta His versus DR4 / hCII DED pre-coated into microtiter wells at the indicated concentrations. Shown is the IL-2 concentration [pg / ml] in the supernatant after activation at the indicated protein concentration of the DR4 / hCII solution used to coat the microtiter plate.

[0031] Figure 11 : Influence of the His-tag in the DR4 / hCII peptide complex and its interaction with chondroitin sulfate (CS) in the bulk phase on the stimulation of the IL-10 response in 3H8 hybridoma cells: 3H8 hybridoma cells were activated by DR4 / hCII at the indicated concentrations in the bulk phase with or without (w / o) chondroitin sulfate (2.5 mg / ml) in microtiter wells with a blocked plastic surface. Shown is the IL-10 concentration [pg / ml] in the supernatant after activation at the indicated protein concentration of the DR4 / hCII solution used to coat the microtiter plate.

[0032] Figure 12 : Comparison of the therapeutic effect of Aq / galCII peptide complexes with or without His-tag on ear swelling induced by DTH response to collagen II in vivo. The effect of Aq / galCII constructs with (His) and without polyhistidine tag (w / o His) is shown compared to the Aq / mCLIPmt control construct comprising a linked mouse mutant CLIP peptide (CLIPmt) in its binding groove (* indicates a p value of < 0.05, ** indicates a p value of < 0.01).

[0033] Figure 13 : Heterogeneity of CII-peptide post-translational modifications in recombinant DR4 / hCII complexes. Shown is the percentage of detectable modifications at the indicated K positions at the corresponding positions analyzed by mass spectrometry. [OH = hydroxylysine, Hex = galactosyl-hydroxylysine, DiHex = glucosyl-galactosyl-hydroxylysine, Ub = ubiquitin, POH = hydroxyproline.

[0034] Figure 14 : Generation of Plod3 gene (LH3) knockdown Expi293 cells. (A) Schematic of the stepwise transfer from lysine to hydroxylysine to Gal-hydroxylysine and Glc-Gal hydroxylysine mediated by the multifunctional collagen-modifying enzyme LH3. (B) Detection of PLOD3 by western blot. Cells from 1 x 106 6Lysates of different Expi293 HEK cell clones transduced with lentivirus encoding Plod3 specific sh-RNA were loaded on SDS-PAGE and PLOD3 was detected on Western Blot using anti-PLOD3 antibody. The theoretical molecular weight of PLOD3 is 84 kDa. Clones #4, #18 and #20 were used for further expansion. (C) Glycans were analyzed by mass spectrometry. After lentiviral transduction with shRNA to knock down plod3 gene, glycans analysis by mass spectrometry was performed to investigate the reduced glycosylation of galactosyl hydroxylysyl residues. Two lysines (K264 and K270) within the collagen type II epitope (SEQ ID NO: 1) shown on top were analyzed. A clear reduction of the glucos-galactosyl hydroxylysyl residues (DiHex) was demonstrated. Unmod = unmodified, OH = hydroxylated, DiOH = dihydroxylated, Hex = galactosylated hydroxylysyl, DiHex = glucos-galactosylated hydroxylysyl.

[0035] Detailed description of preferred embodiments

[0036] The general embodiments "comprising of" or "consisting of" encompass the more specific embodiment "consisting of". Furthermore, the singular and the plural form are used without restriction. As used herein, the singular forms "a", "an" and "the" indicate the singular and the plural, unless the context clearly indicates otherwise.

[0037] The terms "protein" and "amino acid sequence" or "polypeptide" are used interchangeably and refer to an amino acid polymer of any length. The terms also encompass proteins that have been post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation or protein processing. Modifications and variations, such as fusions with other proteins, amino acid sequence substitutions, deletions or insertions, can be made in the structure of a polypeptide and while the molecule retains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid encoding sequence and a protein with the same properties can be obtained.

[0038] The term "polypeptide" generally refers to a sequence of more than 20 amino acids, while the term "peptide" refers to a sequence of up to 20 amino acids. However, these terms can be used interchangeably. Proteins can form multimers such as dimers, where the dimers can be hetero- or homo-dimers. The MHC II / CII peptide complex of the present invention comprises the extracellular region of an MHC class II alpha chain and the extracellular region of an MHC class II beta chain, which typically form a heterodimer, which forms a binding groove to accommodate a collagen type II peptide fused to the N-terminus of one of the chains. However, the skilled person will understand that the two proteins forming the heterodimer can also be produced as a fusion protein to form a single polypeptide chain with domains linked to each other (optionally via a flexible linker), i.e. a single chain heterodimer.

[0039] A "fusion protein" is defined as a protein comprising the entire sequence or any part of the sequence of two or more originally separate natural or modified proteins. Fusion proteins can be constructed by genetic engineering methods using recombinant DNA technology, by fusing two or more genes or cDNAs or parts thereof originally encoding two or more originally separate natural or heterologous proteins or parts thereof. This results in a fusion protein having functional properties derived from each of the original proteins. Thus, a peptide or protein is linked to another protein by a peptide bond or, preferably, a linker peptide.

[0040] The terms "genomic DNA" or "genome" are used interchangeably and refer to the heritable genetic information of a host organism. Genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA), as well as the DNA of other organelles (e.g. mitochondria).

[0041] The term "gene" as used herein refers to a DNA locus of heritable genomic sequence that affects the traits of an organism by being expressed as a functional product or by regulating gene expression. Genes and polynucleotides can include introns and exons in genomic sequence, or only the coding sequences contained in cDNA, such as open reading frames (ORF) including the start codon (methionine codon) and the translation termination codon. Genes and polynucleotides can also include regions that regulate their expression, such as transcription initiation, translation, and transcription termination. Therefore, also included are regulatory elements, such as promoters.

[0042] As used herein, the terms "nucleic acid", "nucleotide" and "polynucleotide" are used interchangeably to refer to a single- or double-stranded polymer of deoxyribonucleotide bases or ribonucleotide bases, read from 5' to 3' end, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), genomic DNA, cDNA, cRNA, recombinant DNA or recombinant RNA, and derivatives thereof, such as those containing modified backbones. Preferably, the polynucleotide, in particular the polynucleotide stably integrated into the mammalian genome, is DNA or cDNA. The polynucleotide of the application can be prepared in different ways (e.g. by chemical synthesis, by gene cloning, etc.), and can take various forms (e.g. straight or linear, single- or double-stranded, or hybrids thereof, primers, probes, etc.). The term "nucleotide sequence" or "nucleic acid sequence" refers to the sense strand and the anti-sense strand of the nucleic acid as a single strand or duplex.

[0043] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide derived from a different cell, organism or different species of the recipient, such as a CHO cell or a HEK293 cell, and introduced into the recipient using recombinant technology. In the context of the present application, the skilled person will understand that it refers to DNA or cDNA. A recombinant polynucleotide can also be referred to as a transgene or a heterologous polynucleotide. It can thus be a gene or an open reading frame (ORF) encoding a recombinant protein. In the context of a mammalian cell, such as a HEK293 or CHO cell, "recombinant polynucleotide" refers to a polynucleotide derived from a different cell or artificially synthesized. The term "recombinant" refers to a molecule, such as a polypeptide or a polynucleic acid molecule, formed by laboratory methods of genetic recombination, such as molecular cloning. Such methods bring together genetic material from multiple sources, or create sequences that do not exist in nature. "Recombinant" also includes, when used in reference to a portion of a nucleic acid, a polynucleotide comprising two or more sequences that are not naturally associated with each other, or a polypeptide encoded by said polynucleotide. A recombinant can thus also refer to a polynucleotide sequence, such as a gene or a transgene or a portion thereof, derived from the same cell line, but inserted into a location in the genome where it does not normally exist, or the introduction of a gene into a cell of an organism where it does not normally exist.

[0044] As used herein, a "recombinant polynucleotide," "recombinant gene," or "recombinant sequence" can be introduced into a target cell or host cell, either directly or preferably by use of an "expression vector," preferably a mammalian expression vector. Methods for constructing vectors are well known to those skilled in the art. Vectors can include, but are not limited to, plasmid vectors, cosmids, artificial / minichromosomes (e.g., ACE), or viral vectors, such as retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus. Eukaryotic expression vectors often also contain prokaryotic sequences that facilitate propagation of the vector in bacteria, such as an origin of replication and antibiotic resistance genes for selection in bacteria. A variety of eukaryotic expression vectors are well known in the art, which contain cloning sites to which a polynucleotide can be operably linked. Often expression vectors also contain an expression cassette that encodes a selectable marker, allowing selection of host cells that harbor the expression marker.

[0045] The term "cytokine" refers to small proteins released by cells that act as intercellular mediators, e.g., that influence the behavior of cells surrounding the secreting cell. Cytokines can be secreted by immune cells or other cells, such as T cells, B cells, NK cells, and macrophages. Cytokines can participate in intercellular signal transduction events, such as autocrine, paracrine, and endocrine signal transduction. They can mediate a range of biological processes, including but not limited to: immunity, inflammation, and hematopoiesis. A cytokine can be a chemokine, an interferon, an interleukin, a lymphokine, or a tumor necrosis factor.

[0046] As used herein, the term "expression" refers to the transcription and / or translation of a nucleic acid sequence within a host cell. The level of expression of a gene product of interest in a host cell can be determined based on the amount of corresponding RNA present in the cell or the amount of polypeptide encoded by the selected sequence. For example, RNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization with cellular RNA, or by PCR, such as qPCR. Protein encoded by a selected sequence can be quantified by a variety of methods, for example, by ELISA, by Western blot, by radioimmunoassay, by immunoprecipitation, by assaying the biological activity of the protein, by FACS analysis following immunostaining of the protein, or by homogeneous time-resolved fluorescence (HTRF) assay. The level of expression of a non-coding RNA, such as a miRNA or shRNA, can be quantified by PCR, such as qPCR.

[0047] The term "gene product" refers to an RNA polynucleotide and a polypeptide encoded by a gene or DNA polynucleotide.

[0048] As used herein, the term "proteinogenic amino acid" refers to all amino acids that are incorporated biosynthetically into a protein during the process of translation. The term "proteinogenic" refers to the production of a protein. In eukaryotes, there are 21 genes that encode amino acids (i.e., proteinogenic amino acids), 20 of which in the standard genetic code and selenocysteine. The 20 amino acids of the standard genetic code are: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0049] As used herein, the term "post-translational modification" or "post-translationally modified" refers to naturally occurring modifications of lysine residues in the CII peptide that can occur when produced in a cell. Post-translational modification of lysine residues can produce hydroxy lysine (Hyl) or O-glycosylated Hyl, such as galactosyl-hydroxy lysine or glucosylgalactosyl-hydroxy lysine, preferably galactosyl-hydroxy lysine.

[0050] As used herein, the term "domain" refers to a folded protein structure that has a tertiary structure independent of the rest of the protein. Generally, a domain is responsible for a discrete functional property of the protein, and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or domain. For example, the al domain of the MHC class II alpha chain and the b l domain of the MHC class II beta chain are each folded polypeptide domains that together form the peptide binding groove of the MHC class II molecule.

[0051] Method for producing recombinant MHC II / CII peptide complexes in mammalian cells

[0052] In one aspect, the present application provides a method of producing an MHC II / CII peptide complex comprising a post-translationally modified CII peptide, comprising: (a) transfecting a mammalian cell with: (i) a polynucleotide encoding an extracellular region of an MHC II a chain comprising at least one al domain; (ii) a polynucleotide encoding an extracellular region of an MHC II β chain comprising at least one β1 domain; and (iii) a polynucleotide encoding a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II a chain or the MHC class II β chain by a linker peptide, wherein the CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG, and AGFKXEQGPXG; (b) culturing the mammalian cell under conditions suitable for producing an MHC II / CII peptide complex; and (c) harvesting the cell supernatant and optionally the cell comprising the MHC II / CII peptide complex, the MHC II / CII peptide complex comprising a post-translationally modified CII peptide. Preferably, the CII peptide comprises a post-translational modification at a lysine residue, preferably at the first lysine residue of the CII peptide. In one embodiment, the first lysine residue of the CII peptide is hydroxylysine (Hyl) or O-glycosylated Hyl. Preferably, the first lysine residue is hydroxylysine or galactosyl-hydroxylysine, even more preferably galactosyl-hydroxylysine. As used herein, the term“first lysine residue” refers to K264 of CII peptide 261-273 (AGFK(264)GEQGPK(270)GEP; SEQ ID NO: 10) or 259-273 (GIAGFK(264)GEQGPK(270) of K264)GEP; SEQ ID NO: 13), corresponding to amino acid position 4 in SEQ ID NOs: 1-12 and amino acid position 6 in SEQ ID NOs: 13-15. The method can further comprise a step of analyzing the post-translational modification (such as the glycosylation profile) of the CII peptide of the MHC II / CII peptide complex. Methods for analyzing the glycosylation profile are well known in the art and include methods such as mass spectrometry. The method of the present application is an in vitro method. Furthermore, the method comprises the use of a mammalian cell line, rather than a primary cell.

[0053] In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG, AGFKGEQGPX1G, AGFKGEX2GPKG, AGFKGX3QGPKG, AGFKX4EQGPKG, AGFKGEX2GPX1G, AGFKGX3QGPX1G, and AGFKX4EQGPX1G, wherein X1 is any protein amino acid except K, preferably R, A, G, or Q, more preferably R; X2 is any protein amino acid except Q, preferably A, R, H, or G; X3 is any protein amino acid except E, preferably A, D, Q, or G; X4 is any protein amino acid except G, more preferably A, S, V, or L. Preferably, X2, X3, or X4 is not K, more preferably, X1, X2, X3, or X4 is not K. In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG or AGFKGEQGPX1G, preferably AGFKGEQGPKGEP or AGFKGEQGPX1GEP, more preferably GIAGFKGEQGPKGEP or GIAGFKGEQGPX1GEP.

[0054] As used herein, the term "MHC II / CII peptide complex" refers to a soluble complex comprising the extracellular domain or a portion thereof of a human MHC II protein forming a peptide binding groove and a collagen II peptide (CII peptide), wherein the peptide is fused (i.e., linked by a linker peptide) to the N-terminus of the alpha chain or the beta chain. Preferably, the CII peptide is fused to the N-terminus of the MHC class II beta chain. The MHC II protein comprises an alpha 1 domain and an alpha 2 domain forming the extracellular domain of the alpha chain, and a beta 1 domain and a beta 2 domain forming the extracellular domain of the beta chain. The terms "extracellular domain" and "extracellular region" are used synonymously herein. The alpha 1 domain and the beta 1 domain form the peptide binding groove, i.e., the site that interacts with and binds to a peptide, such as a CII peptide. Thus, the MHC II / CII peptide complex comprises at least the alpha 1 domain and the beta 1 domain of the MHC II protein. Preferably, the MHC II / CII peptide complex comprises the alpha 1 domain, the alpha 2 domain, the beta 1 domain and the beta 2 domain of the MHC II protein. MHC class II molecules (MHC II proteins) are a class of major histocompatibility complex (MHC) molecules, typically found only on professional antigen presenting cells (APCs) such as dendritic cells, mononuclear phagocytes (such as monocytes and macrophages), and B cells. MHC class II molecules present antigens from extracellular proteins, whereas MHC class I molecules present cytosolic or intracellular peptides. Extracellular proteins are endocytosed, digested and loaded onto MHC II proteins to form MHC II / peptide complexes. The loaded complex is then transferred to the cell surface, where it is presented to effector cells. In humans, MHC proteins are known as human leukocyte antigens (HLA). Thus, as used herein, MHC II proteins include human HLA proteins. The HLA corresponding to MHC class II proteins are HLA-DP, HLA-DM, HLA-DOA, HLA-COB, HLA-DQ and HLA-DR. For RA, there is a genetic association with certain alleles of the HLA-DRB1 locus that encode amino acids in the beta chain of the peptide binding pocket of MHC class II molecules HLA-DR that share the motif (Q / R R / K RAA) (amino acid positions 70-74), the so-called "shared epitope". Examples of RA-associated HLA DRB1 alleles are QKRAA-encoding alleles HLA_DRB1*0401 and 0409, QRRAA-encoding alleles: HLA_DRB1*0404, 0405, 0408, 0101, 0102 and 1402, RRRAA-encoding alleles: HLA_DRB1*1001 and DKRAA-encoding alleles: HLA_DRB1*1303.In one embodiment, the extracellular region of the MHC class II alpha chain and the extracellular region of the MHC class II beta chain are thus derived from HLA-DR, preferably the at least alpha 1 domain is from DRA*0101 and the at least beta 1 domain is from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402 and DRB1*1303, preferably DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001 and DRB1*1402, more preferably DRB1*0401, DRB1*0404 and DRB1*0405, even more preferably DRB1*0401. More preferably the alpha 1 domain and the alpha 2 domain are from DRA*0101 (alpha 1 domain and alpha 2 domain: amino acids 19-200 of SEQ ID NO: 16) and the beta 1 domain and the beta 2 domain are from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402 and DRB1*1303, preferably DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001 and DRB1*1402, more preferably DRB1*0401, DRB1*0404, DRB1*0101 and DRB1*040, even more preferably DRB1*0401 (beta 1 domain and beta 2 domain: amino acids 60-250 of SEQ ID NO: 17). In mice, collagen-induced arthritis (CIA) is associated with the mouse MHC class II A. q alleles (Aq) are associated.

[0055] The specific MHC II / CII peptide complexes used in the examples are abbreviated as follows: Aq / rCII (naturally glycosylated, rat CII), Aq / nCII (naked or unmodified, rat CII), Aq / galCII (galactosylated (Gal-Hyl at K264)), wherein the rat CII peptide used has the amino acid sequence GIAGFKGEQGPKGET (SEQ ID NO: 29) and DR4 / hCII (naturally glycosylated, human), DR4 / nCII (naked or unmodified, human), DR4 / galCII (galactosylated (Gal-Hyl at K264), human), wherein the CII peptide used has the human amino acid sequence GIAGFKGEQGPKGEP (SEQ ID NO: 13).

[0056] The MHC II / CII peptide complex produced according to the method of the present application comprises a post-translationally modified peptide. The CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG and AGFKXEQGPXG. In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG, AGFKGEQGPX1G, AGFKGEX2GPKG, AGFKGX3QGPKG, AGFKX4EQGPKG, AGFKGEX2GPX1G, AGFKGX3QGPX1G and AGFKX4EQGPX1G, wherein X1 is any protein amino acid except K, preferably R, A, G or Q, more preferably R; X2 is any protein amino acid except Q, preferably A, R, H or G; X3 is any protein amino acid except E, preferably A, D, Q or G; and X4 is any protein amino acid except G, more preferably A, S, V or L. Preferably, X2, X3 or X4 is not K, more preferably X1, X2, X3 or X4 is not K. In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG or AGFKGEQGPX1G, preferably AGFKGEQGPKGEP or AGFKGEQGPX1GEP, more preferably GIAGFKGEQGPKGEP or GIAGFKGEQGPX1GEP. Preferably, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG (SEQ ID NO: 1) or AGFKGEQGPXG (SEQ ID NO: 2), preferably AGFKGEQGPKGEP (SEQ ID NO: 10) or AGFKGEQGPXGEP (SEQ ID NO: 11), more preferably GIAGFKGEQGPKGEP (SEQ ID NO: 13) or GIAGFKGEQGPXGEP (SEQ ID NO: 14). The CII peptide GIAGFKGEQGPKGEP corresponds to amino acids 259-273 of the triple helix collagen type II (CII) region. The CII peptide suitable for binding into the binding pocket has a length of 10-20 amino acids, preferably the CII peptide has a length of 11-15 amino acids, more preferably the CII peptide has a length of 13-15 amino acids. In one embodiment, the CII peptide comprises the amino acid sequence AGFKGEQGPKG (SEQ ID NO: 1), more preferably AGFKGEQGPKGEP (SEQ ID NO: 10), and even more preferably GIAGFKGEQGPKGEP (SEQ ID NO: 13).In one embodiment, the second K (K270) can be mutated, preferably to R, A, G or Q, more preferably to R. Thus, also encompassed are embodiments wherein the CII peptide comprises the amino acid sequence AGFKGEQGPXG (SEQ ID NO: 2), AGFKGEQGPXGEP (SEQ ID NO: 11) and GIAGFKGEQGPXGEP (SEQ ID NO: 14), wherein X can be any protein amino acid except K, preferably X is R, A, G or Q, more preferably X is R. Thus, in one embodiment, the CII peptide comprises the amino acid sequence AGFKGEQGPRG (SEQ ID NO: 9), AGFKGEQGPRGEP (SEQ ID NO: 12) and GIAGFKGEQGPRGEP (SEQ ID NO: 15). The CII peptides encompassed by the present application are disclosed in Table 1.

[0057] Table 1

[0058]

[0059] wherein Xi is any protein amino acid except K, preferably R, A, G or Q, more preferably R; X2 is any protein amino acid except Q, preferably A, R, H or G; X3 is any protein amino acid except E, preferably A, D, Q or G; X4 is any protein amino acid except G, more preferably A, S, V or L.

[0060] The MHC II / CII peptide complex produced in the present application comprises a MHC II / CII peptide complex with a CII peptide comprising a post-translational modification. Preferably, the CII peptide comprises a post-translational modification at a lysine residue, preferably at the first lysine residue of the CII peptide. In one embodiment, the first lysine residue of the CII peptide is a hydroxylysine (Hyl) and / or is an O-glycosylated hydroxylysine. K The first lysine (K) residue in GIAGFKGEQGPXGEP is position 264 of the triple helix CII region amino acid sequence (corresponding to the 4th amino acid in SEQ ID NOs: 1-12 and the 6th amino acid in SEQ ID NOs: 13-15). Thus, as used herein, the“first lysine residue” can also be referred to as K264, or lysine at position 264. KThe second lysine (K) residue in the GEP is amino acid position 270 of the triple helical CII region (corresponding to amino acid position 10 in SEQ ID NO: 1, 3-5 or 10, and amino acid position 12 in SEQ ID NO: 13). Thus, as used herein, the“second lysine residue” or“additional lysine residue” can also be referred to as K270, or the lysine at position 270. In a preferred embodiment, the MHC II / CII peptide complex produced according to the present application comprises an MHC II / CII peptide complex, wherein at least the first lysine residue is hydroxylysine and / or galactosyl-hydroxylysine.

[0061] In the CII peptide sequence of the present application, collagen-specific post-translational galactosylation of lysine residues, in particular the first lysine residue, i.e. the lysine residue at position 264, can be involved in T cell recognition by the TCR, and this results in a pharmacological effect. The lysine residue at position 270 is located at the rim of the binding groove of the DR4 molecule, and its modification to galactosyl-hydroxylysine is considered to be less important for TCR recognition. Thus, the second or additional lysine residue (corresponding to K270) can be unmodified hydroxylysine or galactosyl-hydroxylysine. It has been shown that the TCR of a T cell hybridoma recognizing the gal264 epitope is not affected by the K270R mutation. In a preferred embodiment, the CII peptide comprises only the first lysine residue, and any other K is mutated, preferably to R, A, G or Q, more preferably to R. Thus, the present application also encompasses CII peptides AGFKGEQGP R G (SEQ ID NO: 9), preferably AGFKGEQGP R GEP (SEQ ID NO: 12), more preferably GIAGFKGEQGP R GEP (SEQ ID NO: 15). Mutation of the second lysine has the advantage of reducing product heterogeneity. Furthermore, galactosyl-hydroxylysine can be glucosylated to form glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl), which can have a negative impact on TCR recognition due to the bulky size of the disaccharide (Glc-Gal), in particular at position K270. Thus, mutation of K270, in particular K270R, further avoids interference with binding, as no disaccharide modification can be attached at this position.

[0062] Collagen II peptides (CII peptides) are fused to the N-terminus of an MHC class II α chain or an MHC class II β chain via a adaptor peptide, preferably to the N-terminal chain of an MHC class II β chain. The term "adaptor peptide" refers to a polypeptide consisting of multiple amino acid residues. The adaptor peptide can be any peptide, provided it is long enough and flexible enough to allow the peptide to bind to a peptide-binding pocket formed by the MHC II complex. A suitable example of an adaptor peptide is the Gly-Ser adaptor. According to the invention, at least one of the CII peptide, the peptide adaptor, and the extracellular regions of the MHC class II α chain and the MHC class II β chain is expressed as a polypeptide encoded by a polynucleotide. As used herein, the term "fused to" means "linked to," wherein an adaptor peptide is used to link the peptides via peptide bonds, thus generating a fusion protein. This feature structurally distinguishes the MHC II / CII peptide complex produced by the method of the present invention from prior art complexes. In the previous complex, the MHC II protein was produced using a CIIP peptide as a substitute peptide, which is linked to one of the MHC II chains via a linker peptide containing a peptidase cleavage site (such as a thrombin cleavage site). Therefore, after production, this peptide is enzymatically cleaved, and a synthetically prepared galactosylated peptide (i.e., a CII peptide carrying gal-Hyl at position K264) is loaded onto the complex in vitro. Although this synthetically prepared galactosylated peptide can be covalently linked to the MHC II protein, this linking does not occur via a linker peptide.

[0063] Although the MHC II / CII peptide complexes (SEQ ID NO:16 and SEQ ID NO:17) used in the examples and as... Figure 1 As shown, even in the absence of a signal peptide, an enzyme cleavage site (thrombin cleavage site) is still present between the linker and the CII peptide. Figure 1 This is not necessary and is preferably removed from therapeutic products. Connector peptides can improve product stability and prevent peptide loss. Therefore, preferably, the MHC II / CII peptide complex of the present invention does not contain an enzyme cleavage site in the amino acid sequence between the CII peptide and the extracellular region of the MHC II class β chain (or MHC II class α chain). Furthermore, for therapeutic purposes, the MHC II / CII peptide complex (or the polynucleotide-encoded MHC II / CII peptide complex) does not contain: (1) a streptavidin tag (SAWSHPQFEK, SEQ ID NO:30) for purification; (2) a cleavage site (e.g., TEV cleavage site) between the MHC IIα / MHC IIβ chain and the heterodimerization domain; and / or (3) a recognition site for E. coli biotin ligase (BirA) (e.g., an Avi tag), such as... Figure 1The exemplary complexes shown and used in the examples are present. These elements are shown to have no effect on the in vitro and in vivo function of the complex (data not shown). Preferably, the MHC II / CII complex contains a His-tag (polyhistidine-tag) or a functionally equivalent tag at the C-terminus of the polypeptide comprising the HLA-DRα chain and / or the HLA-DRβ chain. An exemplary minimal HLA-DR / CII peptide complex according to the invention may be encoded by the amino acid sequences of SEQ ID NO:18 and SEQ ID NO:19. Those skilled in the art will understand that the peptide sequence may be varied as covered in the claims.

[0064] The sequence of the exemplary complex used in the following examples is as follows:

[0065] 1) DR4-Constructor:

[0066] • DR4 construct α-chain (SEQ ID NO:16), sequence comprising: extracellular α-chain region of DRA*0101 ( underlined The preceding signal peptide, TEV cleavage site (bold), cFos domain ( bold and underlined ) and biotinylation site (BirA, italic and underlined underlined ).

[0067]

[0068]

[0069] • Minimal DR4 construct α-chain (SEQ ID NO:18), sequence comprising: extracellular α-chain region in DRA*0101 ( bold and underlined bold and underlined The preceding signal peptide and cFos domain ( italic and underlined ):

[0070]

[0071] • The DR4 construct β-chain (SEQ ID NO:17) containing the hCII259-273 peptide, the sequence of which includes: immediately following the Strep-tag ( bold and underlined The preceding signal peptide and CII peptide 259-273 ( bold and underlined ), thrombin cleavage sites framed by glycine linkers at each site ( italic and underlined ), DRB*0401 extracellular region ( underlined ), TEV cleavage site (bold), cJun domain ( bold and underlined ) and His- tag (italic).

[0072]

[0073] • DR4 construct β-chain with hCII 259-273 peptide (SEQ ID NO: 19), sequence comprising: signal peptide immediately followed by CII peptide 259-273 (DR4), DRB*0401 ectodomain (DR4), cJun domain (DR4), and His-tag (italic). bold and underlined italic and underlined underlined bold and underlined

[0074]

[0075] 2) DR4-hCLIPmut construct:

[0076] • DR4 construct α-chain as above (SEQ ID NO: 16)

[0077] • DR4 construct β-chain with hCLIPmut (SEQ ID NO: 20), sequence comprising: signal peptide immediately followed by Strep-tag (DR4), mutated hCLIP peptide (DR4), thrombin cleavage site framed by a glycine linker at each site (DR4), DRB*0401 ectodomain (DR4), TEV cleavage site (bold), cJun domain (DR4), and His-tag (italic). bold and underlined italic and underlined underlined bold and underlined underlined 3) Aq-rCII construct:

[0078]

[0079] • Aq construct α-chain (SEQ ID NO: 21), sequence comprising: signal peptide immediately followed by Aq ectodomain α-chain region (Aq), TEV cleavage site (bold), cFos domain (Aq), and biotinylation site (BirA,

[0080] bold and underlined italic and underlined bold and underlined italic and underlined

[0081]

[0082] • Aq construct β-chain with rat CII 259-273 peptide (SEQ ID NO: 22), sequence comprising: signal peptide immediately followed by Strep-tag (Aq), CII peptide 259-273 (Aq), thrombin cleavage site framed by a glycine linker at each site (Aq). bold and underlined italic and underlined bold and underlined ​​​​​​​​​​​​​), Aq extracellular region ( underlined ), TEV cleavage site (bold), cJun domain ( bold and underlined ) and His- tag (italic).

[0083]

[0084] • An Aq construct β-chain (SEQ ID NO:23) of the rat CII259-273 peptide without a His-tag, the sequence of which includes: immediately following the Strep-tag ( underlined The preceding signal peptide and CII peptide 259-273 ( bold and underlined italic and underlined ), thrombin cleavage sites framed by glycine linkers at each site ( bold and underlined ), Aq extracellular region ( italic and underlined bold and underlined ), TEV cleavage site (bold) and cJun domain ( underlined ):

[0085]

[0086] 4) Aq-mCLIPmt constructor:

[0087] The α-chain of the Aq construct is shown above (SEQ ID NO:21).

[0088] • An Aq construct β-chain (SEQ ID NO:24) containing the mCLIP peptide, the sequence of which includes: immediately following the Strep-tag ( bold and underlined The preceding signal peptide and mouse mCLIPmt peptide ( bold and underlined ), thrombin cleavage sites framed by glycine linkers at each site ( italic and underlined ), Aq extracellular region ( bold and underlined ), TEV cleavage site (bold), cJun domain ( italic and underlined ) and His- tag (italic).

[0089]

[0090] • An Aq construct β-chain (SEQ ID NO:25) containing an mCLIP peptide without a His-tag, the sequence of which includes: immediately following a Strep-tag ( bold and underlined The preceding signal peptide and mouse mCLIPmt peptide ( underlined ), thrombin cleavage sites framed by glycine linkers at each site ( bold and underlined ), Aq extracellular region ( bold and underlined ), TEV cleavage site (bold), cJun domain ( italic and underlined ):

[0091]

[0092] Additional amino acid sequences for various elements of the constructs disclosed herein are provided below:

[0093] • cFos domain (SEQ ID NO: 26):

[0094] LTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAH

[0095] • cJun domain (SEQ ID NO: 27):

[0096] RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNH

[0097] • Modified human CLIP peptide (SEQ ID NO: 28): PVSKARMATGALAQA

[0098] • Rat CII-peptide 259-273 (SEQ ID NO: 29): GIAGFKGEQGPKGET

[0099] • Streptavidin tag (SEQ ID NO: 30): SAWSHPQFEK.

[0100] Preferably, the MHC II / CII peptide complex obtained by the method of the present application contains at least one His-tag or functionally equivalent tag at the C-terminus of the polypeptide comprising the HLA-DR alpha chain and / or the HLA-DR beta chain. The His-tag is preferably at least a hexa-histidine tag, more preferably at least a penta-histidine tag. The functionally equivalent tag is for example a chondroitin binding peptide. Thus, the composition can comprise a MHC II / CII peptide complex comprising a chondroitin binding peptide, preferably a chondroitin- and hyaluronan (also known as hyaluronate) binding peptide. In one embodiment, the MHC II / CII peptide complex comprises at least one C-terminal chondroitin binding peptide. Chondroitin binding peptides are known in the art and include, but are not limited to, peptides having the following amino acid sequences: EKRIWFPYRRF (SEQ ID NO: 31), YKTNFRRYYRF (SEQ ID NO: 32), or VLIRHFRKRYY (SEQ ID NO: 33) (Butterfield KC et al., Biochemistry. 2010 Feb 23; 49(7): 1549-55). In one embodiment, the chondroitin binding peptide comprises 5-20 amino acids, preferably 6-20 amino acids, more preferably 6-20 amino acids. To increase binding to hyaluronan, the corresponding sequence comprising the binding consensus motif is defined as follows: B(X7)B, wherein B is R or K, X7 does not comprise an acidic residue and at least one basic amino acid (Yang B et al., EMBO J. 1994 Jan 15; 13(2): 286-96). As disclosed herein, the MHC II / CII peptide complex can also bind chondroitin sulfate via the His-tag. Thus, the chondroitin binding peptide can be a poly-histidine tag, preferably a hexa-histidine tag, or any other amino acid sequence increasing the binding affinity to chondroitin, such as EKRIWFPYRRF (SEQ ID NO: 31), YKTNFRRYYRF (SEQ ID NO: 32), or VLIRHFRKRYY (SEQ ID NO: 33). Both chondroitin and hyaluronan are important components of cartilage.

[0101] The method of the present application comprises transfecting a mammalian cell with (i) a polynucleotide encoding the extracellular region of an MHC II alpha chain comprising at least one alpha 1 domain, (ii) a polynucleotide encoding the extracellular region of an MHC II beta chain comprising at least one beta 1 domain, wherein the CII peptide is further fused to the N-terminus of the MHC II alpha chain or the MHC II beta chain, preferably the MHC II beta chain. As used herein, transfection refers to the introduction of DNA into a mammalian cell using transfection methods known in the art. As used herein, the term "transfection" includes "transduction", which is generally used to describe viral-mediated gene transfer into eukaryotic cells. The polynucleotide can be DNA or RNA, preferably DNA. The transfection can be transient transfection or stable transfection. Preferably, the polynucleotide is present in a vector, preferably an expression vector.

[0102] Methods for stable integration are well known in the art. Briefly, stable integration is generally achieved by transiently introducing into a mammalian host cell at least one recombinant polynucleotide or a vector containing the at least one recombinant polynucleotide, which facilitates stable integration of the recombinant polynucleotide into the mammalian cell genome. Typically, the recombinant polynucleotide is flanked by homology arms, i.e. sequences homologous to the regions upstream and downstream of the integration site. The vector introducing the recombinant polynucleotide into the mammalian cell can be selected from a variety of suitable vector systems, such as plasmids, retroviruses, cosmids, EBV-derived episomes, etc. A variety of shuttle vectors can be used, for example vectors that can replicate autonomously in a variety of host microorganisms, such as E. coli and Pseudomonas sp. Before introducing them into the mammalian host cell, circular vectors can be linearized to facilitate integration into the mammalian cell genome. Methods for introducing vectors into mammalian cells are well known in the art, including transfection with biological methods, such as viral delivery; with chemical methods, such as using cationic polymers, calcium phosphate, cationic lipids or cationic amino acids; with physical methods, such as electroporation or microinjection.

[0103] In one embodiment, the recombinant polynucleotide stably integrated into the genome of a mammalian cell is part of an expression cassette. The expression cassette comprises at least one heterologous polynucleotide encoding a gene product, such as an RNA and / or a protein, operably linked to a promoter and optionally further means controlling the expression of the gene product. Such means include, but are not limited to: enhancers, termination signals, polyadenylation signals and 3' untranslated regions, typically comprising a polyadenylation site. The promoter can be a weak promoter or a strong promoter supporting high levels of expression of the gene product of interest. The promoter includes, but is not limited to: a CMV (cytomegalovirus) promoter, an SV40 (simian vacuolating virus 40) promoter, an RSV (Rous Sarcoma Virus) promoter, an adenovirus promoter (e.g., an adenovirus major late promoter (AdMLP), a CHEF-1 (CHO-derived elongation factor-1) promoter, a polyoma and strong mammalian promoters such as natural immunoglobulin and actin promoters or a natural promoter of the at least one heterologous polynucleotide. Preferably, the promoter is a CMV promoter or an SV40 promoter, most preferably a CMV promoter. Examples of polyadenylation signals are: BGH polyA, SV40 late or early polyA; or, the 3'UTR of an immunoglobulin gene or the like can be used. The skilled person will further understand that the 3' untranslated region can be engineered to support high levels of expression, e.g., by removing instability elements, such as AREs (adenosine-uridine acid-rich elements).

[0104] In one embodiment, the gene product can be placed under the control of an amplifiable genetic selection marker, such as dihydrofolate reductase (DHFR), glutamine synthetase (GS). The amplifiable selection marker gene can be located on the same expression vector as the secreted therapeutic protein expression cassette. Alternatively, the amplifiable selection marker gene and the secreted therapeutic protein expression cassette can be located on different expression vectors but are stably integrated into the genome of the host cell. For example, two or more vectors that are co-transfected simultaneously are typically stably integrated into the genome of the host cell. Amplification of the genetic region comprising the secreted therapeutic protein expression cassette is then mediated by the addition of an amplification agent (e.g., MTX for DHFR, or MSX for GS) to the culture medium.

[0105] For example, a sufficiently high stable level of the gene product in the host cell or production cell can be achieved by cloning multiple copies of the heterologous polynucleotide into the expression vector. As described above, cloning multiple copies of the recombinant polynucleotide into the expression vector, and amplifying the secreted therapeutic protein expression cassette (encoding the MHC II / CII peptide complex) can be further combined.

[0106] In one embodiment, the polynucleotide encoding the extracellular region of the MHC class II alpha chain comprising at least one alpha 1 domain is present in one vector (first polynucleotide), while the polynucleotide encoding the extracellular region of the MHC class II beta chain comprising at least one beta 1 domain (second polynucleotide) can be present in another vector, wherein the CII peptide is further encoded by the first or second polynucleotide to provide the CII peptide fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain. In an alternative embodiment, the first and second polynucleotides can be part of different expression cassettes on the same vector. In yet another alternative embodiment, the first and second polynucleotides form a single polynucleotide encoding a single fusion polypeptide comprising: the extracellular region of the MHC class II alpha chain comprising at least one alpha 1 domain; the extracellular region of the MHC class II beta chain comprising at least one beta 1 domain; and the collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the MHC class II beta chain, via a linker peptide.

[0107] In one embodiment, the extracellular region of the MHC class II alpha chain comprising at least one alpha 1 domain, the extracellular region of the MHC class II beta chain comprising at least one beta 1 domain, and the collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the MHC class II beta chain, via a linker peptide; are encoded by a single polynucleotide to express a single fusion polypeptide (single chain heterodimer).

[0108] In an alternative embodiment, the method comprises a first polynucleotide encoding the extracellular region of the MHC class II alpha chain comprising at least one alpha 1 domain; a second polynucleotide encoding the extracellular region of the MHC class II beta chain comprising at least one beta 1 domain; and a polynucleotide encoding the collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the MHC class II beta chain, via a linker peptide. In one embodiment, the MHC class II alpha chain is fused at its C-terminus (C-terminally) to a first functional domain of a leucine zipper heterodimerization motif, and the MHC class II beta chain is fused at its C-terminus to a second complementary functional domain of a leucine zipper heterodimerization motif. The first functional domain and the second complementary functional domain can be acidic and basic leucine zipper heterodimerization domains, preferably a jun-fos leucine zipper motif. In one embodiment, the first and / or second polynucleotide encodes a polyhistidine tag C-terminally of the functional domain of the leucine zipper heterodimerization motif.

[0109] According to the method of the present application, a mammalian cell is cultured under conditions suitable to produce MHC II / CII peptide complexes, the cell supernatant and / or the cell is collected, wherein the cell supernatant and / or the cell contains MHC II / CII peptide complexes comprising a CII peptide with a post-translational modification, wherein preferably the first lysine residue of the CII peptide is hydroxy lysine (Hyl) or O-glycosylated hydroxy lysine, more preferably the first lysine residue is hydroxy lysine or galactosyl-hydroxy lysine, more preferably galactosyl-hydroxy lysine. The method can further comprise a step of analyzing the post-translational modification of the CII peptide of the MHC II / CII peptide complex, such as the glycosylation profile. Methods for analyzing post-translational modifications and glycosylation profiles are well known in the art and include methods such as mass spectrometry.

[0110] In principle, any mammalian cell suitable for high yield protein production can be used in the present application, as long as it comprises enzymes for post-translational modification of lysine residues in collagen, including hydroxylation of lysine to hydroxy lysine (Hyl), and galactosylation of Hyl to galactosyl hydroxy lysine (Gal-Hyl). The term "galactosylation" as used herein in the context of lysine includes lysine that has been hydroxylated to hydroxy lysine prior to galactosylation. The enzymes can be present endogenously in the cell, or can be recombinantly expressed in the cell. Preferably, the mammalian cell comprises lysyl hydroxylase (EC 1.14.11.4) and collagen galactosyltransferase (EC 2.4.1.50), preferably lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2) and collagen galactosyltransferase GLT25D1 and / or GLT2D2, preferably GLT25D1. These enzymes post-translationally modify collagen. Thus, these enzymes can be present in collagen producing cell lines, such as kidney cells, fibroblasts or osteoclasts, in particular kidney cells such as HEK293 cells or derivatives thereof. HEK293 cells can be grown as adherent cells or in suspension. One example of a HEK293 cell suitable for the method of the present application is a HEK293 cell or a HEK293F cell, such as an Expi293F cell (Gibco, cat. no. A14527, also available as cGMP stock cat. no. 100044202). Other suitable HEK293 cells include HEK293T cells and / or suspension cells thereof. Surprisingly, the small peptides presented by MHC II proteins can also be post-translationally modified in these cells. Although the peptides originate from collagen, they are present in a completely different (non-native) environment in the MHC II complex. In this respect, we note that native MHC II proteins are loaded with peptides from extracellular (post-translationally modified) proteins that are digested in APCs. Thus, the modification is already present on the endocytosed protein and is not added intracellularly. Furthermore, surprisingly, the heterogeneous products resulting from in situ glycosylation are suitable for therapy. Suitable cells can be readily screened for the enzymes required for post-translational modification of lysine residues in collagen, such as by Western blot using suitable antibodies or by RNA expression screening, or by functional screening for their ability to glycosylate type II collagen or MHC II / CII peptide complexes. Methods for detecting gene or protein expression or enzyme activity and glycosylation properties are well known in the art. In one embodiment, the mammalian cell is a kidney cell, a fibroblast or an osteoblast, preferably a HEK293 cell or cell line. HEK293 cells have been previously described to express lysyl hydroxylases PLOD1 and PLOD2 (encoding LH1 and LH2, respectively), galactosyltransferases GLT25D1 and GLT25D2, and additionally PLOD3 (encoding LH3).

[0111] CHO cells, which are commonly used for protein production, have been tested and are not able to add post-translationally modifications at lysine residues sufficiently to obtain galactosyl-hydroxylysine (Gal-Hyl) in the collagen proteins or MHC II / CII peptide complexes described herein. In one embodiment, the mammalian cell is a genetically engineered cell that recombinantly expresses the genes for lysyl hydroxylase and collagen galactosyltransferase. Preferably, the mammalian cell is genetically engineered to recombinantly express lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2) and collagen galactosyltransferase GLT25D1 and / or GLT25D2, preferably GLT25D1. GLT25D2 is only expressed in a few cell types and is therefore less likely to be responsible for normal collagen modification. Any mammalian cell can be genetically engineered to recombinantly express lysyl hydroxylase and collagen galactosyltransferase, preferably lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2) and collagen galactosyltransferase GLT25D1 and / or GLT25D2. Preferably, the genetically engineered mammalian cell is a CHO cell, more preferably a CHO-DG44 cell, a CHO-K1 cell, a CHO-DXB11 cell, a CHO-S cell, a CHO glutamine synthetase (GS) deficient cell, or a derivative of any of these cells.

[0112] The skilled person will understand that the MHC II / CII peptide complexes produced by the method of the application are a heterogeneous mixture of MHC II / CII peptide complexes comprising different post-translational modifications of the CII peptide, in particular at the first and optionally the second lysine residue of the CII peptide. The heterogeneous mixture comprises MHC II / CII peptide complexes comprising K, Hyl, G-Hyl or GG-Hyl at the first lysine and independently K, Hyl, G-Hyl or GG-Hyl at the optional second lysine (where K = lysine, Hyl = hydroxylysine, G-Hyl = galactosyl-hydroxylysine, GG-Hyl = glucosyl-galactosyl-hydroxylysine).

[0113] Thus, in one embodiment, the harvested cell supernatant and optionally the harvested cells also contain MHC II / CII peptide complexes comprising a CII peptide, wherein the first lysine residue of the CII peptide is unmodified or glucosylgalactosyl-hydroxylysine (GG-Hyl), preferably unmodified, and the optional second lysine residue of the CII peptide is independently unmodified, hydroxylysine (Hyl), galactosyl-hydroxylysine (G-Hyl), or glucosylgalactosyl-hydroxylysine (GG-Hyl), preferably unmodified hydroxylysine (Hyl), galactosyl-hydroxylysine (G-Hyl). In one embodiment, the harvested cell supernatant and optionally the harvested cells do not comprise MHC II / CII peptide complexes wherein the second lysine residue is glucosylgalactosyl-hydroxylysine. In another embodiment, the harvested cell supernatant and optionally the harvested cells do not comprise MHC II / CII peptide complexes modified with glucosylgalactosyl-hydroxylysine (GG-Hyl), i.e. MHC II / CII peptide complexes comprising O-glycosylated CII peptides, wherein the first and / or the optional second lysine residue is glucosylgalactosyl-hydroxylysine.

[0114] Preferably, the heterogeneous mixture of MHC II / CII peptide complexes comprises at least 5%, at least 10%, at least 20%, or at least 30% G-Hyl at the first lysine (K264) of the CII peptide of the total MHC II / CII peptide complexes in the mixture or composition. Furthermore, the heterogeneous mixture of MHC II / CII peptide complexes comprises preferably less than 50%, less than 40%, or less than 30% unmodified CII peptides of the total MHC II / CII peptide complexes in the mixture or composition. In some embodiments, the heterogeneous mixture of MHC II / CII peptide complexes comprises less than 20%, less than 10%, less than 5%, and more preferably less than 1% GG-Hyl in the CII peptide of the total MHC II / CII peptide complexes in the mixture or composition. Where percentages refer to the percentage of CII peptides in the MHC II / CII peptide complexes out of the total CII peptides in the MHC II / CII peptide complexes. In a particular preferred embodiment, the second lysine residue (K270) is mutated, e.g. to arginine (K270R). In yet another embodiment, the (optional) second lysine is not post-translationally modified to glucosylgalactosyl-hydroxylysine (GG-Hyl) and is present in the form of unmodified lysine, hydroxylysine, or galactosyl-hydroxylysine.

[0115] It is further advantageous if the mammalian cell lacks galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66) activity in order to reduce the heterogeneity of the MHC II / CII peptide complex mixture and the amount of galactosylhydroxylysine formation. In one embodiment, the mammalian cell thus lacks galactosylhydroxylysyl glucosyltransferase activity. Preferably, the mammalian cell lacks lysyl hydroxylase 3 (LH3). LH3 is a multifunctional enzyme comprising lysyl hydroxylase (LH), galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activity, wherein the main function of the enzyme appears to be GGT activity. LH3 activity can be deleted or reduced using knock-down or knock-out methods. For example, RNA interference such as siRNA or shRNA can be used to reduce enzyme expression.

[0116] The term "RNA interference" (RNAi) refers to sequence-specific or gene-specific inhibition of gene expression (protein synthesis) and not to a general inhibition of protein synthesis. RNAi can involve degradation of messenger RNA (mRNA) by an RNA-induced silencing complex (RISC), preventing translation of the transcribed mRNA. The inhibition of gene expression by RNAi can be transient, but can also be more stable, even permanent. RNAi can be mediated by miRNA, siRNA or shRNA. Preferably, the RNAi according to the application is gene-specific (targets only one gene). Gene-specific RNAi can be mediated by siRNA or shRNA.

[0117] As used herein, the term "small interfering" or "short interfering RNA" or "siRNA" refers to a nucleotide RNA duplex that targets a desired gene and is capable of inhibiting the expression of genes that share homology therewith. It is formed from long double-stranded RNA (dsRNA) or shRNA. The RNA duplex typically comprises two complementary single-stranded RNAs of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides that form 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 base pairs and have a 3' overhang of two nucleotides, preferably the RNA duplex comprises two complementary single-stranded RNAs of 19-27 nucleotides that form 17-25 base pairs and have a 3' overhang of two nucleotides. The siRNA is "targeted" to a gene, wherein the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the mRNA of the targeted gene. The siRNA or its precursor is always introduced into the cell exogenously, e.g. directly or by transfection of a vector having a sequence encoding the siRNA, and the endogenous miRNA pathway is utilized to process the siRNA correctly and to cleave or degrade the target mRNA. The duplex RNA can be expressed in the cell from a single construct.

[0118] As used herein, the term "shRNA" (small hairpin RNA) refers to an RNA duplex in which a portion of the siRNA is part of a hairpin structure (shRNA). The shRNA can be processed within the cell to a functional siRNA. In addition to the duplex portion, the hairpin structure can comprise a loop portion between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the length of the loop is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides. The hairpin structure can also comprise a 3' or 5' overhang portion. In some aspects, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length. In one aspect of the application, the nucleotide sequence comprised in the vector is used as a template for the expression of a small hairpin RNA, including a sense region, a loop region, and an antisense region. Upon expression, the sense and antisense regions form a duplex. The shRNA is always introduced exogenously, for example by transfecting a vector with a sequence encoding the shRNA, and the endogenous miRNA pathway is utilized to properly process the siRNA and cleave or degrade the target mRNA. The use of a vector with a sequence encoding a shRNA has the advantage that the inhibition of the target gene is generally long-term and stable compared to the use of chemically synthesized siRNAs.

[0119] Generally, siRNAs and shRNAs mediate mRNA inhibition by perfect sequence complementarity (i.e. perfect base pairing between the antisense strand of the RNA duplex of the small interfering RNA and the target mRNA), and thus are specific for their target. The antisense strand of the RNA duplex can also be referred to as the active strand of the RNA duplex. As used herein, perfect base pairing of the complete sequence complementarity means that the antisense strand of the RNA duplex of the small interfering RNA has at least 89% sequence identity to at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, and preferably at least 19 consecutive nucleotides of the target mRNA; or preferably at least 93% sequence identity to at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, and preferably at least 19 consecutive nucleotides of the target mRNA. More preferably, the antisense strand of the RNA duplex of the small interfering RNA has 100% sequence identity to at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, and preferably at least 19 consecutive nucleotides of the target mRNA.

[0120] Alternatively, the enzyme or gene can be mutated or deleted. Thus, in an alternative embodiment, the mammalian cell lacks galactosylhydroxylysyl glucosyltransferase activity. In certain embodiments, the mammalian cell lacks the multifunctional enzyme LH3. For example, the gene can be silenced or not expressed sufficiently. In other certain embodiments, the mammalian cell comprises a mutant LH3 enzyme that lacks galactosylhydroxylysyl glucosyltransferase activity.

[0121] In yet another embodiment, the mammalian cell is genetically engineered to have reduced or no galactosylhydroxylysyl glucosyltransferase activity. The PLOD3 gene encoding LH3 can be mutated or deleted; and / or the LH3 enzyme can be a mutant LH3 enzyme that lacks galactosylhydroxylysyl glucosyltransferase activity. Methods of deleting or mutating genes are well known in the art and can include the use of sequence-specific DNA editing enzymes. As used herein, a “sequence-specific DNA editing enzyme” or “site-specific nuclease” is a protein that is capable of cleaving DNA at a defined nucleotide sequence (recognition site). The cleavage can occur in one or both of the complementary DNA strands, thus allowing, for example, targeted mutagenesis, targeted deletion of specific genomic DNA sequences, or site-directed recombination of the cleaved target DNA with a heterologous polynucleotide. The sequence specificity of the editing enzyme can result from one or more sequence-specific DNA binding protein domains within the editing enzyme, or from the enzyme binding a guide polynucleotide (e.g., a guide RNA) that directs it to a DNA sequence having at least partial complementarity to the guide polynucleotide. Thus, the recognition site of the editing enzyme can be altered by engineering the DNA binding protein domains or using alternative guide polynucleotides. A variety of sequence-specific DNA editing enzymes are known in the art, non-limiting examples of which are zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), and CRISPR-associated nucleases.

[0122] Preferably, the genetically engineered mammalian cell lacking galactosylhydroxylysyl glucosyltransferase activity is a HEK293 cell or cell line. An example of a cell line that can benefit from reduced galactosylhydroxylysyl glucosyltransferase to produce the MHCII / CII complex of the application is Expi293F cells (Gibco, Cat. No. A14527, also available as cGMP bank Cat. No. 100044202).

[0123] Galactosylhydroxylysyl glucosyltransferase activity can also be inhibited using carminic acid. Thus, the method of the application can comprise culturing the mammalian cell according to step (b) in carminic acid.

[0124] Mammalian cells are preferably established, adapted and fully cultivated under serum-free conditions and optionally in a medium free of any animal-derived proteins / peptides. Commercially available media, such as PreproGow TM HEK293 medium (PREPROTECH, USA), Expi293 TM Expression medium (ThermoFisher, USA)), HAM's F12 (Sigma, Deisenhofen, Germany), RPPMI (Sigma), HAM's F12 (Sigma, Deisenhofen, Germany), RPPMI-1640 (Sigma), Dulbecco's Modified Eagle Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO medium (Sigma) and protein-free CHO medium (Sigma) are exemplary suitable nutrient solutions. Any of the media can be supplemented with various compounds as needed, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (such as insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics and trace elements. Any other necessary supplements can also be included at suitable concentrations known to those skilled in the art. Suitable selection agents are added to the media for the growth and selection of genetically modified cells expressing selectable genes.

[0125] bold and underlined

[0126] In another aspect, the present application provides a composition comprising a recombinant MHC II / CII peptide complex, said complex comprising: (a) an extracellular region of an MHC class II alpha chain comprising at least an al domain; (b) an extracellular region of an MHC class II beta chain comprising at least a b1 domain; and (c) a collagen II peptide (CII peptide) fused via a linker peptide to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the MHC class II beta chain; wherein the CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG and AGFKXEQGPXG, wherein the MHC II / CII peptide complex comprises a post-translationally modified CII peptide. Preferably, the CII peptide comprises a post-translational modification at a lysine residue, preferably at the first lysine residue of the CII peptide. In one embodiment, the first lysine residue of the CII peptide is a hydroxylysine (Hyl) and / or an O-glycosylated Hyl. Preferably, the first lysine residue is a hydroxylysine (Hyl) and / or a galactosyl-hydroxylysine, more preferably a galactosyl-hydroxylysine.

[0127] The term "MHC II / CII peptide complex" refers to a soluble complex comprising the extracellular domain or a portion thereof of an MHC II protein forming a peptide binding groove and a collagen II peptide (CII peptide), wherein the peptide is fused, i.e. linked, to the N-terminus of the alpha or beta chain. Preferably, the CII peptide is fused to the N-terminus of the MHC class II beta chain. The MHC II protein comprises an al domain and an a2 domain forming the extracellular domain of the alpha chain, and a b1 domain and a b2 domain forming the extracellular domain of the beta chain. The al domain and the b1 domain form the peptide binding groove, i.e. the site that interacts with and binds the peptide, such as the CII peptide. Thus, the MHC II / CII peptide complex comprises at least the al domain and the b1 domain of the MHC II protein. Preferably, the MHC II / CII peptide complex comprises the al domain, the a2 domain, the b1 domain and the b2 domain of the MHC II protein.

[0128] For RA in humans, there is a genetic association with certain alleles of the HLA-DRB1 locus that share a motif (Q / R R / K RAA) of amino acids on the beta chain of the peptide binding pocket of the MHC class II molecule HLA-DR (amino acid positions 70-74, the so-called "shared epitope"). Examples of RA-associated HLA DRB1 alleles are QKRAA-encoding alleles HLA_DRB1*0401 and 0409, QRRAA-encoding alleles: HLA_DRB1*0404, 0405, 0408, 0101, 0102 and 1402, RRRAA-encoding alleles: HLA_DRB1*1001 and DKRAA-encoding alleles: HLA_DRB1*1303. The extracellular region of the MHC class II alpha chain and the extracellular region of the MHC class II beta chain are thus both derived from HLA-DR, preferably at least the al domain is from DRA*0101 and at least the pi domain is from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402 and DRB1*1303, preferably DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001 and DRB1*1402, more preferably DRB1*0401, DRB1*0404 and DRB1*0405. More preferably the al domain and the a2 domain are from DRA*0101 and the pi domain and the p2 domain are from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402 and DRB1*1303, preferably DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001 and DRB1*1402, more preferably DRB1*0401, DRB1*0404, DRB1*1001 and DRB1*0405. In mice, collagen-induced arthritis (CIA) is associated with mouse MHC class II A q alleles. q

[0129] ​The composition comprising the MHC I / CII peptide complex according to the application comprises a post-translationally modified peptide. The CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG and AGFKXEQGPXG. In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG, AGFKGEQGPX1G, AGFKGEX2GPKG, AGFKGX3QGPKG, AGFKX4EQGPKG, AGFKGEX2GPX1G, AGFKGX3QGPX1G and AGFKX4EQGPX1G, wherein X1 is any protein amino acid except K, preferably R, A, G or Q, more preferably R; X2 is any protein amino acid except Q, preferably A, R, H or G; X3 is any protein amino acid except E, preferably A, D, Q or G; and X4 is any protein amino acid except G, more preferably A, S, V or L. Preferably, X2, X3 or X4 is not K, more preferably X1, X2, X3 or X4 is not K. In certain embodiments, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG or AGFKGEQGPX1G, preferably AGFKGEQGPKGEP or AGFKGEQGPX1GEP, more preferably GIAGFKGEQGPKGEP or GIAGFKGEQGPX1GEP. Preferably, the CII peptide comprises an amino acid sequence of AGFKGEQGPKG (SEQ ID NO: 1) or AGFKGEQGPXG (SEQ ID NO: 2), preferably AGFKGEQGPKGEP (SEQ ID NO: 10) or AGFKGEQGPXGEP (SEQ ID NO: 11), more preferably GIAGFKGEQGPKGEP (SEQ ID NO: 13) or GIAGFKGEQGPXGEP (SEQ ID NO: 14). The CII peptide GIAGFKGEQGPKGEP corresponds to amino acids 259-273 of the triple helix CII region. The CII peptide suitable for binding into the binding pocket of MHC II has a length of 10-20 amino acids, preferably the CII peptide has a length of 11-15 amino acids, more preferably the CII peptide has a length of 13-15 amino acids. In one embodiment, the CII peptide comprises the amino acid sequence AGFKGEQGPKG (SEQ ID NO: 1), more preferably AGFKGEQGPKGEP (SEQ ID NO: 10), and even more preferably GIAGFKGEQGPKGEP (SEQ ID NO: 13). In one embodiment, the second K (K270) can be mutated, preferably to R.Thus, also encompassed are embodiments wherein the CII peptide comprises the amino acid sequence AGFKGEQGPXG (SEQ ID NO: 2), AGFKGEQGPXGEP (SEQ ID NO: 11) and GIAGFKGEQGPXGEP (SEQ ID NO: 14), wherein X can be any proteinogenic amino acid except K, preferably X is R, A, G or Q, more preferably X is R. In one embodiment, the CII peptide comprises the amino acid sequence AGFKGEQGPRG (SEQ ID NO: 9), AGFKGEQGPRGEP (SEQ ID NO: 12) and GIAGFKGEQGPRGEP (SEQ ID NO: 15).

[0130] A composition comprising the MHC II / CII peptide complex of the present application, comprising the MHC II / CII peptide complex with a CII peptide; wherein preferably at least the first lysine residue of the CII peptide is hydroxylysine (Hyl) and / or O-glycosylated hydroxylysine. The first lysine (K) residue of the CII peptide corresponds to the amino acid position 264 of the amino acid sequence of the triple helical CII region in GIAGFKGEQGP K KGEP (SEQ ID NO: 13). The optional second lysine (K) residue in the CII peptide corresponds to the second K in GIAGFKGEQGP K GEP, amino acid position 270 of the triple helical CII region. In a preferred embodiment, the MHC II / CII peptide complex according to the present application comprises a CII peptide which is post-translationally modified, wherein at least the first lysine residue is hydroxylysine and / or galactosyl-hydroxylysine. The term "galactosyl-hydroxylysine" can also be referred to as G-Hyl or Gal-Hyl and does not include modifications to glucosyl-galactosyl-hydroxylysine.

[0131] In the CII peptide sequence of the present application, the collagen-specific post- translational galactosylation of the lysine residue, in particular the first lysine residue, i.e. the lysine residue at position 264, can be involved in the T cell recognition by the TCR and thereby the pharmacological effect. The lysine residue at position 270 is located at the rim of the binding groove of the DR4 molecule and its galactosyl-hydroxylysine modification is considered to be less important for the TCR recognition. Thus, the second or further lysine residue (corresponding to K270) can be either of the unmodified hydroxylysine or galactosyl-hydroxylysine, preferably unmodified. It has been shown that the TCR of the T cell hybridoma recognizing the gal264 epitope is not affected by the K270R mutation. In a preferred embodiment, the CII peptide comprises only the first lysine residue and any further K, such as the optional second K, is mutated, preferably to R. Thus, the present application also encompasses a CII peptide comprising the amino terminal sequence AGFKGEQGP RG (SEQ ID NO: 9), preferably AGFKGEQGP R GEP (SEQ ID NO: 12), more preferably GIAGFKGEQGP R CII peptide of GEP (SEQ ID NO: 15). The mutation of the second lysine has the advantage of reducing product heterogeneity, so that the percentage of correctly modified peptides is higher. Furthermore, the galactosyl-hydroxylysine can be glucosylated to form glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl or GG-Hyl), which can have a negative impact on TCR recognition due to the bulky size of the disaccharide (Glc-Gal), especially at position K270. Therefore, the K270 mutation, in particular K270R, further avoids interference with binding, since no disaccharide modification can be attached at this position.

[0132] The collagen II peptide (CII peptide) is fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the N-terminal chain of the MHC class II beta chain, via a linker peptide. The term "linker peptide" refers to a polypeptide consisting of a plurality of amino acid residues. The linker peptide can be any peptide, as long as it is long enough and flexible enough to allow the peptide to bind to the peptide binding pocket formed by the MHC II complex. An example of a suitable linker is a Gly-Ser linker. According to the present application, the CII peptide, the linker peptide, and at least one of the extracellular regions of the MHC class II alpha chain and the MHC class II beta chain are expressed as one polypeptide and are encoded by one polynucleotide. The term "fused to" as used herein means "linked to" with optional use of a linker peptide by a peptide bond and thus generation of a fusion protein. This feature structurally distinguishes the MHC II / CII peptide complex according to the present application from the prior art complexes. In the prior complexes, the MHC II protein was produced with a CII peptide as surrogate peptide, which was linked to one of the MHC II chains via a linker peptide comprising a peptidase cleavage site, such as a thrombin cleavage site. Thus, after production, this peptide was enzymatically cleaved off and a synthetically prepared galactosylated peptide (i.e. a CII peptide carrying gal-Hyl at position K264) was loaded onto the complex in vitro. Although this synthetic galactosylated peptide can be covalently linked to the MHC II molecule, this linkage is not via a linker peptide. Although the MHC II / CII peptide complex used in the examples still comprises an enzymatic cleavage site (thrombin cleavage site, GRRS, SEQ ID NO: 10) between the linker and the CII peptide, the CII peptide is not cleaved off after production, but is part of the complex. Thus, the CII peptide is not a surrogate peptide, but is part of the complex according to the present application. italic and underlined), which is not essential and is preferably removed from the therapeutic product. The linker peptide can improve the stability of the product and prevent loss of the peptide. Thus, preferably, the MHC II / CII peptide complex of the application does not comprise an enzymatic cleavage site in the amino acid sequence between the CII peptide and the extracellular region of the MHC class II beta chain (or MHC class II alpha chain). Furthermore, for therapeutic purposes, the MHC II / CII peptide complex comprised in said composition does not comprise: (1) a streptavidin tag (such as SAWSHPQFEK, SEQ ID NO: 30) for purification; (2) a cleavage site (e.g. TEV cleavage site) between the MHC II alpha / MHC II beta chain and the heterodimerization domain; and / or (3) a recognition site for E. coli biotin ligase (BirA) (e.g. Avi tag), such as bold and underlined present in the exemplary complexes used in the Examples and shown in Figure 1. These elements were shown to have no influence on the in vitro and in vivo function of the complex (data not shown). Preferably, the MHC II / CII complex comprises a His-tag (polyhistidine-tag) or a functionally equivalent tag at the C-terminus of the polypeptide comprising the HLA-DR alpha chain and / or the HLA-DR beta chain. An exemplary minimal HLA-DR / CII peptide complex of the application can be encoded by the amino acid sequences of SEQ ID NO: 18 and SEQ ID NO: 19. The skilled person will understand that the peptide sequences can vary as encompassed by the claims.

[0133] Preferably, the composition of the present application comprises an MHC II / CII peptide complex comprising a His-tag or a functionally equivalent tag C-terminal of the polypeptide comprising the HLA-DR a chain and / or the HLA-DR b chain. A functionally equivalent tag is for example a chondroitin binding peptide. Thus, the composition can comprise an MHC II / CII peptide complex comprising at least one chondroitin binding peptide, preferably a chondroitin- and hyaluronan (also known as hyaluronate) binding peptide. In one embodiment, the MHC II / CII peptide complex comprises at least one C-terminal chondroitin binding peptide. Chondroitin binding peptides are known in the art and include, but are not limited to, peptides having the following amino acid sequence: EKRIWFPYRRF (SEQ ID NO: 31), YKTNFRRYYRF (SEQ ID NO: 32), or VLIRHFRKRYY (SEQ ID NO: 33) (Butterfield KC et al., Biochemistry. 2010 Feb 23; 49(7): 1549-55). In one embodiment, the chondroitin binding peptide comprises 5-20 amino acids, preferably 6-20 amino acids, more preferably 6-12 amino acids. To increase binding to hyaluronan, the corresponding sequence comprising the binding consensus motif is defined as follows: B(X7)B, wherein B is R or K, and X7 does not comprise an acidic residue and at least one basic amino acid (Yang B et al., EMBO J. 1994 Jan 15; 13(2): 286-96). As disclosed herein, the MHC II / CII peptide complex can also bind chondroitin sulfate via the His-tag. Thus, the chondroitin binding peptide can be a polyhistidine tag, preferably a hexahistidine tag, or any other amino acid sequence increasing the binding affinity to chondroitin, such as EKRIWFPYRRF (SEQ ID NO: 31), YKTNFRRYYRF (SEQ ID NO: 32), or VLIRHFRKRYY (SEQ ID NO: 33). Both chondroitin and hyaluronan are important components of cartilage.

[0134] In one embodiment, the extracellular region of the MHC class II a chain comprising at least one a1 domain and the extracellular region of the MHC class II b chain comprising at least one b1 domain; and the collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II a chain or the MHC class II b chain, preferably to the MHC class II b chain, via a linker peptide; are expressed as a single fusion polypeptide (single chain heterodimer).

[0135] In an alternative embodiment, the MHC II / CII peptide complex comprises a first polypeptide, a second polypeptide and a collagen II peptide (CII peptide); wherein the first polypeptide comprises the extracellular region of an MHC class II a chain comprising at least one al domain, the second polypeptide comprises the extracellular region of an MHC class II b chain comprising at least one b1 domain, and the collagen II peptide (CII peptide) is fused to the N-terminus of the MHC class II a chain or the MHC class II b chain, preferably to the MHC class II b chain, via a linker peptide. In one embodiment, the MHC class II a chain is fused at its C-terminus to a first functional domain of a leucine zipper heterodimerization motif and the MHC class II b chain is fused at its C-terminus to a second complementary functional domain of a leucine zipper heterodimerization motif. The first functional domain and the second complementary functional domain can be acidic and basic leucine zipper heterodimerization domains, preferably a jun-fos leucine zipper motif. In one embodiment, the first and / or the second polypeptide comprises a chondroitin sulfate binding peptide, such as a polyhistidine tag, at the C-terminus of the functional domain of the leucine zipper heterodimerization motif.

[0136] The skilled person will understand that the composition according to the present application, comprising MHC II / CII peptide complexes in a heterogeneous mixture of MHC II / CII peptide complexes, the complexes comprising different post-translational modifications of the CII peptide, in particular the first and optionally the second lysine residue of the CII peptide. The heterogeneous mixture can comprise MHC II / CII peptide complexes comprising K, Hyl, G-Hyl or GG-Hyl at the first lysine and independently K, Hyl, G-Hyl or GG-Hyl at the optional second lysine (wherein K = lysine, Hyl = hydroxylysine, G-Hyl = galactosyl-hydroxylysine, GG-Hyl = glucosylgalactosyl-hydroxylysine).

[0137] Thus, in one embodiment, the composition further comprises MHC II / CII peptide complexes comprising a CII peptide, wherein the first lysine residue of the CII peptide is unmodified, hydroxylysine (Hyl) or glucosylgalactosyl-hydroxylysine (GG-Hyl), preferably unmodified or hydroxylysine (Hyl), and the optional second lysine residue of the CII peptide is independently unmodified, hydroxylysine (Hyl), galactosyl-hydroxylysine (G-Hyl) or glucosylgalactosyl-hydroxylysine (GG-Hyl), preferably unmodified, hydroxylysine (Hyl), galactosyl-hydroxylysine (G-Hyl). In one embodiment, the composition does not comprise MHC II / CII peptide complexes modified with glucosyl-galactosyl-hydroxylysine (GG-Hyl), e.g. MHC II / CII peptide complexes comprising O-glycosylated CII peptides, wherein the first and the optional second lysine residue is glucosylgalactosyl-hydroxylysine.

[0138] Preferably, the composition comprises MHC II / CII peptide complexes, wherein at the first lysine (K264) of the CII peptide of the total MHC II / CII peptide complexes in the mixture or composition, the complexes comprise at least 5%, at least 10%, at least 20%, at least 30% of G-Hyl. Furthermore, the composition comprises MHC II / CII peptide complexes, wherein in the total MHC II / CII peptide complexes of the mixture or composition, the complexes comprise preferably less than 50%, less than 40%, less than 30% of unmodified CII peptide. Furthermore, the composition comprises MHC II / CII peptide complexes, wherein in the total MHC II / CII peptide complexes of the mixture or composition, the complexes comprise preferably less than 20%, less than 10%, less than 5% and more preferably less than 1% of GG-Hyl. Wherein the percentages refer to the percentage of the CII peptide in the MHC II / CII peptide complexes of the total CII peptide in the MHC II / CII peptide complexes. In a particularly preferred embodiment, the second lysine residue (K270) is mutated, e.g. to arginine (K270R). In a further embodiment, the (optional) second lysine is not post-translationally modified to glucosylgalactosyl-hydroxylysine (GG-Hyl) and is present as unmodified lysine, hydroxylysine or galactosyl-hydroxylysine.

[0139] In yet another aspect, the present application provides a recombinant MHC II / CII peptide complex obtained or obtainable by the method of the present application. Specifically encompassed is a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide, wherein the first lysine residue of the CII peptide is a hydroxylysine (Hyl) or an O-glycosylated Hyl. Thus, in one embodiment, a recombinant MHC II / CII peptide complex comprising an O-glycosylated CII peptide is obtained by the method of the present application.

[0140] In yet another aspect, the present application provides a composition comprising a recombinant MHC II / CII peptide complex, the complex comprising a post-translationally modified CII peptide obtained by the method of the present application.

[0141] Also contemplated is the use of the composition of the present application or the MHC II / CII peptide complex tetramer of the present application, preferably in vitro, for the detection of antigen-specific T cells.

[0142] The composition of the present application can be a pharmaceutical composition. Thus, the present application also discloses a pharmaceutical composition comprising such a composition comprising a recombinant MHC II / CII peptide complex as described herein and a pharmaceutically acceptable excipient. The composition or pharmaceutical composition can be administered by any route of administration, preferably subcutaneously (s.c.) or intravenously (i.v.). In one embodiment, the composition or pharmaceutical composition is administered using a subcutaneously implanted osmotic pump. The composition or pharmaceutical composition comprising a recombinant MHC II / CII peptide complex according to the present application can be lyophilized or in an aqueous solution. The pharmaceutically acceptable excipient can include carriers as well as stabilizers.

[0143] underlined

[0144] In yet another aspect, the present application relates to the use of a composition comprising a recombinant MHC II / CII peptide complex according to the present application or a recombinant MHC II / CII peptide complex produced by the method of the present application for the treatment of a chronic inflammatory disease. Preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. In one embodiment, the composition comprising a recombinant MHC II / CII peptide complex according to the present application or a recombinant MHC II / CII peptide complex produced by the method according to the present application is used for the treatment of a chronic inflammatory disease in a human subject, in particular arthritis or other chronic inflammatory arthropathy. In one embodiment, the composition is used for the treatment of a chronic inflammatory disease selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, non-radiographic axial spondyloarthritis, ankylosing spondylitis, juvenile idiopathic arthritis, relapsing polychondritis, systemic lupus erythematosus, Lyme disease, Meniere's disease, autoimmune inner ear disease (AIED) or Still's disease.

[0145] The chronic inflammatory disease can be arthritis, preferably an arthritis type selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis or Still's disease, more preferably rheumatoid arthritis, osteoarthritis or psoriatic arthritis, more preferably rheumatoid arthritis. In particular embodiments, the composition according to the application is used for first-line treatment of rheumatoid arthritis, for the treatment of subjects who have an inadequate response to methotrexate and / or conventional synthetic (small molecule) disease-modifying antirheumatic drugs (DMARDs), for the treatment of subjects who have an inadequate response to biologic DMARDs (e.g. anti-TNF, anti-CTLA4 (abatacept), anti-IL-6, anti-CD20 (rituximab) antibodies), for the treatment of subjects who have an inadequate response to targeted synthetic DMARDs (e.g. JAK inhibitors). In alternative embodiments, the composition of the application is used for prophylactic treatment of patients at high risk of developing rheumatoid arthritis, such as anti-ccp antibody positive smokers with emerging musculoskeletal symptoms.

[0146] Preferably, the composition is to be administered subcutaneously or intravenously, more preferably subcutaneously. The composition can be administered in a single dose of about 10 μg to about 250 μg, preferably 20-200 μg, more preferably 50-100 μg. In one embodiment, the treatment comprises a loading and a maintenance phase. The loading phase can comprise 3-10, preferably 6, consecutive administrations on consecutive days. The maintenance dose can be administered weekly or every 3-14 days, preferably weekly, biweekly, monthly, bimonthly or even larger intervals.

[0147] bold and underlined

[0148] In yet another aspect, the present application provides an MHC II / CII peptide complex tetramer comprising a recombinant MHC II / CII peptide complex according to the composition of the present application or a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide obtained according to the method of the present application. Preferably, the CII peptide comprises a post-translational modification at a lysine residue, preferably at the first lysine residue of the CII peptide. In one embodiment, the first lysine residue of the CII peptide is Hyl or O-glycosylated Hyl. In one embodiment, the tetramer comprises a multimerization molecule binding to the recombinant MHC II / CII peptide complex, preferably streptavidin or avidin. In a preferred embodiment, the multimerization molecule is streptavidin. Each recombinant MHC II / CII peptide complex can contain at least one covalently bound N-terminal biotin. The process of biotinylation, i.e. the covalent attachment of biotin to a protein, is preferably site-specific and can be by chemical linkage or by enzymatic linkage. Preferably, the recombinant MHC II / CII peptide complex contains a recognition site for a biotinylation enzyme such as E. coli biotin ligase (BirA). The recognition site for BirA is a 15 amino acid peptide, known as an Avi tag or acceptor peptide (e.g. Avi tag). Enzymatic biotinylation can be performed in vitro or in vivo.

[0149] The multimerization molecule, such as streptavidin, can be conjugated with a label, preferably a fluorescent dye, to allow detection of the tetramer, in particular the bound tetramer. The label can be any label known in the art, such as horseradish peroxidase for detection by enzyme chemiluminescence (ECL) or luciferase. Other examples are fluorescent dyes such as PE, APC, rhodamine (TRITC), FITC, etc.

[0150] The tetramer is produced by a method for preparing an MHC II / CII peptide complex tetramer, the method comprising: (a) providing a composition of the present application or a recombinant MHC II / CII peptide complex comprising an O-glycosylated CII peptide according to the present application, wherein the MHC II / CII peptide complex comprises at least one N-terminal biotinylation, (b) contacting the composition with a multimerization molecule, preferably streptavidin, and, optionally, isolating a tetramer comprising four MHC II / CII peptide complexes bound to streptavidin.

[0151] The tetramers of the application can be used to detect antigen-specific T cells and thus to detect arthritis, in particular rheumatoid arthritis. Thus, in one aspect, the application relates to a method for detecting and / or quantifying T cells specific for a given antigen, wherein the method comprises: (a) providing an MHC II / CII peptide complex tetramer according to the application, wherein the multimerization molecule is conjugated to a label (b) contacting the MHC II / CII peptide complex tetramer with a sample of a subject, preferably a sample containing peripheral blood cells of said subject, and (c) detecting the label of the T cells bound to the MHC II / CII peptide complex tetramer. Preferably, the method is an in vitro detection method. Preferably, the label is a fluorescent dye. The MHC II / CII peptide complex tetramer bound to the T cells can be detected by any suitable method known in the art, e.g. flow cytometry. Preferably, the method is an in vitro method. In one embodiment, the method is a method for diagnosing a patient suffering from arthritis or rheumatoid arthritis, comprising: (a) providing an MHC II / CII peptide complex tetramer according to the application, wherein the multimerization molecule is conjugated to a label (b) contacting the MHC II / CII peptide complex tetramer with a sample of a subject, preferably a sample containing peripheral blood cells of said subject, and (c) detecting the label of the T cells bound to the MHC II / CII peptide complex tetramer. If the MHC II / CII peptide complex tetramer bound to the T cells is detected, the patient is likely to suffer from arthritis or to be at risk of suffering from arthritis, in particular rheumatoid arthritis.

[0152] In view of the above, it is to be understood that the application further encompasses the following:

[0153] 1. A composition comprising a recombinant MHC II / CII peptide complex, said complex comprising:

[0154] (a) an extracellular region of an MHC class II alpha chain comprising at least one alpha 1 domain;

[0155] (b) an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain; and

[0156] (c) a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain, preferably to the N-terminus of the MHC class II beta chain, via a linker peptide;

[0157] wherein the CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG and AGFKXEQGPXG, and wherein the MHC II / CII peptide complex comprises a post-translationally modified CII peptide, preferably wherein the first lysine residue of the CII peptide is a hydroxylysine (Hyl) or an O-glycosylated Hyl.

[0158] 2. The composition of item 1, wherein

[0159] (a) the extracellular region of the MHC class II a chain comprises an al and a 2 domain; and / or

[0160] (b) the extracellular region of the MHC class II b chain comprises a pi and a 2 domain.

[0161] 3. The composition of item 1 or 2, wherein the first lysine residue is galactosyl- hydroxylysine.

[0162] 4. The composition of any one of items 1-3, wherein the CII peptide is fused to the N- terminus of the pi domain via a linker peptide.

[0163] 5. The composition of any one of items 1-4, wherein

[0164] at least the al domain is from DRA*0101 and at least the pi domain is from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402 and DRB1*1303, preferably DRB1*0401.

[0165] 6. The composition of any one of items 1-5, wherein the CII peptide comprises the amino acid sequence of AGFKGEQGPKG, preferably AGFKGEQGPKGEP, more preferably GIAGFKGEQGPKGEP.

[0166] 7. The composition of any one of items 1-6, wherein the CII peptide comprises only the first lysine residue and any additional K is mutated, preferably to R, A, G or Q, more preferably to R.

[0167] 8. The composition of any one of items 1-7, wherein

[0168] (a) the extracellular region of the MHC class II a chain comprises at least one al domain;

[0169] (b) an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain; and

[0170] (c) a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain via a linker peptide;

[0171] each expressed as a single fusion polypeptide.

[0172] 9. The composition of any one of items 1-7, comprising

[0173] (a) a first polypeptide comprising an extracellular region of an MHC class II alpha chain comprising at least one alpha 1 domain;

[0174] (b) a second polypeptide comprising an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain; and

[0175] (c) a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain via a linker peptide.

[0176] 10. The composition of item 9, wherein the MHC class II alpha chain is fused at its C- terminus to a first functional domain of a leucine zipper heterodimerization motif and the MHC class II beta chain is fused at its C-terminus to a second complementary functional domain of a leucine zipper heterodimerization motif.

[0177] 11. The composition of item 10, wherein the first functional domain and the second complementary functional domain are:

[0178] (a) acidic and basic leucine zipper heterodimerization domains; and / or

[0179] (b) a jun-fos leucine zipper motif.

[0180] 12. The composition of any one of items 1-11, further comprising an MHC II / CII peptide complex comprising a CII peptide, wherein the first lysine residue of the CII peptide is unmodified.

[0181] 13. A method of producing an MHC II / CII peptide complex comprising a post-translationally modified (e.g., O-glycosylated) CII peptide, comprising:

[0182] (a) transfecting a mammalian cell with:

[0183] (i) a polynucleotide encoding an extracellular region of an MHC class II alpha chain comprising at least one alpha 1 domain;

[0184] (ii) a polynucleotide encoding an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain; and

[0185] (iii) a polynucleotide encoding a collagen II peptide (CII peptide) fused to the N-terminus of an MHC class II alpha chain or an MHC class II beta chain via a linker peptide, preferably to the MHC class II beta chain, wherein the CII peptide comprises an amino acid sequence selected from the group consisting of AGFKGEQGPKG, AGFKGEQGPXG, AGFKGEXGPKG, AGFKGXQGPKG, AGFKXEQGPKG, AGFKGEXGPXG, AGFKGXQGPXG, and AGFKXEQGPXG;

[0186] (b) cultivating the mammalian cell under conditions suitable for producing MHC II / CII peptide complexes; and

[0187] (c) harvesting the cell supernatant and optionally the cell comprising the MHC II / CII peptide complex, the MHC II / CII peptide complex comprising a post-translationally modified CII peptide, preferably wherein the first lysine residue of the CII peptide is hydroxy lysine (Hyl) or O-glycosylated Hyl.

[0188] 14. The method of item 13 further comprising the step of analyzing the post- translational modification, preferably the glycosylation profile, of the CII peptide of the MHC II / CII peptide complex.

[0189] 15. The method of item 13 or 14, wherein the first lysine residue is galactosyl- hydroxy lysine.

[0190] 16. The method of any one of items 13-15, wherein the mammalian cell

[0191] (a) comprises an enzyme that post-translationally modifies lysine residues in collagen, the modification comprising: hydroxylation of lysine to hydroxy lysine (Hyl) and galactosylation of Hyl to galactosyl hydroxy lysine (Gal-Hyl); and / or

[0192] (b) comprises a lysyl hydroxylase and a collagen galactosyltransferase, preferably lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2) and collagen galactosyltransferase GLT25D1 and / or GLT25D2, preferably GLT25D1.

[0193] 15. The method of item 16, wherein the cell is

[0194] (a) a kidney cell, a fibroblast cell, or an osteoblast cell, preferably a kidney cell, more preferably a HEK293 cell line; or

[0195] (b) genetically engineered cells recombinantly expressing lysyl hydroxylase and collagen galactosyltransferase, preferably lysyl hydroxylase 1 (LH1) and / or lysyl hydroxylase 2 (LH2) and collagen galactosyltransferase GLT25D1 and / or GLT25D2.

[0196] 16. The method of any one of items 13-17, wherein the mammalian cells

[0197] (a) lack galactosyl-hydroxylysyl glucosyltransferase activity;

[0198] (b) lack the multifunctional enzyme LH3; or

[0199] (c) comprise a mutant LH3 enzyme that lacks galactosyl-hydroxylysyl glucosyltransferase activity.

[0200] 19. The method of item 18, wherein the mammalian cells are genetically engineered to have reduced or no galactosyl-hydroxylysyl glucosyltransferase activity, preferably wherein

[0201] (a) the PLOD3 gene encoding LH3 is mutated or deleted;

[0202] (b) the LH3 enzyme is a mutant LH3 enzyme that lacks galactosyl-hydroxylysyl glucosyltransferase activity; or

[0203] (c) LH3 expression is inhibited by RNA interference.

[0204] 20. The method of any one of items 13-16, further comprising adding alizarin acid during culturing the mammalian cells according to step (b).

[0205] 21. The method of any one of items 13-20, wherein

[0206] (a) the extracellular region of the MHC class II alpha chain comprising alpha 1 and alpha 2 domains; and / or

[0207] (b) the extracellular region of the MHC class II beta chain comprising beta 1 and beta 2 domains.

[0208] 22. The method of any one of items 13-21, wherein the CII peptide is fused to the N- terminus of the beta 1 domain.

[0209] 23. The method of any one of items 13-22, wherein

[0210] at least the a1 domain is from DRA*0101 and at least the b1 domain is from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303, preferably DRB1*0401.

[0211] 24. The method of any one of items 13-23, wherein the CII peptide comprises the amino acid sequence AGFKGEQGPKG, preferably AGFKGEQGPKGEP, more preferably GEPGIAGFKGEQGPKGEP.

[0212] 25. The method of any one of items 13-24, wherein the CII peptide comprises only the first lysine residue, and any additional K is mutated, preferably to R, A, G, or Q, more preferably to R.

[0213] 26. The method of any one of items 13-25, wherein

[0214] (a) an extracellular region of an MHC class II a chain comprising at least one a1 domain;

[0215] (b) an extracellular region of an MHC class II b chain comprising at least one b1 domain; and

[0216] (c) a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II a chain or the MHC class II b chain by a linker peptide;

[0217] are encoded by a single polynucleotide to express a single fusion polypeptide.

[0218] 27. The method of any one of items 13-25, comprising:

[0219] (a) a first polynucleotide encoding an extracellular region of an MHC class II a chain comprising at least one a1 domain;

[0220] (b) a second polynucleotide encoding an extracellular region of an MHC class II b chain comprising at least one b1 domain; and

[0221] (c) a polynucleotide encoding a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II a chain or the MHC class II b chain by a linker peptide.

[0222] 28. The method of item 27, wherein the MHC class II alpha chain is fused at its C- terminus to a first domain of a leucine zipper heterodimerization motif and the MHC class II beta chain is fused at its C-terminus to a second, complementary domain of a leucine zipper heterodimerization motif.

[0223] 29. The method of item 28, wherein the first domain and second, complementary domain are:

[0224] (a) acidic and basic leucine zipper heterodimerization domains; and / or

[0225] (b) a jun-fos leucine zipper motif.

[0226] 30. The method of any one of items 13-29, wherein the harvested cell supernatant and optionally the harvested cells further comprise MHC II / CII peptide complexes comprising a CII peptide, wherein the first lysine residue of the CII peptide is unmodified.

[0227] 31. A recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide obtained by the method of any one of items 13-30, preferably wherein the first lysine residue of the CII peptide is a hydroxylysine (Hyl) or an O-glycosylated Hyl.

[0228] 32. A composition comprising a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide obtained by the method of any one of items 13-30, preferably wherein the first lysine residue of the CII peptide is a hydroxylysine (Hyl) or is an O-glycosylated Hyl.

[0229] 33. The composition of any one of items 1-12 and 32 for use in the treatment of a chronic inflammatory disease.

[0230] 34. The recombinant MHC II / CII peptide complex of item 31 for use in the treatment of a chronic inflammatory disease.

[0231] 35. The composition for use according to item 33 or the recombinant MHC I / CII peptide complex for use according to item 34, wherein the chronic inflammatory disease is rheumatoid arthritis, osteoarthritis, psoriatic arthritis, non-radiographic axial spondyloarthritis, ankylosing spondylitis, juvenile idiopathic arthritis, relapsing polychondritis, systemic lupus erythematosus, Lyme disease, Meniere's disease, autoimmune inner ear disease (AIED), or Still's disease.

[0232] 36. An MHC II / CII peptide complex tetramer comprising: a recombinant MHC II / CII peptide complex according to the composition of any one of items 1-12 and 32 or a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide according to item 31.

[0233] 37. The MHC II / CII peptide complex tetramer according to item 36, wherein the tetramer comprises a multimerization molecule, preferably streptavidin, binding the recombinant MHC II / CII peptide complex.

[0234] 38. The MHC II / CII peptide complex tetramer according to item 37, wherein each recombinant MHC II / CII peptide complex contains at least one covalently bound N-terminal biotin.

[0235] 39. The MHC II / CII peptide complex tetramer according to item 37 or 38, wherein the multimerization molecule is conjugated to a label, preferably a fluorescent dye.

[0236] 40. A method of preparing an MHC II / CII peptide complex tetramer comprising:

[0237] (a) providing a composition according to any one of items 1-12 and 32 or a recombinant MHC II / CII peptide complex comprising a post-translationally modified CII peptide according to item 31, wherein the MHC II / CII peptide complex comprises at least one N-terminal biotinylation,

[0238] (b) contacting the composition with a multimerization molecule, preferably streptavidin, and

[0239] (c) optionally isolating a tetramer comprising four MHC II / CII peptide complexes bound to streptavidin.

[0240] 41. The method of item 40, wherein the multimerization molecule is conjugated to a label, preferably a fluorescent dye.

[0241] 42. An in vitro method for detecting and / or quantifying T cells specific for a given antigen, wherein the method comprises:

[0242] (a) providing an MHC II / CII peptide complex tetramer according to item 39,

[0243] (b) contacting the MHC II / CII peptide complex tetramer with a sample of a subject, preferably a sample containing peripheral blood cells of the subject, and

[0244] (c) detecting the label of the MHC II / CII peptide complex tetramer bound to the T cells.

[0245] 43. The in vitro method of item 42, wherein the label is a fluorescent dye, and T cells binding to the MHC II / CII peptide complex tetramer are detected by flow cytometry.

[0246] 44. Use of the compositions according to items 1-12 or the MHC II / CII peptide complex tetramers according to items 36-39 for in vitro detection of antigen-specific T cells. Example

[0247] bold and underlined

[0248] Aq / galCII or DR4 / galCII (loaded with synthetic Gal-peptide: GIAGFK(Gal-Hyl)GEQGPKGEP) and Aq / nCII or DR4 / nCII (loaded with unmodified peptide: GIAGFKGEQGPKGEP; SEQ ID NO: 13): Aq-mCLIPmt protein, by transient transfection into HEK293 cell line (Expi293F cells, Gibco, catalog number A14527) or CHO cells ( italic and underlined The Gal-peptide was expressed in [a specific format], purified using His-tagged immobilized metal ion affinity chromatography (IMAC) combined with size exclusion chromatography (SEC). The covalently bound propeptide was then cleaved with thrombin and replaced with excess Gal peptide or unmodified peptide. Finally, SEC was performed to remove the cleaved propeptide and excess Gal peptide or unmodified peptide. The Gal-peptide (GIAGFK(Gal-Hyl)GEQGPKGEP) was synthesized, purified, and characterized as described in Diogo, D. et al., Curr Opin Rheumatol. 2014; 26:85-92; Gregersen PK et al., Arthritis Rheum. 1987; 30:1205-1213; Duke O et al., Clin Exp Immunol. 1982; 49:22-30.

[0249] Native Glycosylated Mouse Aq / rCII and Human DR4 / hCII: Native glycosylated mouse Aq / rCII and human DR4 / hCII proteins were purified in HEK293 cell line (Expi293F cells, Gibco, catalog number A14527) via transient transfection and purified using His-tagged immobilized metal ion affinity chromatography (IMAC) combined with size exclusion chromatography (SEC). For in vivo experiments, the MHC II-peptide complex was diluted to the desired concentration in sterile PBS (Gibco), filtered using a DynaGard 0.2 pm syringe tip filter, and 100 pI of protein solution was aseptically loaded into an ALZET microosmotic pump (DURECT, model 1007D, 0.5 pI / h, 7 days). The pump was handled with surgical gloves. To ensure immediate delivery of material, the pre-loaded pump was incubated overnight in PBS at 4°C prior to implantation.

[0250] More specifically, encoding bold and underlined The cDNA of the two strands of the complex shown is synthesized in Eurofins and contains KpnI and XhoI restriction sites at the 5' and 3' ends. The restriction enzymes KpnI and XhoI (FastDigest) were used. TM The synthesized cDNA was digested by Thermo Fisher Scientific. After digestion with the same restriction enzyme, the digested DNA fragments were cloned into the mammalian expression vector pCEP4 (Life Technologies). After sequence verification, the two recombinant plasmids encoding the two strands of the complex were used to clone the cDNA. TM DNA transfection reagent (Polyplus-transfection) co-transfected into Expi393F TM In cells. The supernatant was harvested 6 days post-transfection. The recombinant protein was first captured using a 5 ml HisTrap Excel (GE Healthcare Life Sciences) affinity column, followed by size exclusion chromatography on a Superdex 200 pg (GE Healthcare Life Sciences) column. The recombinant protein was purified to a single peak and concentrated, dialyzed and filtered into biotinylation buffer (20 mM Tris-HCl, 50 mM NaCl, pH 8.0) using an Amicon centrifuge with a 10 kDa MWCO. Biotinylation was performed using biotin-protein ligase according to the manufacturer's instructions (Avidity), with the reaction carried out at 30°C for 2 hours. Free biotin was removed by size exclusion chromatography on a Superdex 200 pg column.

[0251] underlined

[0252] Male QB mice (B10.Q x BALB / c, n = 9) F1, 12-16 weeks of age, were used for experiments. The B10.Q mouse was originally provided by J. Klein (Tubingen, Germany) and the BALB / c mice were purchased from The Jackson Laboratory. All mice were housed and kept at the animal facility of the Medical Inflammation Research of Karolinska Institute. All animals used were fed standard rodent chow and had free access to water. Different experimental groups were housed together to minimize experimental bias. All animal experiments were approved by the local ethical committee (Stockholms Norra Djurforsoksetiska Namnd, Stockholm, Sweden). All in vivo arthritis experiments were covered by the ethical numbers N213 / 14 and N35 / 16. Animals were anesthetized by inhalation of isoflurane and sacrificed with CO2.

[0253] bold and underlined

[0254] Rat collagen type II (rCII) was prepared from Swarm ratchondosarcoma (SRC) by limited pepsin digestion and further purification, which is described in Chavele KM and Ehrenstein MR, FEBS Lett. 2011; 585:3603-10. Prepared rCII was stored at 4°C until use. To induce collagen-induced arthritis (CIA), each mouse was injected with 100 μg of rCII emulsified 1 : 1 in CFA (Difco) in a total volume of 100 μl at the base of the tail. After 35 days, mice were given a booster injection of 50 μg of rat CII emulsified 1 : 1 in IFA (Difco) in a total volume of 50 μl. From 2 weeks after immunization and until the end of the experiment, the clinical arthritis was followed by visually scoring the animals based on the number of inflamed joints in each paw. The extended scoring scheme adopted is described in Klareskog Let al., Annu Rev Immunol. 2008; 26:651-75, varying from 1-15 for each paw, with a maximum score of 60 for each mouse. Mice were examined 2-4 times per week for 90 days after immunization.

[0255] composition comprising recombinant MHC II / CII peptide complexes

[0256] On day 7 after immunization, ALZET micro-osmotic pumps diffusing different amounts of native glycosylated Aq / rCII (n=9) or PBS (control group, n=9) were implanted s.c. into the QB mice using sterile technique. For s.c. placement, a small incision was made in the skin between the scapulae, a small pocket was created, the pump was inserted into the pocket, and the flow regulator was pointed away from the incision. The skin incision was closed using wound clips.

[0257] A single s.c. injection of 100 mg in mice was almost as effective as a continuous 7-day s.c. pump infusion of 15 mg / day in the treatment of clinical arthritis. However, prolonged treatment appeared to be more effective in inducing regulatory TR1 cells, as evidenced by FACS analysis of T cells from the draining lymph nodes of treated mice.

[0258] Figure 1

[0259] On day 8 after immunization, QB mice pre-immunized with rat CII / CFA (rCII) were injected i.d. with 10 μg of rCII in phosphate-buffered saline (PBS) into the left ear. As a control, the right ear was injected with the solvent, and 24 h later ear swelling was measured using calipers by an investigator blinded to the treatment of the animal. The osmotic pumps were implanted 4 days after immunization with rCII, and treatment was performed by 24 h application of 100 μg of Aq / peptide complex. Groups: Aq / mCLIPmt (n=6), Aq / galCII with His-tag (His, n=5) and without His-tag (w / o His, n=5).

[0260] Figure 1

[0261] MHC II / peptide complexes were diluted in sterile PBS and coated onto plates by incubation overnight at 4°C, or added directly in soluble form to T-cell hybridomas. MHC II / peptide complex-coated plates were then washed twice with sterile PBS to remove unbound complexes, and 5x10 4 T-cell hybridomas 3H8 and mDRl. l, which are specific for Gal OK264 and unmodified CII259-273 (K264), respectively, were used. After 24 h, IL-2 or IL-10 (in some experiments) in the culture supernatant was measured by sandwich ELISA (BioLegend). Mouse rIL-2 or rI-10 were used as positive control and standard, respectively.

[0262] Stimulation experiments were performed in microtiter wells using T-hybridoma cells under different conditions: 1) pre-coated with recombinant DR4 / CII-peptide complex, 2) coated with hyaluronic acid (Sigma Aldrich (#H7630)) or chondroitin sulfate (Sigma Aldrich (#C9819)) followed by the addition of DR4 / CII-peptide complex in the fluid phase to them. This design was chosen to investigate the influence of potential interactions of the two components on T cell activation by DR4 / CII-peptide complex and to mimic the interaction of DR4 / CII-peptide complex with connective tissue components physiologically expressed in the extracellular matrix (ECM) of tissues and the draining lymphatic system, or 3) with blocked surfaces, where the solute ECM components hyaluronic acid, chondroitin sulfate or heparin sulfate and DR4 / CII-peptide complex were added to investigate their influence on T-hybridoma cells as a model for modulating T cell function in the body fluids of diseased tissue compartments (e.g. joint effusion or lymphatic fluid).

[0263] therapeutic use

[0264] MHC II / peptide tetramer complexes were freshly prepared by adding PE-labeled streptavidin and APC-labeled streptavidin (Biolegend) in a 1:4 molar ratio to the recombinant proteins and incubating for 1 hour at +4°C. To identify peptide-specific T lymphocytes, cells were incubated with DR4 / peptide tetramer complexes (20 pg / ml) in the presence of 50 nM dasatinib (small molecule protein tyrosine kinase inhibitor) for 1 hour at +37°C before staining of cell surface markers. A viability stain solution (Zombie NIR; Biolegend) was added right before acquisition to exclude dead cells from analysis. Samples were acquired by using a LSR Fortessa flow cytometer with FacsDiVa software (BD Biosciences) and data were analyzed using FlowJo software (v10, FlowJo LLC).

[0265] tetramers comprising recombinant MHC II / CII peptide complexes

[0266] PBMCs were incubated in TexMACS (Biolegend) at 1.5 x 10 6Cells were stimulated with 1 pg / mL anti-CD3 (Biolegend, Cat# 317304) and 100 ng / mL IL-27 (Peprotech, Cat# 200-38B) (positive control, Tr1), 3.6 pg / mL DR4 / nCII, 3.6 pg / mL DR4 / galCII, 3.6 pg / mL DR4 / hCII or left unstimulated (negative control, w / o) for 8 days. Stimulation was performed in duplicates. On day 8, culture supernatants were collected and released cytokines were analyzed using a custom panel to detect human cytokines in a multiplex microbead-based LEGENDplex assay according to the manufacturer’s protocol.

[0267] test substances and preparations

[0268] In vitro assays were performed to analyze the induction / differentiation of regulatory T cell function, e.g. upregulation of the Tr1 phenotype associated cytokine IL-10 after an extended latent period of DR4 / CII monomer stimulation for several days. Therefore, PBMC were isolated by density gradient centrifugation and 1.2 x 10 6 Cells were stimulated with 1 pg / mL anti-CD3 (Biolegend, Cat# 317304) and 100 ng / mL IL-27 (Peprotech, Cat# 200-38B) (positive control, Tr1), 3.6 pg / mL DR4 / nCII, 3.6 pg / mL DR4 / galCII, 3.6 pg / mL DR4 / hCII or left unstimulated (negative control, w / o) for 8 days. Stimulation was performed in duplicates. On day 8, culture supernatants were collected and released cytokines were analyzed using a custom panel to detect human cytokines in a multiplex microbead-based LEGENDplex TM assay according to the manufacturer’s protocol.

[0269] Results:

[0270] Figure 4

[0271] It has been demonstrated in previous experiments that two intravenous injections of Aq molecules loaded with synthetic galactosylated CII259-273peptides can protect mice from developing arthritis. However, the synthesis of galactosylated CII259-273is both time-consuming and expensive. In addition, loading synthetic peptides onto recombinant MHC class II molecules is neither simple nor cost-effective. Therefore, it would be a significant advantage to establish a biosynthetic process that allows the one-step production of MHC II molecules comprising a covalently bound CII259-273peptide fused to one of the MHC II chains in the host cell Figure 1 ), to ensure the appropriate post-translational modification of the lysine side chain at position 264 in the CII peptide by hydroxylation and subsequent in situ galactosylation. However, post-translational collagen peptide modifications depend on the presence of the respective enzyme activities, i.e. lysyl hydroxylase activity and collagen beta galactosyltransferase activity. For example, proteins produced by E. coli typically do not exhibit such modifications, although some insect cells have the ability to hydroxylate lysine residues, they do not produce CII peptides comprising O-linked glycosylation of hydroxy lysine. Furthermore, it is unknown whether a host cell providing the required enzyme activities is indeed able to provide the required modifications at the selected amino acid positions of the CII-peptide within the framework of a non-collagen MHC II protein sequence.

[0272] It was first shown below that the Aq / rCII(259-273) complex can be expressed in HEK293 cells and that the purified complex contains the CII peptide (CII259-273) covalently linked, with a post-translational modification of the lysine at position 264. We analyzed the type of modification of the lysine side chain in the CII peptide and whether the purified in situ galactosylated Aq / rCII(259-273) complex has a protective effect in the CIA mouse model, in a similar way to that observed for recombinant Aq molecules loaded with galactosylated CII1259-273peptides. To test the therapeutic potential of the HEK293-produced Aq / rCII(259-273) complex, we used osmotic pumps implanted subcutaneously one week after immunization. Osmotic pumps are superior to intravenous injections because the Aq / rCII(259-273) complex remains at constant levels in the circulation and can be used for in vivo tolerance induction over a longer period of time. When comparing three different types of pumps that release their content over 24 hours, 7 days or 6 weeks, it was found that all three pumps mediated protection from arthritis when containing Aq molecules loaded with synthetic galactosylated CII peptides (data not shown). However, it was found that the pump-mediated protection using pumps releasing over 7 days was more closely associated with the development of CII-specific T cells with regulatory capacity than the protection using 24-hour pumps (data not shown). Without being bound by theory, a slow release rate at low doses can lead to the development of regulatory T cells, whereas a faster release at higher doses can lead to the exhaustion of pathogenic T cells. However, the observed differences can also be explained by the prolonged exposure, which increases the likelihood that CII-specific T cells (which occur at low frequency) interact with the Aq / rCII(259-273) complex before it is eliminated from the circulation. The experiments described below were performed using osmotic pumps that release their content over 7 days.

[0273] To evaluate which post-translational modifications are present when the Aq / rCII(259-273) complex is produced in HEK293 cells, Aq-restricted T cell hybridoma clones with different specificities for the CII259-273epitope were stimulated in vitro with the purified complex. animals ).

[0274] The ability to produce post-translational modifications in terms of O-linked glycosylation of lysine side chains is severely impaired in S2 insect cells producing MHC II / CII complexes. As expected, only the HCQ.4 clone, which recognizes the CII259-273peptide with an unmodified or hydroxylated lysine at position 264, reacted with the Aq / rCII(259-273) complex produced in S2 insect cells. In contrast, all CII-specific clones reacted with the Aq / rCII(259-273) complex produced in HEK293 cells. The other specificities of the T cell hybridoma clones used were as follows: HCQ3 (CII, Gal-HK264), HCQ.4 (CII, unmodified and HK264), HCQ.11 (Glc-Gal-HK264), HM1R.2 (CII, Gal-HK264 and Gal-HK264+270), HP3 (Aq-restricted pepsin peptide). This indicates that position 264 can indeed become post-translationally modified when produced in HEK293 cells. Moreover, the resulting complex is heterogeneous with position 264 comprising unmodified and / or hydroxylated lysine as well as glycosylated lysine with mono- and di-saccharides. The Aq-restricted clone HP3, which has specificity for pepsin peptides, did not react with any of the Aq / rCII(259-273) complexes.

[0275] induction and clinical evaluation of arthritis

[0276] Seven days after the initial immunization (and a booster immunization 35 days later), mice immunized with CII in adjuvant were implanted with osmotic pumps loaded with three different amounts of HEK293-produced Aq-rCII(259-273) complex and followed for the development of arthritis. Mice implanted with pumps containing only PBS served as negative controls. As shown in Figure A, the Aq-rCII(259-273) complex conferred protection in a dose-dependent manner, with mice treated with the highest amount of Aq-rCII(259-273) complex (100 μg) being completely protected from developing arthritis. Mice treated with the intermediate amount (50 μg) of Aq-rCII(259-273) complex showed some protection, whereas treatment with the lowest amount (10 μg) resulted in a frequency of arthritis comparable to the PBS-treated control group. treatment regimen

[0277] DTH

[0278] ​We have shown that it is possible to produce functional mouse MHC II / CII complexes (Aq / rCII) in HEK293 cells using the in situ glycosylation machinery of the host cell. We next investigated whether it is possible to produce human MHC II / CII complexes in HEK293 cells using the in situ glycosylation machinery of the cell. Complexes were produced in HEK293 cells as described above. Control complexes were expressed in CHO cells and loaded with unmodified peptide (DR4 / nCII) or galactosylated peptide (DR4 / galCII). Two activated restricted human T cell hybridomas (3H8: unmodified CII epitope, mDR1.1: galactosylated CII epitope) were used to examine the galactosylation status of native glycosylated DR4 / peptide complexes (DR4 / hCII) compared to DR4 / peptide complexes loaded with unmodified or galactosylated peptides. As shown in Figure 1 T cell hybridoma assay A, T cell hybridoma mDR1.1 was activated upon stimulation with DR4 / galCII complexes, whereas stimulation with DR4 / nCII remained almost negative. DR4 / covalently linked CII (DR4 / hCII) was a heterogenous product in terms of galactosylation status compared to DR4 / galCII and DR4 / nCII. This means that the composition comprising DR4 / hCII complexes contains peptides with galactosylated and unmodified lysine residues at position 264. The level of activation of cells stimulated with DR4 / hCII was slightly lower compared to DR4 / galCII complexes and very similar to DR4 / nCII complexes detection of antigen-specific T cells by staining with MHC II tetramers B, using 3H8 cells).

[0279] activation of human T cells after CII peptide stimulation

[0280] The aim was to establish a tetramer-based method to directly detect antigen-specific T cells in peripheral blood (PBMCs) of RA patients and healthy donors. Therefore, biotinylated DR4 / CII peptide complexes were incubated with streptavidin-PE or streptavidin-APC. To reduce non-specific binding of tetramers, double tetramer staining was performed using two fluorescent dyes. Antigen-specific T cells using DR4 / galCII tetramers (CII259-273, K264gal) could be detected in RA patients and healthy donors in vitro stimulation / differentiation of T cells from peripheral blood of HLA-DRB1*0401 positive RA patients A). In addition, T cells specific for unmodified CII peptide could be detected using DR4 / nCII tetramers Example 1 : Production of functionally active Aq / rCII in HEK293 cells B). The average frequency of antigen-specific T cells using native glycosylated DR4 / hCII tetramers was higher compared to DR4 / galCII tetramers or DR4 / nCII tetramers Figure 1B). The numbers observed were as expected due to the very low frequency of antigen-specific T cells in the peripheral blood (0.01-0.1%).

[0281] Using CD154 (CD40L) surface staining as a marker for T cell activation, activated CD4+ T cells were also detected in PBMC of HLA-DRB1*0401 RA patients after galCII peptide stimulation by flow cytometry Figure 2 ). As a positive control, cells were also incubated with the superantigen SEB, leading to a strong upregulation of the activation marker CD154 (data not shown). In contrast, cells incubated with antibodies against the costimulatory molecule CD28 were mainly negative (data not shown). Since the expected frequency of antigen-specific T cell populations in the peripheral blood is very low, it was satisfactory to detect 0.01-0.1% CD154+ T cells (out of the parental population: CD3 / CD4 live T cells). It is noteworthy that the degree of T cell activation was lower using the unmodified (naked) CII peptide. Since the number of antigen-specific T cells stained with DR4 / galCII or DR4 / nCII tetramers was similar in PBMC of HLA-DRB1*0401 RA patients Example 2: In situ glycosylated Aq / rCII in a mouse CIA model ), the differences observed after peptide activation seem to be due to differences in the activity or functional state of the individual T cells.

[0282] Figure 3

[0283] In vitro studies were performed to investigate the induction / differentiation of regulatory T cell functions, e.g. the IL-10 cytokine associated with the Tr1 phenotype was upregulated after a latency of up to 8 days of stimulation with DR4 / CII monomers. Therefore, PBMC isolated from genotyped HLA-DRB1*0401 positive RA patients were stimulated under Tr1 cell induction conditions with anti-CD3 and IL-27 (positive control, Tr1), with DR4 / nCII (3.6 pg / mL), DR4 / galCII (3.6 pg / mL) or left unstimulated (negative control, K1) for 8 days. Stimulation was performed in duplicates. At day 8, culture supernatants were collected and analyzed for cytokine release using a custom-made human cytokine panel in a multiplex bead-based LEGENDplex TM determination format according to the manufacturer’s protocol. Example 3: Production of functionally active DR4 / hCII in HEK293 cellsThe results shown in Figure 6 clearly demonstrate the ability of DR4 / nCII and DR4 / galCII to induce the release of the anti-inflammatory cytokine IL-10 in PBMCs of RA patients, with levels even slightly higher compared to the positive control incubated for 8 days under regular TR1 inducing conditions. There is no evidence for concomitant pathway activation leading to increased production of pro-inflammatory cytokines (e.g. TNF-a, IL-2, IL-17a, IL-17f or IFN-γ).

[0284] Figure 4

[0285] It has been investigated whether sequences not directly necessary for the MHC II / CII complex can be omitted from the construct, including the contribution of the polyhistidine tag (His-tag), the biotinylation site, the TEV cleavage site, the thrombin cleavage site and the Strep-tag for T cell activation properties of the recombinant complex. Generally, in a standard hybridoma activation assay, the DR4 / CII peptide complex as well as an anti-CD3 antibody (positive control) are coated to the plastic surface of microtiter wells. In an initial experiment, we used the internal TEV cleavage site of the DR4 / nCII peptide complex to investigate the impact of proteolytic cleavage of the His-tag on T hybridoma cell (3H8) activation measured by IL-2 secretion compared to the uncleaved DR4 / hCII peptide complex coated to the microtiter wells. The efficacy of proteolytic cleavage was controlled by Western blot analysis. In addition, the efficacy of equivalent coating of the microtiter wells using equimolar solutions of cleaved and uncleaved complex was confirmed by ELISA using DR4 specific antibodies and peroxidase coupled secondary antibodies Figure 4 ). This also confirmed that the complex did not dissociate and existed as a heterodimer. Our data show that although the functional domain of the DR4 / nCII complex recognized by the TCR of the T hybridoma cell was coated with similar efficacy to the plastic surface, the cleaved construct was significantly reduced in activating the T hybridoma cell Example 4: Detection of CII peptide-specific T cells in humans ). The possibility that the zip cleavage leads to dissociation of the complex is very small. The zip is mainly required for the complex to be formed during biosynthesis, whereas the formed MHC II-peptide-complex is rather stable on its own at least in vitro due to the stabilizing effect of the peptide bound in the binding groove formed by the variable regions of both chains.

[0286] We conclude that the His-tag in the uncleaved DR4 / nCII complex is essential for the complex to be properly oriented on the surface in a polymerized arrangement, thereby exposing the peptide-binding groove to T cells by preferentially contacting charged contact regions on the plastic surface. To confirm this conclusion, the resulting DR4 / hCIIΔHis complex lacked only the His-tag (6xHis) at the C-terminus of the MHC class II β chain, but was otherwise identical to the DR4 / hCII complex, containing a JUN / FOS heterodimerization domain (compared to...). Figure 5 As an additional control involving the electrostatic interaction of the positively charged functional imidazole group of histidine, further mutant recombinant variants of the DR4 / hCII complex were prepared, in which the His-tag was replaced by the negatively charged amino acid residue triplet Asp-Glu-Asp(DED) (DR4 / hCII_DED). Furthermore, we exchanged non-physiological plastic materials with charged extracellular matrix (ECM) components (chondroitin sulfate, heparin sulfate, acetylated hyaluronic acid) from cell surfaces, extracellular fluids (such as synovial fluid or lymph), and tissues (such as articular cartilage or synovium). For this purpose, we first coated the microtiter wells with a highly concentrated solution of chondroitin sulfate, heparin sulfate, and hyaluronic acid (10 mg / ml). The coated surfaces were thoroughly washed, and the IL-2 concentration in the supernatant was used as a reading for in vitro stimulation experiments of 3H8 hybridoma cells by adding the DR4 / CII peptide complex to the liquid phase. For controls, parallel experiments were performed in microtiter plates with sealed surfaces in the absence of ECM components. Figure 5 The results shown in A demonstrate that under these conditions, only the His-tagged complex induced a strong IL-2 response, and its ability to activate T cells appears to depend critically on chondroitin sulfate coated onto the surface of the microburette wells, while the effect of hyaluronic acid (HA) remains less pronounced. Figure 5 B), and heparin sulfate was almost undetectable. Figure 6 C). The soluble DR4 / hCII_DED control complex does not induce an IL-2 reaction when present on the surface of a chondroitin sulfate-coated microtiter well. Figure 5 A). However, the observed effect of the His-tagged complex cannot be simply explained by the electrostatic interaction of the polysulfated anionic glycosaminoglycan via the positively charged imidazole group of the polyhistidine tag, since heparan sulfate also contains highly negatively charged sulfate groups but does not appear to significantly promote the IL-2 response in 3H8 hybridoma cells stimulated by the solute His-tagged DR4 / hCII complex. Therefore, the results indicate that the specific interaction between the polyhistidine tag and the chondroitin sulfate matrix increases the IL-2 response in 3H8 hybridoma cells stimulated by the dissolved DR4 / hCII complex.

[0287] The initial results obtained with the DR4 / nCII complex cleaved with Tev were confirmed in parallel T hybridoma cell stimulation experiments in which different DR4 / hCII constructs were coated directly onto plastic surfaces at three different concentrations (0.01 mg / ml, 0.1 mg / ml and 1 mg / ml). The ability of the DR4 / hCII ΔHis complex as well as the mutated DR4 / hCII_DED complex to induce an IL-2 response was significantly reduced when coating was performed using 0.1 mg / ml and 1 mg / ml. However, at a comparable level to the unmodified complex (DR4 / hCII) at 10-fold lower coating concentration, a response was observed when coating was performed using 1 mg / ml (Fig. 2). Example 5: In vitro stimulation / differentiation of T cells from peripheral blood of HLA-DRB1*0401 positive RA patients

[0288] However, the experiments also showed that all constructs have a functional peptide in the DR4 binding groove as a prerequisite for TCR activation of 3H8 hybridoma cells. Thus, our studies provide clear evidence that the His-tag in the DR4 / CII complex improves the activity of the complex. Without being bound by theory, the His-tag appears to provide an improved spatial orientation of the peptide binding groove for TCR recognition by influencing the interaction with the ECM component chondroitin sulfate. Furthermore, Figure 7 Subsequent studies shown in Figure 3 demonstrate that the DR4 / CII complex containing the His-tag can enhance its ability to stimulate IL-10 production by T hybridoma cells in the presence of chondroitin sulfate or hyaluronic acid in the solid phase with a microtiter well surface blocked with plastic.

[0289] The in vitro data support a key functional role of the His-tag in the DR4 / CII complex as it has immunomodulatory pharmacological effects. In addition, in vivo studies were performed using the T cell dependent CII-induced hypersensitivity model in Aq-expressing QB mice. T cell dependent inflammatory swelling was triggered in CII-primed mice on day 8 post-immunization by intradermal injection of CII to one ear, with control triggered by vehicle applied to the contralateral ear. Prior to induction of the DTH response, mice received a 24 hour subcutaneous infusion of a pump containing the His-tag containing Aq / galCII complex, the Aq / galCII ΔHis complex without the His-tag, or the control Aq / CLIP complex containing a control peptide [MHC class II associated invariant chain: CLIP] linked in its binding groove on day 4 post-immunization. Example 6: His-tag in DR4 / CII peptide complexes: contribution to pharmacological effect ​The results shown in Figure 6 provide clear evidence for the functional impact of the His-tag on the therapeutic reduction of T cell dependent ear swelling induced by experimental CII specific DTH responses. Thus, our studies consistently demonstrate an improved function of the MHC II / CII peptide complex containing a polyhistidine sequence for the immunomodulatory therapeutic effect on T cells, most likely mediated through its impact on ECM components interacting in the context of targeted structures on cell surfaces, tissue components and body fluids available in large amounts in vivo.

[0290] Figure 8

[0291] Post-translational modification of the CII sequence of the peptide in the binding groove of the recombinant DR4 complex involves several consecutive steps of different enzymes. These collagen-specific post-translational modifications preferentially affect the lysine residues at positions 264 and 270. The initial step is lysyl hydroxylation mediated by lysyl hydroxylases, followed by galactosylation of the hydroxylated lysine mediated by galactosyltransferases. In addition, a single glucose residue can be added to the galactosylated hydroxylysine. All these steps occur during biosynthesis in cells with collagen post-translational machinery, such as in HEK cells, resulting in a heterologous recombinant product comparable to the native ECM protein in cartilage in vivo. In humans, this mixture can be advantageous to increase the range of potential T cells that can be recruited from the entire repertoire for conversion into regulatory cells to produce anti-inflammatory mediators such as IL-10 to suppress immune-mediated joint disease. Studies on in vitro activation of IL-2 and IL-10 responses in peripheral blood T cells of RA patients in response to recombinant DR4 / CII complexes containing galactosylated (DR4 / galCII) or non-modified CII (DR4 / CII) provide experimental support in this direction. However, mass spectrometric analysis of several batches of CR4 / hCII produced in HEK cells showed that a considerable amount of the recombinant protein exhibited a high content of disaccharide (Glc-Gal-Hyl) at both lysine residues Figure 8 , which can be detrimental for TCR recognition as the peptide binding groove is covered by bulky carbohydrate structures interfering with TCR recognition.

[0292] Collagen-specific post-translational galactosylation of the lysine residue in the CII peptide sequence of the recombinant DR4 / hCII construct, especially at residue 264, is important for the recognition by TCR and the resulting pharmacological effects. Since the lysine residue at position 270 is located at the rim of the binding groove of the DR4 molecule, it is generally assumed that its carbohydrate modification is not involved in TCR recognition. To reduce heterogeneity and the potential risk of a negative interference of the carbohydrate linkage to the hydroxylated lysine residue at position 270 (K270) with the TCR recognition of the CII peptide in the binding groove of DR4, K270 can be mutated to an arginine residue (R). This mutation has previously been shown not to affect binding to the TCR of antigen-specific T cell hybridomas.

[0293] More importantly, the prevention of the final transfer of the glucose residue to the galactosylated hydroxylysine at position 264. This carbohydrate moiety can have a negative impact on TCR recognition, since the bulky and flexible disaccharide (Glc-Gal) can interfere with TCR binding. In vitro stimulation of T cells from the peripheral blood of RA patients showed that both unmodified (nCII) as well as mono-galactosylated peptides (galCII) can be recognized. The reaction catalyzing the transfer of the glucose residue to the galactosylated hydroxylysine is catalyzed by galactosyl-hydroxylysylglucosyltransferase (synonym: procollagen lysyl hydroxylase 3 (LH3)). LH3 is a multifunctional enzyme, which is also able to catalyze the initial step of the above lysine modification, i.e. the hydroxylation to produce hydroxylysine (Hyl) and the galactosyl transfer to produce galactosyl-hydroxylysine (Gal-Hyl) (A). However, its non-redundant activity is the final transfer of the glucose to the galactosyl-hydroxylysine. Figure 1 A). However, its non-redundant activity is the final transfer of the glucose to the galactosyl-hydroxylysine.

[0294] Therefore, we genetically engineered Expi293F cells to knock down the LH3 enzyme. The HEK cell line used for the production of the DR4 / hCII complex is expected to be advantageous to increase the efficacy of the recombinantly produced DR4 / hCII complex, which is selectively made defective in the final galactosyl-hydroxylysine to glucose transfer into the CII peptide. This can be achieved by generating HEK293 LH3 knock-out cells, e.g. by introducing a mutation disrupting the gene into the plod3 gene encoding the lysyl hydroxylase 3 gene using the CRISPR / CAS gene editing method.

[0295] In the first step, we generated Expi293F cells with plod3 knockdown by lentiviral transfection of specific shRNAs to investigate the potential of this strategy to obtain less heterogeneous products with increased specific T cell activation activity by improving the recombinant expression system. For transduction of Expi293 cells, 200,000 cells / well were seeded in 12-well plates followed by 3 hours incubation at 37°C, 8% CO2 and 120 rpm shaking of the plates. 200 uL lentiviral particles (custom lentiviral particles from Sigma) were mixed with 10 uL PEIpro transfection reagent (Polyplus) and added to the cells and incubated for another 4 hours at 37°C, 120 rpm and 8% CO2 shaking, 1 mL fresh medium was added and incubation was continued for 3 days before transduction efficiency was analyzed.

[0296] Three days after lentiviral transduction with shRNAs targeting Plod3, the cells were split into two. To one, puromycin was added to a final concentration of 2 ug / mL to kill non-transduced cells, the other was analyzed by flow cytometry to check transduction efficiency. Cells were under antibiotic selection pressure until non-transduced cells died and transduced cells divided for about 18 days to a survival rate of more than 90%. These stable transduced pools were scaled up to 500 mL and transfected with DR4 / hCII as described above. After purification, glycan analysis by mass spectrometry was performed to investigate the reduced glucosylation of galactosyl-hydroxylysyl residues in plod3 knockdown Expi293F cells and Expi293F control cells. Two lysines within the collagen type II epitope (K264 and K270) were analyzed. A clear reduction of the glucose-galactosyl-hydroxylysyl residue (DiHex) could be seen in Expi293 KO cells Figure 9 Figure 9 Figure 9 Figure 9 Figure 10 Figure 11 Figure 12 Example 7: Obstacles for recombinant production of DR4 / qalCII complexes in HEK cells Figure 13 Figure 14 Figure 14 C).

[0297] At the same time, the stable transduced cells were diluted and seeded on 96-well plates for mini-pool generation. During the seeding process, the cells were grown under selection pressure and 40 mini-pools were isolated. Cells were expanded and PLOD3 expression in cell lysates was determined by Western blot to verify efficient knockdown of Plod3. Lysates from 1 x 105 6 Expi293F mini-pools from lentiviral transduction were loaded on SDS-PAGE. Western blot analysis for successful knockdown was performed using an anti-PLOD3 antibody (Thermo Fisher PAS-48435) and a secondary rabbit HRP antibody. The theoretical molecular weight of PLOD3 is 84 kDa. Clones #4, #18 and #20 showed efficient Plod3 knockdown and were therefore used for further expansion and experiments. Clone 18 was lost due to decreased viability, clones 4 and 20 were selected for further production, glycan analysis was also performed.

[0298] SEQUENCE LISTING:

[0299] SEQ ID NO: 1 AGFKGEQGPKG

[0300] SEQ ID NO: 2 AGFKGEQGPX1G

[0301] SEQ ID NO: 3 AGFKGEX2GPKG

[0302] SEQ ID NO: 4 AGFKGX3QGPKG

[0303] SEQ ID NO: 5 AGFKX4EQGPKG

[0304] SEQ ID NO: 6 AGFKGEX2GPX1G

[0305] SEQ ID NO: 7 AGFKGX3QGPX1G

[0306] SEQ ID NO: 8 AGFKX4EQGPX1G

[0307] SEQ ID NO: 9 AGFKGEQGPRG

[0308] SEQ ID NO: 10 AGFKGEQGPKGEP

[0309] SEQ ID NO: 11 AGFKGEQGPX1GEP

[0310] SEQ ID NO: 12 AGFKGEQGPRGEP

[0311] SEQ ID NO: 13 GIAGFKGEQGPKGEP

[0312] SEQ ID NO: 14 GIAGFKGEQGPX1GEP

[0313] SEQ ID NO: 15 GIAGFKGEQGPRGEP

[0314] SEQ ID NO: 16 DR4 Construct α-chain

[0315] SEQ ID NO: 17 DR4 Construct β-chain with hCII259-273 peptide

[0316] SEQ ID NO: 18 Minimal DR4 Construct α-chain

[0317] SEQ ID NO: 19 Minimal DR4 Construct β-chain with hCII259-273 peptide

[0318] SEQ ID NO: 20 DR4 construct β-chain with hCLIPmut

[0319] SEQ ID NO: 21 Aq construct α-chain

[0320] SEQ ID NO: 22 Aq construct β-chain with rat CII 259-273 peptide

[0321] SEQ ID NO: 23 Aq construct β-chain with rat CII 259-273 peptide without His-tag

[0322] SEQ ID NO: 24 Aq construct β-chain with mCLIP peptide

[0323] SEQ ID NO: 25 Aq construct β-chain with mCLIP peptide without His-tag

[0324] SEQ ID NO: 26 cFos domain

[0325] SEQ ID NO: 27 cJune domain

[0326] SEQ ID NO: 28 modified human CLIP-peptide

[0327] SEQ ID NO: 29 rat CII-peptide 259-273

[0328] SEQ ID NO: 30 Streptavidin tag

[0329] SEQ ID NO: 31 EKRIWFPYRRF

[0330] SEQ ID NO: 32 YKTNFRRYYRF

[0331] SEQ ID NO: 33 VLIRHFRKRYY

[0332] SEQ ID NO: 34 SAWSHPQFEKGIAGFKGEQGPKGEPSGGGS SEQUENCE LIST <110> Fraunhofer-Gesellschaft <120> Generation of MHC II / CII peptide complexes <130> TPE01753A <150> EP19191094.2 <151> 2019-08-09 <160> 34 <170> BiSSAP 1.3.6 <210> 1 <211> 11 <212> PRT <213> Homo sapiens <220> <223> CII peptide 261‑271 <400> 1 Ala Gly Phe Lys Gly Glu Gln Gly Pro Lys Gly 1 5 10 <210> 2 <211> 11 <212> PRT <213> Homo sapiens <220> <223> CII peptide 261-271, Lys = Xaa <220> <221> UNSURE <222> 10 <223> Xaa = any protein amino acid except Lys; preferred Xaa = Arg, Ala, Gly, or Gln <400> 2 Ala Gly Phe Lys Gly Glu Gln Gly Pro Xaa Gly 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CII peptide 261‑271; Gln = Xaa <220> <221> UNSURE <222> 7 <223> Xaa = any protein amino acid except Gln; preferably Xaa = Ala, Arg, His, or Gly <400> 3 Ala Gly Phe Lys Gly Glu Xaa Gly Pro Lys Gly 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CII peptide 261-271; Glu = Xaa <220> <221> UNSURE <222> 6 <223> Xaa = any protein amino acid except Glu, preferably Xaa = Ala, Asp, Gln, or Gly <400> 4 Ala Gly Phe Lys Gly Xaa Gln Gly Pro Lys Gly 1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CII peptide 261-271; Gly = Xaa <220> <221> UNSURE <222> 5 <223> Xaa = any protein amino acid except Gly; preferred Xaa = Ala, Ser, Val, or Leu <400> 5 Ala Gly Phe Lys Xaa Glu Gln Gly Pro Lys Gly 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CII peptide 261‑271; Gln = Xaa and Lys = Xaa <220> <221> UNSURE <222> 7 <223> Xaa = any protein amino acid except Gin; preferably Xaa = Ala, Arg, His or Gly <220> <221> UNSURE <222> 10 <223> Xaa = any protein amino acid except Lys; preferably Xaa = Arg, Ala, Gly or Gin <400> 6 Ala Gly Phe Lys Gly Glu Xaa Gly Pro Xaa Gly 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 261-271; Glu = Xaa and Lys = Xaa <220> <221> UNSURE <222> 6 <223> Xaa = any protein amino acid except Gin; preferably Xaa = Ala, Asp, Gin or Gly <220> <221> UNSURE <222> 10 <223> Xaa = any protein amino acid except Lys, preferably Xaa = Arg, Ala, Gly or Gin <400> 7 Ala Gly Phe Lys Gly Xaa Gin Gly Pro Xaa Gly 1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 261-271; Gly = Xaa and Lys = Xaa <220> <221> UNSURE <222> 5 <223> Xaa = any protein amino acid except Gly; preferably Xaa = Ala, Ser, Val or Leu <400> 8 Ala Gly Phe Lys Xaa Glu Gln Gly Pro Xaa Gly 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 261-271; Lys = Arg <400> 9 Ala Gly Phe Lys Gly Glu Gln Gly Pro Arg Gly 1 5 10 <210> 10 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CII peptide 261-273 <400> 10 Ala Gly Phe Lys Gly Glu Gln Gly Pro Lys Gly Glu Pro 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 261-273; Lys = Xaa <220> <221> UNSURE <222> 10 <223> Xaa = any protein amino acid except Lys; preferably Lys = Arg, Ala, Gly or Gin Arg, Ala, Gly or Gin <400> 11 Ala Gly Phe Lys Gly Glu Gin Gly Pro Xaa Gly Glu Pro 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 261-273; Lys = Arg <400> 12 Ala Gly Phe Lys Gly Glu Gin Gly Pro Arg Gly Glu Pro 1 5 10 <210> 13 <211> 15 <212> PRT <213> Homo sapiens <220> <223> CII peptide 259-273 <400> 13 Gly lie Ala Gly Phe Lys Gly Glu Gin Gly Pro Lys Gly Glu Pro 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 259-273; Lys = Xaa <220> <221> UNSURE <222> 12 <223> Xaa = any protein amino acid except Lys; preferably Xaa = Arg, Ala, Gly or Gin <400> 14 Gly lie Ala Gly Phe Lys Gly Glu Gin Gly Pro Xaa Gly Glu Pro 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CII peptide 259-273; Lys = Arg <400> 15 Gly lie Ala Gly Phe Lys Gly Glu Gin Gly Pro Arg Gly Glu Pro 1 5 10 15 <210> 16 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> DR4 construct alpha chain <400> 16 Met Lys Leu Cys lie Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly lie Lys Glu Glu His Val lie lie Gin Ala Glu Phe Tyr Leu 20 25 30 Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu 35 40 45 lie Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu 50 55 60 Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn 65 70 75 80 Ile Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn 85 90 95 Tyr Thr Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn 100 105 110 Ser Pro Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp 115 120 125 Lys Phe Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys 130 135 140 Pro Val Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp 145 150 155 160 His Leu Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu 165 170 175 Asp Val Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu 180 185 190 Leu Lys His Trp Glu Phe Asp Ala Ser Gly Gly Gly Glu Asn Leu Tyr 195 200 205 Phe Gln Gly Gly Gly Gly Ser Leu Thr Asp Thr Leu Gln Ala Glu Thr 210 215 220 Asp Gln Leu Glu Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala Asn 225 230 235 240 Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala His Gly 245 250 255 Gly Gly Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu 260 265 270 Trp His Glu 275 <210> 17 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> DR4 Construct Beta Chain has hCII 259-273 Peptide <400> 17 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Ile Ala Gly 20 25 30 Phe Lys Gly Glu Gln Gly Pro Lys Gly Glu Pro Ser Gly Gly Gly Ser 35 40 45 Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Gly Asp Thr Arg Pro 50 55 60 Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly Thr 65 70 75 80 Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu Tyr 85 90 95 Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu 100 105 110 Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu Glu 115 120 125 Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val 130 135 140 Gly Glu Ser Phe Thr Val Gln Arg Arg Val Tyr Pro Glu Val Thr Val 145 150 155 160 Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu Val Cys 165 170 175 Ser Val Asn Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg 180 185 190 Asn Gly Gln Glu Glu Lys Thr Gly Val Val Ser Thr Gly Leu Ile Gln 195 200 205 Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro 210 215 220 Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Leu Thr 225 230 235 240 Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Gly Gly Gly Glu Asn 245 250 255 Leu Tyr Phe Gin Gly Gly Gly Gly Ser Arg He Ala Arg Leu Glu Glu 260 265 270 Lys Val Lys Thr Leu Lys Ala Gin Asn Ser Glu Leu Ala Ser Thr Ala 275 280 285 Asn Met Leu Arg Glu Gin Val Ala Gin Leu Lys Gin Lys Val Met Asn 290 295 300 His His His His His His His 305 310 <210> 18 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Minimal DR4 Construct Alpha Chain <400> 18 Met Lys Leu Cys He Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu 20 25 30 Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu 35 40 45 He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu 50 55 60 Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn 65 70 75 80 Ile Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn 85 90 95 Tyr Thr Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn 100 105 110 Ser Pro Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp 115 120 125 Lys Phe Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys 130 135 140 Pro Val Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp 145 150 155 160 His Leu Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu 165 170 175 Asp Val Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu 180 185 190 Leu Lys His Trp Glu Phe Asp Ala Ser Gly Gly Gly Gly Gly Gly Ser 195 200 205 Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Glu Lys 210 215 220 Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys 225 230 235 240 Leu Glu Phe Ile Leu Ala Ala His 245 <210> 19 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Minimal DR4 Construct Beta Chain with hCII 259-273 Peptide <400> 19 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Gly Ile Ala Gly Phe Lys Gly Glu Gln Gly Pro Lys Gly Glu 20 25 30 Pro Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Thr Arg Pro 35 40 45 Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly Thr 50 55 60 Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu Tyr 65 70 75 80 Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu 85 90 95 Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu Glu 100 105 110 Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val 115 120 125 Gly Glu Ser Phe Thr Val Gln Arg Arg Val Tyr Pro Glu Val Thr Val 130 135 140 Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu Val Cys 145 150 155 160 Ser Val Asn Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg 165 170 175 Asn Gly Gln Glu Glu Lys Thr Gly Val Val Ser Thr Gly Leu Ile Gln 180 185 190 Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro 195 200 205 Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Leu Thr 210 215 220 Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Gly Gly Gly Gly Gly 225 230 235 240 Gly Ser Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala 245 250 255 Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val 260 265 270 Ala Gln Leu Lys Gln Lys Val Met Asn His His His His His His His His 275 280 285 <210> 20 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> DR4 construct beta chain has hCLIPmut <400> 20 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Pro Val Ser Lys 20 25 30 Ala Arg Met Ala Thr Gly Ala Leu Ala Gln Ala Ser Gly Gly Gly Ser 35 40 45 Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Gly Asp Thr Arg Pro 50 55 60 Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly Thr 65 70 75 80 Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu Tyr 85 90 95 Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu 100 105 110 Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu Glu 115 120 125 Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val 130 135 140 Gly Glu Ser Phe Thr Val Gin Arg Arg Val Tyr Pro Glu Val Thr Val 145 150 155 160 Tyr Pro Ala Lys Thr Gin Pro Leu Gin His His Asn Leu Leu Val Cys 165 170 175 Ser Val Asn Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Arg 180 185 190 Asn Gly Gin Glu Glu Lys Thr Gly Val Val Ser Thr Gly Leu Ile Gin 195 200 205 Asn Gly Asp Trp Thr Phe Gin Thr Leu Val Met Leu Glu Thr Val Pro 210 215 220 Arg Ser Gly Glu Val Tyr Thr Cys Gin Val Glu His Pro Ser Leu Thr 225 230 235 240 Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Gly Gly Gly Glu Asn 245 250 255 Leu Tyr Phe Gin Gly Gly Gly Gly Ser Arg Ile Ala Arg Leu Glu Glu 260 265 270 Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala 275 280 285 Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met Asn 290 295 300 His His His His His His His 305 310 <210> 21 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> Aq Construct alpha chain <400> 21 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Glu Asp Asp Ile Glu Ala Asp His Val Gly Phe Tyr Gly Ile 20 25 30 Val Val Tyr Gln Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe 35 40 45 Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Val 50 55 60 Trp Met Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly 65 70 75 80 Gly Leu Gln Asn Ile Ala Thr Gly Lys His Asn Leu Gly Gly Trp Thr 85 90 95 Lys Arg Ser Asn Phe Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr 100 105 110 Val Phe Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu Ile 115 120 125 Cys Phe Val Asp Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu 130 135 140 Arg Asn Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu 145 150 155 160 Val Asn Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe Ile 165 170 175 Pro Ser Asp Asp Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu 180 185 190 Asp Glu Pro Val Leu Lys His Trp Glu Pro Glu Ile Pro Ala Thr Met 195 200 205 Ser Glu Leu Thr Glu Thr Val Ser Gly Gly Gly Glu Asn Leu Tyr Phe 210 215 220 Gln Gly Gly Gly Gly Ser Leu Thr Asp Thr Leu Gin Ala Glu Thr Asp 225 230 235 240 Gln Leu Glu Asp Glu Lys Ser Ala Leu Gin Thr Glu Ile Ala Asn Leu 245 250 255 Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala His Gly Gly 260 265 270 Gly Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp 275 280 285 His Glu 290 <210> 22 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Aq Constructed beta Chain has Rat CII 259-273 Peptide <400> 22 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Ile Ala Gly 20 25 30 Phe Lys Gly Glu Gln Gly Pro Lys Gly Glu Thr Ser Gly Gly Gly Ser 35 40 45 Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Glu Arg His Phe Val 50 55 60 Ala Gln Leu Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gln Arg Ile 65 70 75 80 Arg Ser Val Asn Arg Tyr Ile Tyr Asn Arg Glu Glu Trp Val Arg Phe 85 90 95 Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro 100 105 110 Asp Ala Glu Tyr Trp Asn Ser Gln Pro Glu Ile Leu Glu Arg Thr Arg 115 120 125 Ala Glu Val Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr 130 135 140 His Thr Ser Leu Arg Arg Leu Glu Gln Pro Asn Val Ala Ile Ser Leu 145 150 155 160 Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser Val 165 170 175 Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe Arg Asn Gly 180 185 190 Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gln Leu Ile Arg Asn Gly 195 200 205 Asp Trp Thr Phe Gln Val Leu Val Met Leu Glu Met Thr Pro His Cys 210 215 220 Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser Pro 225 230 235 240 Ile Thr Val Glu Trp Arg Ala Gln Ser Glu Ser Ala Arg Ser Lys Ser 245 250 255 Gly Gly Gly Glu Asn Leu Tyr Phe Gin Gly Gly Gly Gly Ser Arg He 260 265 270 Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gin Asn Ser Glu 275 280 285 Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gin Val Ala Gin Leu Lys 290 295 300 Gln Lys Val Met Asn His His His His His His His 305 310 315 <210> 23 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Aq Constructed beta chain has rat CII 259-273 peptide without His-tag <400> 23 Met Lys Leu Cys He Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Ser Ala Trp Ser His Pro Gin Phe Glu Lys Gly He Ala Gly 20 25 30 Phe Lys Gly Glu Gin Gly Pro Lys Gly Glu Thr Ser Gly Gly Gly Ser 35 40 45 Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Glu Arg His Phe Val 50 55 60 Ala Gin Leu Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gin Arg Ile 65 70 75 80 Arg Ser Val Asn Arg Tyr Ile Tyr Asn Arg Glu Glu Trp Val Arg Phe 85 90 95 Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro 100 105 110 Asp Ala Glu Tyr Trp Asn Ser Gin Pro Glu Ile Leu Glu Arg Thr Arg 115 120 125 Ala Glu Val Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr 130 135 140 His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Ala Ile Ser Leu 145 150 155 160 Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser Val 165 170 175 Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe Arg Asn Gly 180 185 190 Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu Ile Arg Asn Gly 195 200 205 Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr Pro His Gin 210 215 220 Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser Pro 225 230 235 240 Ile Thr Val Glu Trp Arg Ala Gln Ser Glu Ser Ala Arg Ser Lys Ser 245 250 255 Gly Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly Gly Gly Ser Arg Ile 260 265 270 Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu 275 280 285 Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys 290 295 300 Gln Lys Val Met Asn His 305 310 <210> 24 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Aq Constructed beta Chain with mCLIP Peptide <400> 24 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Pro Val Ser Gln 20 25 30 Ala Arg Met Ala Thr Pro Leu Leu Met Arg Pro Ser Gly Gly Gly Ser 35 40 45 Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Glu Arg His Phe Val 50 55 60 Ala Gln Leu Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gln Arg Ile 65 70 75 80 Arg Ser Val Asn Arg Tyr Ile Tyr Asn Arg Glu Glu Trp Val Arg Phe 85 90 95 Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro 100 105 110 Asp Ala Glu Tyr Trp Asn Ser Gln Pro Glu Ile Leu Glu Arg Thr Arg 115 120 125 Ala Glu Val Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr 130 135 140 His Thr Ser Leu Arg Arg Leu Glu Gln Pro Asn Val Ala Ile Ser Leu 145 150 155 160 Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser Val 165 170 175 Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe Arg Asn Gly 180 185 190 Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gln Leu Ile Arg Asn Gly 195 200 205 Asp Trp Thr Phe Gln Val Leu Val Met Leu Glu Met Thr Pro His Gln 210 215 220 Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser Pro 225 230 235 240 Ile Thr Val Glu Trp Arg Ala Gln Ser Glu Ser Ala Arg Ser Lys Ser 245 250 255 Gly Gly Gly Glu Asn Leu Tyr Phe Gln Gly Gly Gly Gly Ser Arg Ile 260 265 270 Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu 275 280 285 Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys 290 295 300 Gln Lys Val Met Asn His His His His His His His 305 310 315 <210> 25 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> The β-chain of the Aq construct has the mCLIP peptide without His-tag <400> 25 Met Lys Leu Cys Ile Leu Leu Ala Val Val Ala Phe Val Gly Leu Ser 1 5 10 15 Leu Gly Ser Ala Trp Ser His Pro Gin Phe Glu Lys Pro Val Ser Gin 20 25 30 Ala Arg Met Ala Thr Pro Leu Leu Met Arg Pro Ser Gly Gly Gly Ser 35 40 45 Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser Glu Arg His Phe Val 50 55 60 Ala Gin Leu Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gin Arg Ile 65 70 75 80 Arg Ser Val Asn Arg Tyr Ile Tyr Asn Arg Glu Glu Trp Val Arg Phe 85 90 95 Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro 100 105 110 Asp Ala Glu Tyr Trp Asn Ser Gin Pro Glu Ile Leu Glu Arg Thr Arg 115 120 125 Ala Glu Val Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr 130 135 140 His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Ala Ile Ser Leu 145 150 155 160 Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser Val 165 170 175 Thr Asp Phe Tyr Pro Ala Lys lie Lys Val Arg Trp Phe Arg Asn Gly 180 185 190 Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu lie Arg Asn Gly 195 200 205 Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr Pro His Gin 210 215 220 Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser Pro 225 230 235 240 lie Thr Val Glu Trp Arg Ala Gin Ser Glu Ser Ala Arg Ser Lys Ser 245 250 255 Gly Gly Gly Glu Asn Leu Tyr Phe Gin Gly Gly Gly Gly Ser Arg lie 260 265 270 Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gin Asn Ser Glu 275 280 285 Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gin Val Ala Gin Leu Lys 290 295 300 Gln Lys Val Met Asn His 305 310 <210> 26 <211> 40 <212> PRT <213> Homo sapiens <220> <223> cFos domain <400> 26 Leu Thr Asp Thr Leu Gin Ala Glu Thr Asp Gin Leu Glu Asp Glu Lys 1 5 10 15 Ser Ala Leu Gin Thr Glu lie Ala Asn Leu Leu Lys Glu Lys Glu Lys 20 25 30 Leu Glu Phe lie Leu Ala Ala His 35 40 <210> 27 <211> 40 <212> PRT <213> Homo sapiens <220> <223> cJun domain <400> 27 Arg lie Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gin Asn 1 5 10 15 Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gin Val Ala Gin 20 25 30 Leu Lys Gin Lys Val Met Asn His 35 40 <210> 28 <211> 15 <212> PRT <213> Homo sapiens <220> <223> Mutated human CLIP-peptide <400> 28 Pro Val Ser Lys Ala Arg Met Ala Thr Gly Ala Leu Ala Gin Ala 1 5 10 15 <210> 29 <211> 15 <212> PRT <213> Homo sapiens <220> <223> Rat CII-peptide 259-273 <400> 29 Gly Ile Ala Gly Phe Lys Gly Glu Gin Gly Pro Lys Gly Glu Thr 1 5 10 15 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Streptavidin-tag <400> 30 Ser Ala Trp Ser His Pro Gin Phe Glu Lys 1 5 10 <210> 31 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Chondrocalcin-binding peptide <400> 31 Glu Lys Arg lie Trp Phe Pro Tyr Arg Arg Phe 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Chondrocalcin-binding peptide <400> 32 Tyr Lys Thr Asn Phe Arg Arg Tyr Tyr Arg Phe 1 5 10 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Chondrocalcin-binding peptide <400> 33 Val Leu Ile Arg His Phe Arg Lys Arg Tyr Tyr 1 5 10 <210> 34 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> hCII259-273 with N-terminal and C-terminal sequences <400> 34 Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Ile Ala Gly Phe Lys 1 5 10 15 Gly Glu Gln Gly Pro Lys Gly Glu Pro Ser Gly Gly Gly Ser 20 25 30

Claims

1. A composition comprising a recombinant MHC II / CII peptide complex, said complex comprising: (a) Extracellular region of MHC class II α chain containing at least one α1 domain; (b) The extracellular region of an MHC class II β chain containing at least one β1 domain; and (c) CII peptides, which are fused to the N-terminus of the MHC class II α chain or the MHC class II β chain via a linker peptide; The CII peptide has the following amino acid sequence: AGFKGEQGPXG, and the MHC II / CII peptide complex comprises a post-translational modified CII peptide. The first lysine residue of the CII peptide is an O-glycosylated Hyl, and Wherein at least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303, and / or wherein the MHC II / CII peptide complex does not contain an enzyme cleavage site in the amino acid sequence between the CII peptide and the extracellular region of the MHC class II α or β chain.

2. The composition according to claim 1, wherein the CII peptide has the following amino acid sequence: AGFKGEQGPKG.

3. The composition according to claim 1, wherein, The CII peptide is fused to the N-terminus of the MHC class II β chain via a linker peptide.

4. The composition according to any one of claims 1-3, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) The CII peptide is fused to the N-terminus of the β1 domain via a linker peptide; (c) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from an HLA-DR allele selected from the group consisting of: DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303; (d) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated.

5. The composition according to any one of claims 1-3, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) The CII peptide is fused to the N-terminus of the β1 domain via a linker peptide; (c) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from an HLA-DR allele selected from the group consisting of: DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303; (d) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated to R.

6. The composition according to any one of claims 1-3, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) The CII peptide is fused to the N-terminus of the β1 domain via a linker peptide; (c) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from DRB1*0401; (d) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated.

7. The composition according to any one of claims 1-3, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) The CII peptide is fused to the N-terminus of the β1 domain via a linker peptide; (c) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from DRB1*0401; (d) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated to R.

8. The composition according to any one of claims 4-7, wherein (a) Extracellular region of MHC class II α chain containing at least one α1 domain; (b) The extracellular region of an MHC class II β chain containing at least one β1 domain; and (c) A CII peptide fused to the N-terminus of the MHC class II α chain or the N-terminus of the MHC class II β chain via a linker peptide. The complex containing (a), (b), and (c) is a single fusion polypeptide.

9. The composition according to any one of claims 4-7, comprising: (a) A first polypeptide containing an extracellular region of an MHC class II α chain comprising at least one α1 domain; (b) A second polypeptide containing an extracellular region of an MHC class II β chain comprising at least one β1 domain; and (c) CII peptides, which are fused to the N-terminus of the MHC class II α chain or the MHC class II β chain via a linker peptide.

10. The composition of claim 9, wherein the MHC class II α chain is fused at its C-terminus to a first functional domain of a leucine zipper heterodimerization motif, and the MHC class II β chain is fused at its C-terminus to a second complementary functional domain of a leucine zipper heterodimerization motif.

11. The composition of claim 10, wherein the MHC class II α chain is fused at its C-terminus to a first functional domain of a leucine zipper heterodimerization motif, and the MHC class II β chain is fused at its C-terminus to a second complementary functional domain of a leucine zipper heterodimerization motif; wherein the first functional domain and the second complementary functional domain are: (a) Acidic and basic leucine zipper heterodimerization domains; and / or (b)jun-fos leucine zipper motif.

12. A method for generating an MHC II / CII peptide complex comprising a post-translational O-glycosylated Hyl-modified CII peptide, comprising: (a) Transfection of mammalian cells with a polynucleotide comprising the following complex: (i) A polynucleotide encoding an extracellular region of an MHC IIα chain containing at least one α1 domain; (ii) a polynucleotide encoding an extracellular region of an MHC IIβ chain containing at least one β1 domain; and (iii) A polynucleotide encoding a CII peptide fused to the N-terminus of an MHC class II α chain or an MHC class II β chain via a linker peptide, wherein the CII peptide has the following amino acid sequence: AGFKGEQGPXG; (b) Culture mammalian cells under conditions suitable for the production of MHC II / CII peptide complexes; and (c) Harvest the cell supernatant, wherein the MHC II / CII peptide complex comprises a post-translational modified CII peptide; Wherein at least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from an HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303, and / or wherein the MHC II / CII peptide complex does not contain an enzyme cleavage site in the amino acid sequence between the CII peptide and the extracellular region of the MHC class II α or β chain.

13. The method according to claim 12, wherein, The CII peptide has the following amino acid sequence: AGFKGEQGPKG.

14. The method according to claim 12 or 13, wherein, (a) Transfection of mammalian cells with a polynucleotide comprising the following complex: (i) A polynucleotide encoding an extracellular region of an MHC IIα chain containing at least one α1 domain; (ii) a polynucleotide encoding an extracellular region of an MHC IIβ chain containing at least one β1 domain; and (iii) A polynucleotide encoding a CII peptide that fuses with the N-terminus of an MHC class II β chain via a linker peptide.

15. The method according to claim 12 or 13, wherein, In (c), the cell supernatant and cells containing the MHC II / CII peptide complex were harvested.

16. The method according to claim 15, wherein, The procedure further includes a step of analyzing the post-translational modifications of the CII peptide in the MHC II / CII peptide complex.

17. The method according to claim 12 or 13, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from an HLA-DR allele selected from the group consisting of: DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303; (c) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated.

18. The method according to claim 12 or 13, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from an HLA-DR allele selected from the group consisting of: DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303; (c) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated to R.

19. The method according to claim 12 or 13, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from DRB1*0401; (c) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated.

20. The method according to claim 12 or 13, wherein (a) The first lysine residue is galactosyl-hydroxylysine; (b) At least the α1 domain is derived from DRA*0101, and at least the β1 domain is derived from DRB1*0401; (c) The amino acid sequence of the CII peptide is AGFKGEQGPKG, and any additional K is mutated to R.

21. The method according to any one of claims 12-20, wherein the mammalian cells (a) An enzyme that includes post-translational modifications of lysine residues in collagen, the modifications including: Hydroxylated lysine to hydroxylysine and galactosylated hyalyl to galactosyl hydroxylysine; and / or (b) Contains lysyl hydroxylase and collagen galactosyltransferase.

22. The method according to any one of claims 12-20, wherein the mammalian cells (a) An enzyme that includes post-translational modifications of lysine residues in collagen, the modifications including: Hydroxylated lysine to hydroxylysine and galactosylated hyalyl to galactosyl hydroxylysine; and / or (b) Contains lysyl hydroxylase 1 and / or lysyl hydroxylase 2, and collagen galactosyltransferase GLT25D1 and / or GLT25D2.

23. The method according to any one of claims 12-20, wherein the mammalian cells (a) An enzyme that includes post-translational modifications of lysine residues in collagen, the modifications including: Hydroxylated lysine to hydroxylysine and galactosylated hyalyl to galactosyl hydroxylysine; and / or (b) Contains lysyl hydroxylase 1 and / or lysyl hydroxylase 2, as well as collagen galactosyltransferase GLT25D1.

24. The method of claim 21, wherein the cell is (a) Kidney cells, fibroblasts, or osteoblasts; or (b) Genetically engineered cells that recombinantly express lysyl hydroxylase and collagen galactosyltransferase.

25. The method of claim 21, wherein the cell is (a) Kidney cells; or (b) Genetically engineered cells that recombinantly express lysyl hydroxylase and collagen galactosyltransferase.

26. The method of claim 21, wherein the cell is (a) HEK293 cell line; or (b) Genetically engineered cells that recombinantly express lysyl hydroxylase and collagen galactosyltransferase.

27. The method of claim 21, wherein the cell is (a) Kidney cells, fibroblasts, or osteoblasts; or (b) Genetically engineered cells that recombinantly express lysyl hydroxylase 1 and / or lysyl hydroxylase 2, and collagen galactosyltransferase GLT25D1 and / or GLT25D2.

28. The method of claim 21, wherein the cell is (a) Kidney cells; or (b) Genetically engineered cells that recombinantly express lysyl hydroxylase 1 and / or lysyl hydroxylase 2, and collagen galactosyltransferase GLT25D1 and / or GLT25D2.

29. The method of claim 21, wherein the cell is (a) HEK293 cell line; or (b) Genetically engineered cells that recombinantly express lysyl hydroxylase 1 and / or lysyl hydroxylase 2, and collagen galactosyltransferase GLT25D1 and / or GLT25D2.

30. The method according to any one of claims 24-29, wherein the mammalian cells (a) Lack of galactosylhydroxylysylglucosyltransferase activity; (b) Lack of the multifunctional enzyme LH3; or (c) Includes mutant LH3 enzymes lacking galactosyl hydroxylysyl glucosyltransferase activity.

31. The method according to any one of claims 24-29, comprising: (a) The first polynucleotide encoding the extracellular region of an MHC class II α chain containing at least one α1 domain; (b) A second polynucleotide encoding an extracellular region of an MHC class II β chain containing at least one β1 domain; and (c) Polynucleotides encoding CII peptides fused to the N-terminus of the MHC class II α chain or the N-terminus of the MHC class II β chain via a linker peptide. The MHC class II α chain is fused to the first functional domain of the leucine zipper heterodimerization motif at its C-terminus, and the MHC class II β chain is fused to the second complementary functional domain of the leucine zipper heterodimerization motif at its C-terminus.

32. The method according to any one of claims 24-29, comprising: (a) The first polynucleotide encoding the extracellular region of an MHC class II α chain containing at least one α1 domain; (b) A second polynucleotide encoding an extracellular region of an MHC class II β chain containing at least one β1 domain; and (c) Polynucleotides encoding CII peptides fused to the N-terminus of the MHC class II α chain or the N-terminus of the MHC class II β chain via a linker peptide. The MHC class II α chain is fused to the first functional domain of the leucine zipper heterodimerization motif at its C-terminus, and the MHC class II β chain is fused to the second complementary functional domain of the leucine zipper heterodimerization motif at its C-terminus. Wherein the first functional domain and the second complementary functional domain are: (a) Acidic and basic leucine zipper heterodimerization domains; and / or (b)jun-fos leucine zipper motif.

33. A recombinant MHC II / CII peptide complex comprising a post-translational modified CII peptide, obtained by the method of any one of claims 12-32, wherein the first lysine residue of the CII peptide is an O-glycosylated Hyl.

34. Use of the composition according to any one of claims 1-11 or the recombinant MHC II / CII peptide complex according to claim 33 in the preparation of a medicament for the treatment of rheumatoid arthritis.

35. A tetramer of MHC II / CII peptide complex, comprising: a recombinant MHC II / CII peptide complex of any one of claims 1-11 or a recombinant MHC II / CII peptide complex comprising a post-translational modified CII peptide as claimed in claim 33.

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