Methods for stratifying diabetes patients

Abstract

To provide methods for stratifying diabetes patients regarding likelihood of favorable response.SOLUTION: A method for predicting a response of a type 1 diabetic patient to treatment with an immunogenic peptide containing an insulin MHC class II T cell epitope and an oxidoreductase motif includes a step of determining MHC class II HLA haplotype of the patient.SELECTED DRAWING: None

Description

[Background technology]

[0001] Several strategies have been described to prevent the generation of unwanted immune responses to antigens. WO2008 / 017517 describes a new strategy using peptides containing an MHC class II T cell epitope and an oxidoreductase motif from a given antigenic protein. These peptides convert CD4+ T cells into a cell type with cytolytic properties called cytolytic CD4+ T cells. These cells are capable of killing antigen-presenting cells (APCs) that present the antigen from which the peptides are derived through apoptosis induction. WO2008 / 017517 demonstrates this concept for allergies and autoimmune diseases, such as type 1 diabetes. In this document, insulin can act as an autoantigen.

[0002] WO2009101207 and Carlier et al. (2012) Plos one 7,10 e45366 further describe antigen-specific cytolytic cells in more detail.

[0003] WO2016059236 discloses further modified peptides in which an additional histidine is present adjacent to the oxidoreductase motif.

[0004] WO2018162498 further discloses peptides comprising an oxidoreductase motif with an additional histidine and an MHCII T cell epitope derived from insulin, and their use in the treatment of type 1 diabetes (T1D). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2008 / 017517 [Patent Document 2] WO2009101207 [Patent Document 3] WO2016059236 [Patent Document 4] WO2018162498

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[0007] However, even with the above in mind, there remains a need for methods to stratify and select T1D patient subpopulations that will benefit most from a treatment, and to tailor said treatment as needed for other patient subpopulations that are less responsive.To date, no information is available regarding the effects of, and patient response to, the use of immunogenic peptides containing insulin T cell epitopes coupled to oxidoreductase motifs in T1D patients.

[0008] The present invention has revealed a method for stratifying T1D patients with regard to the likelihood of a successful response. [Means for solving the problem]

[0009] The present invention provides stratification methods and tools for predicting the responsiveness of type 1 diabetic patients to treatment with immunogenic peptides comprising an insulin antigen and an oxidoreductase motif, as well as methods for treating such type 1 diabetic patients with said immunogenic peptides.

[0010] Furthermore, the inventors have found that in patients, the level of responsiveness can depend on the MHC class II haplotype.

[0011] Thus, the present invention provides the following aspects:

[0012] 1. An in vitro method for predicting the response of a type 1 diabetes patient to treatment with an immunogenic peptide having a length of 12 to 50 amino acids that contains an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from this motif by 0 to 7 amino acids, the method comprising a step of determining the patient's MHC class II HLA haplotype, and patients who are HLA-DR4 positive (HLA-DR4+) are predicted to be responsive to the treatment.

[0013] The term "HLA DR4 positive" encompasses both heterozygous and homozygous HLA DR4 positive patients.

[0014] In one embodiment, the patient is also HLA-DR3 negative (HLA-DR3-).

[0015] The term "HLA-DR3 negative" refers to patients who are homozygous HLA-DR3 negative.

[0016] More particularly, the stratification method identifies patients who would benefit particularly well from treatment with the immunogenic peptides according to the present invention.

[0017] More particularly, the stratification method identifies patients with an HLA-DR4 positive haplotype and optionally an HLA-DR3 negative haplotype as more likely to respond than patients who are HLA-DR4 negative.

[0018] This responsiveness can be achieved, for example: - by counting the total daily insulin dose per kg, where a reduction in this total for untreated or non-responsive patients indicates a positive response to treatment, or - by using an MMTT test measuring C-peptide secretion, where responding patients show a trend towards improvement (median C-peptide decrease lower than the reference model, delta ratio greater than 0) when compared to untreated or non-responding patients. It can be measured.

[0019] In some embodiments, the haplotyping in the patient is performed using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis, or through antibody testing.

[0020] 2. A method for reducing an immune response to an autoimmune antigen selected from (pro)insulin or C-peptide in a patient, comprising administering an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from said motif by 0 to 7 amino acids, wherein said patient has been selected based on the presence of a DR4-positive (HLA-DR4+) and optionally an HLA-DR3-negative (HLA-DR3-) MHC class II HLA haplotype.

[0021] 3. An immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from this motif by 0 to 7 amino acids, for use in the treatment or prevention of type 1 diabetes in patients selected on the basis of the presence of a DR4 positive (HLA-DR4+) and optionally an HLA-DR3 negative (HLA-DR3-) MHC class II HLA haplotype.

[0022] 4. A method for the treatment or prevention of type 1 diabetes, comprising administering an effective dose of an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from the motif by 0 to 7 amino acids to a patient selected on the basis of the presence of a DR4-positive (HLA-DR4+) and optionally an HLA-DR3-negative (HLA-DR3-) MHC class II HLA haplotype.

[0023] 5. In certain embodiments of aspects 2-4, the MHC class II haplotype of the patient is determined before or during treatment.

[0024] In some embodiments, the haplotyping is performed using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis, or through antibody testing.

[0025] 6. In certain embodiments of any one of aspects 1-5, the oxidoreductase motif has the general formula: Zm[CST]XnC or ZmCXn[CST] (In the formula, n is an integer of 0 to 6, preferably 0 to 3, and more preferably 0, 1, 2, or 3, m represents an integer of 0 to 2; C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. may include:

[0026] In some embodiments, when m is 0 and the oxidoreductase motif is an N-terminal oxidoreductase motif (the oxidoreductase motif is located at the N-terminus of the immunogenic peptide), the first cysteine, threonine, or serine of the motif may be chemically modified through N-acetylation, N-methylation, N-ethylation, or N-propionylation.

[0027] In some embodiments, when m is 0 and the oxidoreductase motif is a C-terminal oxidoreductase motif (where the oxidoreductase motif is located at the C-terminal end of the immunogenic peptide), the final cysteine, threonine, or serine of the motif may be chemically modified through C-terminal substitution of its C-terminal amide or acid group with an acetyl, methyl, ethyl, or propionyl group.

[0028] These motifs are exemplified below.

[0029] In a preferred embodiment, the oxidoreductase motif may comprise the tetrapeptide sequence Cxx[CST][SEQ ID NO: 1] or [CST]xxC[SEQ ID NO: 2], optionally preceded by one or more basic amino acids such as HCXXC, KCXXC, RCXXC, KHCXXC, HKCXXC, RHCXXC, HRCXXC, KRCXXC, or RKCXXC.

[0030] In a preferred embodiment, the oxidoreductase motif is not naturally occurring within the 11 amino region at the N- or C-terminus adjacent to the T cell epitope, and more preferably, the oxidoreductase motif is not naturally occurring in the T cell epitope.

[0031] In certain embodiments, the MHC class II T cell insulin epitope may be defined by the sequence LALEGSLQK [SEQ ID NO: 3].

[0032] 7. The method or use according to any one of aspects 1 to 6, wherein said peptide comprises the sequence Cxx[CST]SLQPLALEGSLQK [SEQ ID NO: 4] or [CST]xxCSLQPLALEGSLQK [SEQ ID NO: 5].

[0033] 8. The method or use according to any one of aspects 1 to 7, wherein said peptide comprises the sequence CxxCSLQPLALEGSLQK [SEQ ID NO: 6].

[0034] 9. The method or use according to any one of aspects 1 to 8, wherein said peptide comprises the sequence HCxx[CST]SLQPLALEGSLQK [SEQ ID NO: 7] or H[CST]xxCSLQPLALEGSLQK [SEQ ID NO: 8].

[0035] 10. The method or use according to any one of aspects 1 to 9, wherein said peptide comprises the sequence HCxxCSLQPLALEGSLQK [SEQ ID NO: 9].

[0036] 11. The method or use according to any one of aspects 1 to 10, wherein the peptide comprises the sequence Cxx[CST] [SEQ ID NO: 1] or [CST]xxC [SEQ ID NO: 2] redox motif sequence and the sequence SLQPLALEGSLQKRG [SEQ ID NO: 20].

[0037] 12. The method or use according to any one of aspects 1 to 11, wherein said peptide comprises or consists of the amino acid sequence HCPYCSLQPLALEGSLQKRG [SEQ ID NO: 26].

[0038] 13. The method or use according to any one of aspects 1 to 12, wherein said peptide is administered as a pharmaceutical composition comprising said peptide and a pharmaceutically acceptable carrier.

[0039] 14. The method or use according to any one of aspects 2 to 13, wherein the peptide is administered in a dosage regimen of 50 to 1500 μg, preferably 100 to 1200 μg.

[0040] 15. The method or use according to any one of aspects 2 to 14, wherein said peptide is administered in a single dose, or in two, three, four, five or more doses, simultaneously or sequentially.

[0041] 16. The peptide according to the following scheme: 1) a first subcutaneous injection of 50 μg of the peptide, followed by three consecutive subcutaneous injections of 25 μg of the peptide, each administered two weeks apart; 2) a first subcutaneous injection of 150 μg of the peptide, followed by three consecutive subcutaneous injections of 75 μg of the peptide, each administered two weeks apart; and 3) A first subcutaneous injection of 450 μg of the peptide, followed by three consecutive subcutaneous injections of 225 μg of the peptide, each administered two weeks apart. 16. The method or use according to any one of aspects 2 to 15, wherein the compound is administered via four biweekly subcutaneous or intramuscular injections according to any one of aspects 2 to 15.

[0042] 17. The method or use according to any one of aspects 2 to 16, wherein said patient is further HLA-DR3 negative (HLA-DR3-).

[0043] 18. The method or use according to any one of aspects 2 to 17, wherein said peptide is administered as a pharmaceutical composition comprising said peptide and a pharmaceutically acceptable carrier.

[0044] 19. The method or use according to any one of aspects 2 to 18, wherein said peptide is administered as a pharmaceutical composition comprising said peptide and an adjuvant.

[0045] Other particular embodiments of the immunogenic peptide used in any one of the embodiments or aspects disclosed herein include the immunogenic peptide having the following sequence: Cxx[CST]SLQPLALEGSLQKRG [SEQ ID NO: 10], [CST]xxCSLQPLALEGSLQKRG [SEQ ID NO: 11], CxxCSLQPLALEGSLQKRG [SEQ ID NO: 12], HCxx[CST]SLQPLALEGSLQKRG [SEQ ID NO: 13], H[CST]xxCSLQPLALEGSLQKRG [SEQ ID NO: 14], or HCxxCSLQPLALEGSLQKRG [SEQ ID NO: 15] It consists of one of the following.

[0046] In particular embodiments of such immunogenic peptide sequences, Cxx[CST][SEQ ID NO:1] is CPY[CST][SEQ ID NO:16] and / or [CST]xxC[SEQ ID NO:2] is [CST]PYC[SEQ ID NO:17], and more particularly CxxC[SEQ ID NO:18] is CPYC[SEQ ID NO:19].

[0047] In certain embodiments, the peptide consists of the sequence HCPYCVRSLQPLALEGSLQKRG [SEQ ID NO: 25] or HCPYCSLQPLALEGSLQKRG [SEQ ID NO: 26].

[0048] In any one of the above aspects or embodiments, the redox motif is present N-terminal to the epitope.

[0049] In an alternative set of the above aspects or embodiments, the peptide has a redox motif C-terminal to the epitope.

[0050] 20. Another aspect of the present invention relates to any one of the peptides as disclosed above for use as a medicament, particularly in the treatment or prevention of type 1 diabetes or for reducing the symptoms of type 1 diabetes, wherein the patient or subject has been determined to be positive for the DR4 HLA haplotype of MHC class II molecules and optionally HLA-DR3 negative (HLA-DR3-).

[0051] In some embodiments of aspect 20, the haplotyping in the patient is performed using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis, or through antibody testing.

[0052] In some embodiments of aspect 20, patients who are homozygous for the HLA type DR4+ are considered to be the most responsive, and / or patients who are heterozygous for the HLA type DR4+, e.g., patients who are DR4+ and DR3+, are considered to be intermediate responsive.

[0053] 21. Another aspect relates to a pharmaceutical composition comprising any one of the peptides as disclosed above and a pharmaceutically acceptable carrier for use in the treatment or prevention of type 1 diabetes or for reducing the symptoms of type 1 diabetes, wherein the patient or subject has been determined to be positive for the DR4 HLA haplotype of MHC class II molecules and optionally HLA-DR3 negative (HLA-DR3-).

[0054] 22. In an alternative embodiment, a patient or subject who is or has been determined to be positive for the DR4 HLA haplotype of MHC class II molecules and optionally HLA-DR3 negative (HLA-DR3-) is - providing peripheral blood cells; - contacting said cells in vitro with any one of the immunogenic peptides as disclosed above; - expanding the cells in the presence of IL-2; The treatment can be performed using a population of cytolytic CD4+ T cells against APCs presenting an insulin epitope, which is obtained by the above-mentioned in vitro method for generating a population of cytolytic CD4+ T cells against APCs presenting an insulin epitope, comprising:

[0055] 23. Another aspect relates to a population of cytolytic CD4+ T cells against insulin-presenting APCs obtainable by the above method for use in the treatment or prevention of type 1 diabetes or for reducing the symptoms of type 1 diabetes, wherein the patient or subject has been determined to be positive for the DR4 HLA haplotype of MHC class II molecules and optionally HLA-DR3 negative (HLA-DR3-).

[0056] In some embodiments of aspect 22 or 23 above, the haplotyping in the patient is performed using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis, or through antibody testing.

[0057] In some embodiments of aspect 22 or 23 above, patients who are homozygous for the HLA type DR4+ are considered to be the most responsive, and / or patients who are heterozygous for the HLA type DR4+, e.g., patients who are DR4+ and DR3+, are considered to be intermediate responsive. [Brief explanation of the drawings]

[0058] [Figure 1] 1 depicts binding of two test peptides defined by the sequences HCPYCVRSLQPLALEGSLQKRG (SEQ ID NO: 25) and HCPYCSLQPLALEGSLQKRG (SEQ ID NO: 26) to DRB1*0301 or DRB1*0401 recombinant MHC II protein. Test peptide binding is demonstrated in a dose-dependent manner by a decrease in fluorescent signal (RFU) due to competition with a fluorescently tagged control high-affinity binder peptide. [Figure 2]FIG. 1 depicts the reactivity of different T1D patients as measured by enumerating viable responder CD4+ T cells after 1, 4 and 6 specific restimulations with an immunogenic peptide having the sequence HCPYCVRSLQPLALEGSLQKRG (SEQ ID NO: 25) presented by autologous dendritic cells. [Figure 3] FIG. 1 depicts a scheme of Phase Ib study design with the immunogenic peptide defined by HCPYCSLQPLALEGSLQKRG (SEQ ID NO: 26). [Figure 4] Figure 1 shows box plots of C-peptide AUC 2 hours after MMTT challenge in different HLA-genotype subgroups. P1 = placebo, C1 = cohort 1, C2 = cohort 2, C3 = cohort 3. Data are expressed as percent variation in response ((V8-V2) / V2) 6 months after entering the Phase 1b study. [Figure 5] Figure 1 shows a box plot of insulin dose per Kg in different HLA-genotype subgroups. P1 = placebo, C1 = cohort 1, C2 = cohort 2, C3 = cohort 3. Data are expressed as percent variation in response ((V8-V2) / V2) 6 months after entering the Phase Ib study. [Figure 6] Figure 1 shows a box plot of measured vs. predicted C-peptide AUC after 2 hours of MMTT challenge in different HLA-genotype subgroups. P1 = placebo, C1 = cohort 1, C2 = cohort 2, C3 = cohort 3. Data are expressed as percent change in response (delta ratio) 3 months (V6) and 6 months (V8) after entering the Phase Ib study. [Figure 7] Figure 1 depicts a box plot of insulin dose per Kg in different HLA-genotype subgroups across V3 to V8. Data are expressed as percent variation in response ((Visit X-V2) / V2) in a Phase Ib study. DETAILED DESCRIPTION OF THE INVENTION

[0059] While the present invention will be described with reference to specific embodiments, the present invention is not limited thereto, but only by the claims. Any reference signs in the claims should not be construed as limiting the scope of the claims. The following terms or definitions are provided solely to aid in the understanding of the present invention. Unless otherwise specified herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. The definitions provided herein should not be construed to have a claim scope less than that understood by a person skilled in the art.

[0060] Unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, as would be apparent to one skilled in the art, reference being again made to, for example, standard handbooks, the general background art mentioned above and further citations therein.

[0061] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. The term "any," as used herein, when used in connection with an aspect, claim, or embodiment, refers to any single thing (i.e., anyone) as well as all combinations of said aspect, claim, or embodiment being referenced.

[0062] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms also encompass the embodiments "consisting essentially of" and "consisting of."

[0063] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0064] As used herein, the term "about" when referring to a measurable value, e.g., a parameter, amount, duration, etc., is intended to encompass variations of + / - 10% or less from the specified value, preferably + / - 5% or less, more preferably + / - 1% or less, and even more preferably + / - 0.1% or less, insofar as such variations are appropriate for the practice of the disclosed invention. It should be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

[0065] As used herein, the term "for use" as used in "a composition for use in the treatment of a disease" is intended to also disclose the corresponding method of treatment and the corresponding use of the preparation for the manufacture of a medicament for the treatment of a disease.

[0066] As used herein, the term "peptide" refers to a molecule that comprises an amino acid sequence of 12 to 200 amino acids linked by peptide bonds, but which may include non-amino acid structures.

[0067] Peptides according to the invention can contain any of the conventional 20 amino acids or modified versions thereof, or can contain non-naturally occurring amino acids incorporated by chemical peptide synthesis or by chemical or enzymatic modification.

[0068] The term "antigen" as used herein refers to a macromolecule, generally a structure made of a protein (with or without polysaccharides) or proteinaceous composition that contains one or more haptens and contains T-cell epitopes.

[0069] As used herein, the term "antigenic protein" refers to a protein that contains one or more T cell epitopes. As used herein, an autoantigen or autoantigenic protein refers to a human or animal protein present in the body that elicits an immune response within the same human or animal body.

[0070] The term "epitope" refers to one or several parts of an antigenic protein (which can define conformation-dependent epitopes), which are specifically recognized and bound by an antibody or part thereof (Fab', Fab2', etc.) or a receptor displayed on the cell surface of B- or T-cell lymphocytes, which is capable of inducing an immune response by said binding.

[0071] In the context of the present invention, the term "T cell epitope" refers to a dominant, subdominant, or minor T cell epitope, i.e., a portion of an antigenic protein that is specifically recognized and bound by receptors expressed on the cell surface of T lymphocytes when complexed with an MHC class II molecule. Whether an epitope is dominant, subdominant, or minor depends on the immune response elicited against the epitope. Dominance depends on the frequency with which such an epitope is recognized by T cells and is able to activate them among all possible T cell epitopes of a protein.

[0072] A T cell epitope is an epitope recognized by and bound to MHC class II molecules, consisting of a sequence of + / - 9 amino acids that fits into the groove of the MHC II molecule. In a peptide sequence representing a T cell epitope, the amino acids of the epitope are numbered P1 to P9, the amino acids at the N-terminus of the epitope are numbered P-1, P-2, etc., and the amino acids at the C-terminus of the epitope are numbered P+1, P+2, etc. Peptides recognized by MHC class II molecules but not by MHC class I molecules are called MHC class II-restricted T cell epitopes.

[0073] The term "MHC" refers to "major histocompatibility complexes." In humans, MHC genes are known as HLA ("human leukocyte antigen") genes. Although no rule is consistently followed, some literature uses HLA to refer to HLA protein molecules and MHC to refer to the genes encoding HLA proteins. Thus, as used herein, the terms "MHC" and "HLA" are equivalent. The human HLA system has its equivalent in mice, the H2 system. The most intensively studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. In humans, MHC is divided into three regions: class I, II, and III. The A, B, and C genes belong to MHC class I, while the six D genes belong to class II. MHC class I molecules are composed of a single polymorphic chain containing three domains (alpha 1, 2, and 3) that associates with beta 2 microglobulin on the cell surface. Class II molecules are composed of two polymorphic chains, each containing two chains (alpha 1 and 2, and beta 1 and 2).

[0074] Class I MHC molecules are expressed on virtually all nucleated cells.

[0075] Peptide fragments presented in the context of class I MHC molecules are recognized by CD8+ T lymphocytes (cytolytic T lymphocytes or CTLs). CD8+ T lymphocytes frequently mature into cytolytic effectors capable of lysing cells bearing stimulatory antigens. Class II MHC molecules are expressed primarily on activated lymphocytes and antigen-presenting cells. CD4+ T lymphocytes (helper T lymphocytes or Th) are activated upon recognition of specific peptide fragments presented by class II MHC molecules typically found on antigen-presenting cells such as macrophages or dendritic cells. CD4+ T lymphocytes proliferate and secrete cytokines such as IL-2, IFN-gamma, and IL-4, which support antibody- and cell-mediated responses.

[0076] Functional HLA is characterized by a deep binding groove in which endogenous and foreign, potentially antigenic peptides bind. The groove is further characterized by a distinct shape and physicochemical properties. HLA class I binding sites are closed, in that the peptide termini are pinned at the ends of the groove. They also participate in a network of hydrogen bonds with conserved HLA residues. Given these constraints, the length of binding peptides is limited to 8, 9, or 10 residues. However, it has been demonstrated that peptides of up to 12 amino acid residues can also bind to HLA class I. Comparison of the structures of different HLA complexes confirmed a general mode of binding, in which peptides can adopt a relatively linear, elongated conformation or contain central residues that protrude from the groove.

[0077] In contrast to HLA class I binding sites, class II sites are open at both ends. This allows peptides to extend beyond the actual binding region, thereby "overhanging" at both ends. Thus, class II HLAs can bind peptide ligands of various lengths, from 9 to over 25 amino acid residues. Similar to HLA class I, the affinity of class II ligands is determined by "constant" and "variable" components. The constant portion again results from a network of hydrogen bonds formed between conserved residues in the HLA class II groove and the backbone of the bound peptide. However, this hydrogen-bonding pattern is not restricted to the N- and C-terminal residues of the peptide but is distributed throughout the entire chain. The latter is important because it restricts the conformation of complex peptides to binding in a strictly linear fashion. This is common to all class II allotypes. The second component, which determines peptide binding affinity, varies due to specific positions of polymorphism within the class II binding site. Different allotypes form different complementary pockets within the groove, thereby explaining subtype-dependent peptide selection or specificity. Importantly, the constraints on amino acid residues retained within the class II pocket are generally "softer" than those for class I. There is much more cross-reactivity of peptides between different HLA class II allotypes. The sequence of + / - 9 amino acids (i.e., 8, 9, or 10) of an MHC class II T-cell epitope that fits into the groove of the MHC II molecule is usually numbered P1 through P9. Additional amino acids at the N-terminus of the epitope are numbered P-1, P-2, etc., and amino acids at the C-terminus of the epitope are numbered P+1, P+2, etc.

[0078] At the genetic level, the MHC class II cluster is located on the short arm of chromosome 6 (6p21). The cluster contains three classical class II genes (HLA-DP, HLA-DQ, and HLA-DR) and two non-classical class II genes (HLA-DM and HLA-DO). The structure of MHC class II is achieved by the combination of two membrane-bound chains, termed α and β, which create the antigen-binding cleft of MHC class II. Both the α and β chains are encoded by separate loci, namely, DRα / DRβ, DQα / DQβ, and DPα / DPβ, which are closely related as pairs of α and β genes. The HLA-DP, HLA-DQ, and HLA-DR loci are highly polymorphic, particularly in the antigen-binding pocket of class II molecules. HLA-DP and HLA-DQ contain polymorphisms in both the α and β chain genes (DPA, DPB, DQA, and DQB). In HLA-DR, polymorphisms are associated only with the DR β chain (DRB gene). Although there are nine DRB loci (numbered DRB1 to DRB9), only the DRB1 locus is found in all haplotypes and therefore constitutes the major determinant of classical DR serology (McCluskey et al., Current Protocols in Immunology (2017), 118, A.1S.1-A.1S.6).

[0079] Taking the HLA-DRB1 group as an example, the existence of more than 40 different haplotypes has been reported in the literature (Marsh et al., Tissue Antigens (2010), 75, 291). The most relevant across the human population are the DRB1*03 and DRB1*04 haplotype groups. In the DRB1*03 group, two alleles, namely, DRB1*0301 and DRB1*0302, are common, but other alleles such as DRB1*0303, DRB1*0304, and DRB1*0307 have been reported. In the DRB1*04 group, ten major alleles can be found: DRB1*0401, DRB1*0402, DRB1*0403, DRB1*0404, DRB1*0405, DRB1*0406, DRB1*0407, DRB1*0408, DRB1*0410 and DRB1*0411. As used throughout this application, the term "DR4 positive" or "DR4+" indicates that a subject is positive for one of the DRB1*04 haplotypes. Similarly, as used throughout this application, the term "DR3 positive" or "DR3+" indicates that a subject is positive for one of the DRB1*03 haplotypes. As used throughout this application, the term "DR4 negative" or "DR4-" indicates that a subject does not have any of the DRB1*04 haplotypes. Similarly, the term "DR3 negative" or "DR3-" as used throughout this application indicates that the subject does not have any of the DRB1*03 haplotypes.

[0080] HLA typing can be performed using techniques known in the art, including, but not limited to, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis. A non-limiting example of a PCR-based analysis is the Taqman® allelic discrimination assay available from Applied Biosystems. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing, solid-phase sequencing, sequencing using mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, and hybridization sequencing. Non-limiting examples of electrophoretic analysis include rub gel electrophoresis, e.g., agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Other methods for genotyping individuals at polymorphic sites in markers include, for example, Third Wave Technologies' INVADER® assay, restriction fragment length polymorphism (RFLP) analysis, allele-specific oligonucleotide hybridization, heteroduplex mobility assay, and single-strand conformation polymorphism (SSCP) analysis.

[0081] Alternatively, HLA typing can be performed by antibody testing.

[0082] The term "homologue" as used herein with respect to an epitope used in the context of the present invention refers to a molecule that has at least 50%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% amino acid sequence identity with a naturally occurring epitope, thereby maintaining the ability of the epitope to bind to an antibody or cell surface receptor of a B and / or T cell. A particular homologue of an epitope corresponds to a naturally occurring epitope that is altered by at most three, particularly at most two, and especially one amino acid.

[0083] The term "derivative" as used herein with respect to the peptides of the invention refers to a molecule that contains at least the peptide active portion (i.e., the redox motif and the MHC class II epitope capable of eliciting cytolytic CD4+ T cell activity) and additionally includes a complementary portion that may have a different purpose, such as stabilizing the peptide or altering the pharmacokinetic or pharmacodynamic properties of the peptide.

[0084] As used herein, the term "sequence identity" of two sequences refers to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter sequence when the two sequences are aligned. In particular, the sequence identity is between 70% and 80%, between 81% and 85%, between 86% and 90%, between 91% and 95%, between 96% and 100%, or 100%.

[0085] As used herein, the terms "polynucleotide (or nucleic acid) encoding a peptide" and "polynucleotide (or nucleic acid) encoding a peptide" refer to a nucleotide sequence that, when expressed in an appropriate environment, results in the production of the relevant peptide sequence or a derivative or homolog thereof. Such polynucleotides or nucleic acids include normal sequences encoding the peptide, as well as derivatives and fragments of these nucleic acids that are capable of expressing a peptide having the required activity. Nucleic acids encoding peptides or fragments thereof according to the present invention are sequences that encode peptides or fragments thereof that are of mammalian origin or correspond to mammalian, particularly human, peptide fragments.

[0086] The term "immune disorder" or "immune disease" refers to a disease in which the immune system's response is responsible for or maintains a dysfunctional or non-physiological condition in an organism. Immune disorders include, among others, allergic disorders and autoimmune diseases.

[0087] The term "autoimmune disease" or "autoimmune disorder" refers to a disease resulting from an abnormal immune response of an organism against its own cells and tissues due to the organism's inability (even to the submolecular level) to recognize its own component parts as "self." The group of diseases can be divided into two categories: organ-specific and systemic diseases. An "allergen" is defined as a substance, usually a macromolecule or proteinaceous composition, that elicits the production of IgE antibodies in predisposed individuals, especially in genetically predisposed (atopic) patients. A similar definition is presented in Liebers et al. (1996) Clin. Exp. Allergy 26, 494-516.

[0088] The term "type 1 diabetes" (T1D) or "type 1 diabetes" (also known as "type 1 diabetes mellitus" or "immune-mediated diabetes," or formerly known as "juvenile-onset diabetes" or "insulin-dependent diabetes") is an autoimmune disorder that usually develops in susceptible individuals during childhood. The destruction of most insulin-producing pancreatic beta cells by autoimmune mechanisms underlies T1D pathogenesis. Briefly, the organism loses immune tolerance to the pancreatic beta cells that govern insulin production and induces an immune response, primarily a cell-mediated immune response, associated with the production of autoantibodies that leads to the self-destruction of the beta cells.

[0089] The term "therapeutically effective amount" refers to an amount of the peptide of the present invention or a derivative thereof that produces a desired therapeutic or preventive effect in a patient. For example, with respect to a disease or disorder, it is an amount that reduces to some extent one or more symptoms of the disease or disorder, in particular an amount that partially or completely normalizes physiological or biochemical parameters associated with or causing the disease or disorder. Generally, a therapeutically effective amount is an amount of the peptide of the present invention or a derivative thereof that leads to an improvement or restoration of normal physiological conditions. For example, when used to therapeutically treat a mammal affected by an immune disorder, it is the daily amount of peptide per kg of body weight of the mammal. Alternatively, when administration is by gene therapy, the amount of naked DNA or viral vector is adjusted to ensure local production of an adequate dosage of the peptide of the present invention, its derivative, or homolog.

[0090] The term "natural" when referring to a peptide relates to the fact that the sequence is identical to a fragment of a naturally occurring protein (wild-type or mutant). In contrast, the term "artificial" refers to a sequence that does not occur in nature as such. Artificial sequences are derived from natural sequences by limited modifications, such as changing / deleting / inserting one or more amino acids in the naturally occurring sequence or by adding / removing amino acids at the N- or C-terminus of the naturally occurring sequence.

[0091] Amino acids are referred to herein by their full name, their three letter abbreviation or their one letter abbreviation.

[0092] Herein, amino acid sequence motifs are written according to the Prosite format. Motifs are used to describe certain sequence variations at specific portions of a sequence. The symbol X is used for positions where any amino acid is acceptable. Alternatives are indicated by listing the allowed amino acids for a given position between square brackets ("[]"). For example, [CST] represents an amino acid selected from Cys, Ser, or Thr. Amino acids that are excluded as alternatives are indicated by listing them between curly brackets ("{}"). For example, {AM} represents any amino acid except Ala and Met. Optionally, different elements within a motif are separated from each other by a hyphen (-). Repetition of the same element within a motif can be indicated by placing a number or range of numbers between brackets after the element. For example, X(2) corresponds to XX or XX, X(2,5) corresponds to 2, 3, 4, or 5 X amino acids, and A(3) corresponds to AAA or AAA.

[0093] To identify the amino acid X, the amino acids between H and C are referred to as exterior amino acids X (single underlined in the sequence above) and the amino acids within the redox motif are referred to as interior amino acids X (double underlined in the sequence above). X represents any amino acid, in particular an L-amino acid, more particularly one of the 20 naturally occurring L-amino acids.

[0094] Peptides containing modified peptide motif sequences with T cell epitopes and reducing activity can generate a population of antigen-specific cytolytic CD4+ T cells directed against antigen-presenting cells.

[0095] Thus, in its broadest sense, the present invention relates to the use of peptides comprising at least one T cell epitope of an antigen (self or non-self) capable of eliciting an immune response and an "oxidoreductase," "thioreductase," "thioredox," or "oxidation reduction" (all terms may be used interchangeably herein) sequence motif with reduction activity in peptide disulfide bonds. The MHC class II T cell epitope and the modified redox motif sequence can be immediately adjacent to each other in the peptide or, optionally, separated by one or more amino acids (so-called linker sequences). Optionally, the peptide further comprises an endosomal targeting sequence and / or additional "flanking" sequences.

[0096] The peptides disclosed herein contain an MHC class II T cell epitope of the insulin antigen and an engineered redox motif capable of eliciting an immune response. The reducing activity of the motif sequence within the peptide can be tested for its ability to reduce sulfhydryl groups, for example, in an insulin solubility assay, in which insulin solubility is altered after reduction, or by a fluorescently labeled substrate such as insulin. An example of such an assay uses fluorescent peptides and is described in Tomazzolli et al. (2006) Anal. Biochem. 350, pp. 105-112. Two peptides bearing an FITC label are self-quenched when they are covalently linked to each other through a disulfide bridge. Reduction by the peptides of the present invention results in the individual reduced peptides becoming fluorescent again.

[0097] The (modified) redox motif can be placed amino-terminal to the T cell epitope or carboxy-terminal to the T cell epitope.

[0098] Peptide fragments with reducing activity are encountered by thioreductases, small disulfide reductases that include glutaredoxins, nucleoredoxins, thioredoxins, and other thiol / disulfide oxidoreductases (Holmgren (2000) Antioxid. Redox Signal. 2, 811-820; Jacquot et al. (2002) Biochem. Pharm. 64, 1065-1069). Peptide fragments with reducing activity are multifunctional and ubiquitous, found in many prokaryotes and eukaryotes. The reducing peptide fragments exert their reducing activity on disulfide bonds in proteins (e.g., enzymes) through redox-active cysteines within the conserved activity domain consensus sequences: CXXC [SEQ ID NO: 18], CXXS [SEQ ID NO: 23], CXXT [SEQ ID NO: 24], SXXC [SEQ ID NO: 21], and TXXC [SEQ ID NO: 22] (Fomenko et al. (2003) Biochemistry 42, pp. 11214-11225; Fomenko et al. (2002) Prot. Science 11, pp. 2285-2296), where X represents any amino acid. Such domains are also found in larger proteins, such as protein disulfide isomerase (PDI) and phosphoinositide-specific phospholipase C.

[0099] Four amino acid redox motifs, such as those known from Fomenko and WO 2008 / 017517, contain cysteines at positions 1 and / or 4, and thus the motif is either CXX[CST][SEQ ID NO:1] or [CST]XXC[SEQ ID NO:2]. Such tetrapeptide sequences are referred to as "motifs." The motif within a peptide can be any of the alternatives CXXC [SEQ ID NO:18], SXXC [SEQ ID NO:21], TXXC [SEQ ID NO:22], CXXS [SEQ ID NO:23], or CXXT [SEQ ID NO:24]. In particular, the peptide contains the sequence motif CXXC [SEQ ID NO:18].

[0100] As will be further described, peptides used in the present invention can be produced by chemical synthesis, which allows for the incorporation of unnatural amino acids. Thus, the "C" in the redox-engineered redox motifs listed above represents cysteine ​​or another amino acid bearing a thiol group, such as mercaptovaline, homocysteine, or other natural or unnatural amino acids bearing a thiol functional group. To be redox-active, the cysteine ​​present in the engineered redox motif should not be part of a cysteine ​​disulfide bridge. Nevertheless, the redox-engineered redox motif may contain an engineered cysteine, such as a methylated cysteine, which is converted in vivo to a cysteine ​​bearing a free thiol group. X can be any of the 20 natural amino acids, including S, C, or T, or an unnatural amino acid. In certain embodiments, X is an amino acid with a small side chain, such as Gly, Ala, Ser, or Thr. In further particular embodiments, X is not an amino acid with a bulky side chain, such as Trp. In further particular embodiments, X is not cysteine. In further particular embodiments, at least one X in the engineered redox motif is His. In other further particular embodiments, at least one X in the engineered redox motif is Pro.

[0101] The peptides may further include modifications to enhance stability or solubility, for example, modifications of the N-terminal NH2 group or the C-terminal COOH group (eg, modification of a COOH to a CONH2 group).

[0102] The terms "oxidoreductase motif," "thiol-oxidoreductase motif," "thioreductase motif," "thiooxiredox motif," or "redox motif" are used synonymously herein and refer to a motif involved in the transfer of electrons from one molecule (also called the reductant, hydrogen or electron donor) to another (also called the oxidant, hydrogen or electron acceptor).

[0103] In particular, the term "oxidoreductase motif" can refer to the known [CST]XXC or CXX[CST] motifs, but in particular to the more common sequence motifs Zm[CST]XnC or ZmCXn[CST] (In the formula, n is an integer of 0 to 6, for example, 0, 1, 2, 3, 4, 5, or 6, m represents an integer of 0 to 2, for example, 0, 1, or 2; C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. Refers to...

[0104] To have reducing activity, the cysteines present in the modified oxidoreductase motif should not be present as part of a cysteine ​​disulfide bridge.

[0105] Typically, the oxidoreductase motif has the general formula ZmCXnC (wherein X is any amino acid) Z is a basic amino acid preferably selected from H, K or R, n is an integer from 0 to 3, m is an integer from 0 to 2. may include:

[0106] The term "basic amino acid" refers to any amino acid that behaves like a Bronsted-Lowry and Lewis base, including natural basic amino acids such as arginine (R), lysine (K) or histidine (H), or unnatural basic amino acids such as, but not limited to: - lysine variants, such as Fmoc-β-Lys(Boc)-OH (CAS number 219967-68-7), Fmoc-Orn(Boc)-OH (CAS number 109425-55-0), also called L-ornithine or ornithine, Fmoc-β-Homolys(Boc)-OH (CAS number 203854-47-1), Fmoc-Dap(Boc)-OH (CAS number 162558-25-0) or Fmoc-Lys(Boc)OH(DiMe)-OH (CAS number 441020-33-3); - tyrosine / phenylalanine variants, such as Fmoc-L-3Pal-OH (CAS No. 175453-07-3), Fmoc-β-HomoPhe(CN)-OH (CAS No. 270065-87-7), Fmoc-L-β-HomoAla(4-pyridyl)-OH (CAS No. 270065-69-5) or Fmoc-L-Phe(4-NHBoc)-OH (CAS No. 174132-31-1); - proline variants, such as Fmoc-Pro(4-NHBoc)-OH (CAS number 221352-74-5) or Fmoc-Hyp(tBu)-OH (CAS number 122996-47-8); - arginine variants, such as Fmoc-β-Homoarg(Pmc)-OH (CAS number 700377-76-0).

[0107] Thus, in addition to the commonly known thioredox motif CXXC and its variants disclosed herein, there are also motifs in which two cysteine ​​moieties are adjacent to each other (CC) or separated by 1, 3, 4, 5, or 6 amino acids, e.g., CXC, CXXXC, CXXXXC, CXXXXXC, or CXXXXXXC. In any one of the above embodiments, one of the cysteines may also be changed to S or T.

[0108] Typically, one or more basic amino acids "Z", selected from, for example, H, K, or R, can be added to a thioredox motif such as an H-motif, K-motif, R-motif, KH-motif, HK-motif, RH-motif, HR-motif, KR-motif, or RK-motif.

[0109] Particularly interesting examples of thioredox motifs that can be used in the present invention are: CC, HCC, KCC, RCC; CXC, HCXC, KCXC, RCXC, KHCXC, HKCXC, RHCXC, HRCXC, RKCXC, KRCXC; CXXC、HCXXC、KCXXC、RCXXC、KHCXXC、HKCXXC、RHCXXC、HRCXXC、RKCXXC、KRCXXC; CXXXC、HCXXXC、KCXXXC、RCXXXC、KHCXXXC、HKCXXXC、RHCXXXC、HRCXXXC、RKCXXXC、KRCXXXC; CXXXC、HCXXXC、KCXXXC、RCXXXC、KHCXXXC、HKCXXXC、RHCXXXC、HRCXXXC、RKCXXXC、KRCXXXC; CXXXXC、HCXXXXC、KCXXXXC、RCXXXXC、KHCXXXXC、HKCXXXXC、RHCXXXXC、HRCXXXXC、RKCXXXXC、KRCXXXXC; CXXXXXXC、HCXXXXXXC、KCXXXXXXC、RCXXXXXXC、KHCXXXXXXC、HKCXXXXXXC、RHCXXXXXXC、HRCXXXXXXC、RKCXXXXXXC、KRCXXXXXXC; CXXXXXXC、HCXXXXXXC、KCXXXXXXC、RCXXXXXXC、KHCXXXXXXC、HKCXXXXXXC、RHCXXXXXXC、HRCXXXXXXC、RKCXXXXXXC、KRCXXXXXXC; is.

[0110] Specific examples of CXC are CHC, CKC, CRC, CGC, CAC, CVC, CLC, CIC, CMC, CFC, CWC, CPC, CSC, CTC, CYC, CNC, CQC, CDC, and CEC. m) where m is an integer from 0 to 3, preferably 0 or 1, and Z is any amino acid, preferably a basic amino acid, such as H, K, or R, or a non-natural basic amino acid as defined herein. Preferred examples of such motifs are KCHC, KCKC, KCRC, KCGC, KCAC, KCVC, KCLC, KCIC, KCMC, KCFC, KCWC, KCPC, KCSC, KCTC, KCYC, KCNC, KCQC, KCDC, KCEC, HCHC, HCKC, HCRC, HCGC, HCAC, HCVC, HCLC, HCIC, H CMC, HCFC, HCWC, HCPC, HCSC, HCTC, HCYC, HCNC, HCQC, HCDC, HCEC, RCHC, RCKC, RCRC, RCGC, RCAC, RCVC, RCLC, RCIC, RCMC, RCFC, RCWC, RCPC, RCSC, RCTC, RCYC, RCNC, RCQC, RCDC, and RCEC.

[0111] In a preferred embodiment, the oxidoreductase motif is CX3C, i.e., CXXXC, typically CX 1 X 2 X 3 C (where X 1 , X 2 , and X 3 are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein. 1 , X 2 , and X 3 is any amino acid except C, S, or T. In certain embodiments, X in the above motifs 1 , X 2 , or X 3 At least one of is a basic amino acid, eg, H, K, or R, or a non-natural basic amino acid as defined herein.

[0112] Specific examples of CXXXC motifs are CXPYC, CPXYC, and CPYXC (wherein X can be any amino acid), more preferably CXPYC, such as: CKPYC, CRPYC (SEQ ID NO: 55), CHPYC, CGPYC, CAPYC, CVPYC, CLPYC, CIPYC, CMPYC, CFPYC, CWPYC, CPPYC, CSPYC, CTPYC, CCPYC, CYPYC, C NPYC, CQPYC, CDPYC, and CEPYC; or CPXYC, for example: CPKYC, CPRYC, CPHYC, CPGYC, CPAYC, CPVYC, CPLYC, CPIYC, CPMYC, CPFYC, CPWYC, CPPYC, CPSYC, CPTYC, CPCYC, CPYYC, CPNYC, CPQYC, CPDYC, CPEYC, and CPLYC; or CPYXC, for example: CPYKC, CPYRC, CPYHC, CPYGC, CPYAC, CPYVC, CPYLC, CPYIC, CPYMC, CPYFC, CPYWC, CPYPC, CPYSC, CPYTC, CPYCC, CPYYC, CPYNC, CPYQC, CPYDC, CPYEC, and CPYLC.

[0113] Further specific examples of CXXXC motifs are CXHGC, CHXGC, and CHGXC (wherein X can be any amino acid), more preferably CXHGC, such as: CKHGC, CRHGC, CHHGC, CGHGC, CAHGC, CVHGC, CLHGC, CIHGC, CMHGC, CFHGC, CWHGC, CPHGC, CSHGC, CTHGC, CCHGC, CYHGC, CNHGC, CQHGC, CDHGC, CEHGC, and CKHGC; or CGXHC, such as: CGKHC, CGRHC, CGHHC, CGGHC, CGAH C, CGVHC, CGLHC, CGIHC, CGMHC, CGFHC, CGWHC, CGPHC, CGSHC, CGTHC, CGCHC, CGYHC, CGNHC, CGQHC, CGDHC, CGEHC, and CGLHC; or CHGXC, such as: CHGKC, CHG RC, CHGHC, CHGGC, CHGAC, CHGVC, CHGLC, CHGIC, CHGMC, CHGFC, CHGWC, CHGPC, CHGSC, CHGTC, CHGCC, CHGYC, CHGNC, CHGQC, CHGDC, CHGEC, and CHGLC.

[0114] Further specific examples of the CXXXC motif are CXGPC, CGXPC, and CGPXC (wherein X can be any amino acid), more preferably CXGPC, such as: CKGPC, CRGPC, CHGPC, CGGPC, CAGPC, CVGPC, CLGPC, CIGPC, CMGPC, CFGPC, CWGPC, CPGPC, CSGPC, CTGPC, CCGPC, CYGPC, CNGPC, CQGPC, CDGPC, CEGPC, and CKGPC; or CGXPC, such as: CGKPC, CGRPC, CGHPC, CGGPC, CGAP C, CGVPC, CGLPC, CGIPC, CGMPC, CGFPC, CGWPC, CGPPC, CGSPC, CGTPC, CGCPC, CGYPC, CGNPC, CGQPC, CGDPC, CGEPC, ​​and CGLPC; or CGPXC, e.g.: CGPKC, CGP RC, CGPHC, CGPGC, CGPAC, CGPVC, CGPLC, CGPIC, CGPMC, CGPFC, CGPWC, CGPPC, CGPSC, CGPTC, CGPCC, CGPYC, CGPNC, CGPQC, CGPDC, CGPEC, and CGPLC.

[0115] Further specific examples of CXXXC motifs are CXGHC, CGXHC, and CGHXC (wherein X can be any amino acid), more preferably CXGHC, such as: CKGHC, CRGHC, CHGHC, CGGHC, CAGHC, CVGHC, CLGHC, CIGHC, CMGHC, CFGHC, CWGHC, CPGHC, CSGHC, CTGHC, CCGHC, CYGHC, CNGHC, CQGHC, CDGHC, CEGHC, and CKGHC; or CGXFC, such as: CGKFC, CGRFC, CGHFC, CGGFC, CGAF C, CGVFC, CGLFC, CGIFC, CGMFC, CGFFC, CGWFC, CGPFC, CGSFC, CGTFC, CGCFC, CGYFC, CGNFC, CGQFC, CGDFC, CGEFC, and CGLFC; or CGHXC, for example: CGHKC, CGHRC, CGHHC, CGHGC, CGHAC, CGHVC, CGHLC, CGHIC, CGHMC, CGHFC, CGHWC, CGHPC, CGHSC, CGHTC, CGHCC, CGHYC, CGHNC, CGHQC, CGHDC, CGHEC, and CGHLC.

[0116] Further specific examples of the CXXXC motif are CXGFC, CGXFC, and CGFXC (wherein X can be any amino acid), more preferably CXGFC, such as: CKGFC, CRGFC, CHGFC, CGGFC, CAGFC, CVGFC, CLGFC, CIGFC, CMGFC, CFGFC, CWGFC, CPGFC, CSGFC, CTGFC, CCGFC, CYGFC, CNGFC, CQGFC, CDGFC, CEGFC, and CKGFC; or CGXFC, such as: CGKFC, CGRFC, CGHFC, CGGFC, CGAF C, CGVFC, CGLFC, CGIFC, CGMFC, CGFFC, CGWFC, CGPFC, CGSFC, CGTFC, CGCFC, CGYFC, CGNFC, CGQFC, CGDFC, CGEFC, and CGLFC; or CGFXC, such as: CGFKC, CGF RC, CGFHC, CGFGC, CGFAC, CGFVC, CGFLC, CGFIC, CGFMC, CGFFC, CGFWC, CGFPC, CGFSC, CGFTC, CGFCC, CGFYC, CGFNC, CGFQC, CGFDC, CGFEC, and CGFLC.

[0117] Further specific examples of CXXXC motifs are CXRLC, CRXLC, and CRLXC (wherein X can be any amino acid), more preferably CXRLC, such as: CKRLC, CRRLC, CHRLC, CGRLC, CARLC, CVRLC, CLRLC, CIRLC, CMRLC, CFRLC, CWRLC, CPRLC, CSRLC, CTRLC, CCRLC, CYRLC, CNRLC, CQRLC, CDRLC, CERLC, and CKRLC; or CRXLC, such as: CRKLC, CRRLC, CRHLC, CRGLC, CRAL C, CRVLC, CRLLC, CRILC, CRMLC, CRFLC, CRWLC, CRPLC, CRSLC, CRTLC, CRCLC, CRYLC, CRNLC, CRQLC, CRDLC, CRELC, and CRLLC; or CRLXC, for example: CRLKC, CRLRC, CRLHC, CRLGC, CRLAC, CRLVC, CRLLC, CRLIC, CRLMC, CRLFC, CRLWC, CRLPC, CRLSC, CRLTC, CRLCC, CRLYC, CRLNC, CRLQC, CRLDC, CRLEC, and CRLLC.

[0118] Further specific examples of the CXXXC motif are CXHPC, CHXPC, and CHPXC (wherein X can be any amino acid), more preferably CXHPC, such as: CKHPC, CRHPC, CHHPC, CGHPC, CAHPC, CVHPC, CLHPC, CIHPC, CMHPC, CFHPC, CWHPC, CPHPC, CSHPC, CTHPC, CCHPC, CYHPC, CNHPC, CQHPC, CDHPC, CEHPC, and CKHPC; or CHXPC, such as: CHKPC, CHRPC, CHHPC, CHGPC, CHAP C, CHVPC, CHLPC, CHIPC, CHMPC, CHFPC, CHWPC, CHPPC, CHSPC, CHTPC, CHCPC, CHYPC, CHNPC, CHQPC, CHDPC, CHEPC, and CHLPC; or CHPXC, such as: CHPKC, CHPRC, CHPHC, CHPGC, CHPAC, CHPVC, CHPLC, CHPIC, CHPMC, CHPFC, CHPWC, CHPPC, CHPSC, CHPTC, CHPCC, CHPYC, CHPNC, CHPQC, CHPDC, CHPEC, and CHPLC.

[0119] Any one of these exemplary CXXXC motifs may be one or more amino acids (Z m ) where m is an integer from 0 to 3, preferably 0 or 1, and Z is any amino acid, preferably a basic amino acid, such as H, K, or R, or a non-natural basic amino acid as defined herein.

[0120] In a preferred embodiment, the oxidoreductase motif is CX4C, i.e., CXXXXC, typically CX 1 X 2 X 3 X 4 C (where X 1 , X 2 , X 3 and X 4are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein. 1 , X 2 , X 3 and X 4 is any amino acid except C, S, or T. In certain embodiments, X in the above motifs 1 , X 2 , X 3 or X 4 At least one of is a basic amino acid, eg, H, K, or R, or a non-natural basic amino acid as defined herein.

[0121] Specific examples of CXXXXC motifs are CLAVLC, CTVQAC or CGAVHC and C and variants thereof, e.g., CX 1 AVLC, CLX 2 VLC, CLAX 3 LC or CLAVX 4 C;CX 1 VQAC, CTX 2 QAC, CTVX 3 AC or CTVQX 4 C;CX 1 AVHC, CGX 2 VHC, CGAX 3 HC or CGAVX 4 C (where X 1 , X 2 , X 3 and X 4 are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein.

[0122] Any one of these exemplary CXXXXC motifs may be one or more amino acids (Z m) where m is an integer from 0 to 3, preferably 0 or 1, and Z is any amino acid, preferably a basic amino acid, such as H, K, or R, or a non-natural basic amino acid as defined herein.

[0123] In a preferred embodiment, the oxidoreductase motif is CX5C, i.e., CXXXXXC, typically CX 1 X 2 X 3 X 4 X 5 C (where X 1 , X 2 , X 3 , X 4 and X 5 are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein. 1 , X 2 , X 3 , X 4 and X 5 is any amino acid except C, S, or T. In certain embodiments, X in the above motifs 1 , X 2 , X 3 , X 4 or X 5 At least one of is a basic amino acid, eg, H, K, or R, or a non-natural basic amino acid as defined herein.

[0124] Specific examples of the CXXXXXC motif are CPAFPLC or CDQGGEC and variants thereof, e.g., CX 1 AFPLC, CPX 2 FPLC, CPAX 3 PLC, CPAFX 4 LC or CPA FPX 5 C;CX 1 QGGEC, CDX 2 GGEC, CDQX 3 GEC, CDQGX 4 EC or CDQGGX5 C (where X 1 , X 2 , X 3 , X 4 and X 5 are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein. Any one of these exemplary CXXXXXC motifs may be one or more amino acids (Z m ) where m is an integer from 0 to 3, preferably 0 or 1, and Z is any amino acid, preferably a basic amino acid, such as H, K, or R, or a non-natural basic amino acid as defined herein.

[0125] In a preferred embodiment, the oxidoreductase motif is CX6C, i.e., CXXXXXXC, typically CX 1 X 2 X 3 X 4 X 5 X 6 C (where X 1 , X 2 , X 3 , X 4 , X 5 and X 6 are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein. 1 , X 2 , X 3 , X 4 , X 5 and X 6 is any amino acid except C, S, or T. In certain embodiments, X in the above motifs 1 , X 2 , X 3 , X 4 , X 5 or X 6At least one of is a basic amino acid, eg, H, K, or R, or a non-natural basic amino acid as defined herein.

[0126] Specific examples of CXXXXXXC motifs are CDIADKYC or variants thereof, such as CX 1 IADKYC, CDX 2 ADKYC, CDIX 3 DKYC, CDIAX 4 KYC, CDIADX 5 YC or CDIA DKX 6 C (where X 1 , X 2 , X 3 , X 4 and X 5 are each independently any amino acid selected from the group consisting of G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, and H, or may be a non-natural basic amino acid as defined herein.

[0127] Any one of these exemplary CXXXXXXC motifs may be one or more amino acids (Z m ) where m is an integer from 0 to 3, preferably 0 or 1, and Z is any amino acid, preferably a basic amino acid, such as H, K, or R, or a non-natural basic amino acid as defined herein.

[0128] Particularly preferred examples of such oxidoreductase motifs are C[KHR]C, CX[KHR]XC, CXX[KHR]C, C[KHR]XXC, [KHR]CC, [KHR]CXC, [KHR]XXXC CC[KHR], CXC[KHR], CXXXC[KHR], [KHR]CC[KHR], [KHR]CXC[KHR], [KHR]CXXXC[KHR], [KHR]C[KHR]C, C[KHR]C[KHR] , [KHR]CXX[KHR]C, [KHR]CX[KHR]XC, [KHR]C[KHR]XXC, CXX[KHR]C[KHR], CX[KHR]XC[KHR], C[KHR]XXC[KHR], etc.

[0129] In any one of the motif embodiments herein, when m is 0 and it is an N-terminal oxidoreductase motif (the oxidoreductase motif is located at the N-terminus start of the immunogenic peptide), the first cysteine, threonine, or serine of the motif can be chemically modified through N-acetylation, N-methylation, N-ethylation, or N-propionylation.

[0130] In any one of the motif embodiments herein, when m is 0 and it is a C-terminal oxidoreductase motif (the oxidoreductase motif is located at the C-terminal end of the immunogenic peptide), the last cysteine, threonine, or serine of the motif may be chemically modified through C-terminal substitution of its C-terminal amide or acid group with an acetyl, methyl, ethyl, or propionyl group.

[0131] In peptides used in the present invention that contain a modified redox motif, the motif is positioned so that, when the epitope fits into the MHC groove, the motif remains outside the MHC binding groove. The modified redox motif is placed in the peptide immediately adjacent to the epitope sequence (i.e., a linker sequence of zero amino acids between the motif and the epitope) or is separated from the T cell epitope by a linker comprising an amino acid sequence of seven or fewer amino acids. In particular, the linker comprises 1, 2, 3, 4, 5, 6, or 7 amino acids. Particular embodiments are peptides with a linker of 0, 1, 2, 3, or 4 amino acids between the epitope sequence and the modified redox motif sequence. Preferably, the linker comprises an amino acid sequence of four amino acids. In peptides in which the modified redox motif is adjacent to the epitope sequence, this is designated as positions P-4 to P-1 or P+1 to P+4 relative to the epitope sequence. In addition to peptide linkers, other organic compounds can be used as linkers to connect portions of the peptide to each other (eg, the modified redox motif sequence and the T cell epitope sequence).

[0132] The peptides used in the present invention may further comprise an additional short amino acid sequence at the N- or C-terminus of the sequence comprising the T-cell epitope and the modified redox motif. Such amino acid sequences are generally referred to herein as "flanking sequences." The flanking sequence may be located between the epitope and the endosomal targeting sequence, and / or between the modified redox motif and the endosomal targeting sequence. In certain peptides that do not contain an endosomal targeting sequence, a short amino acid sequence may be present at the N- and / or C-terminus of the modified redox motif and / or epitope sequence in the peptide. In particular, the flanking sequence is a sequence of 1 to 7 amino acids, most particularly a sequence of 2 amino acids.

[0133] The modified redox motif may be located N-terminally from the epitope.

[0134] In certain embodiments of the present invention, peptides are provided that include a single epitope sequence and a modified redox motif sequence. In further particular embodiments, the modified redox motif appears several times (1, 2, 3, 4 or more times) in the peptide, for example, as repeats of the modified redox motif that may be spaced apart from each other by one or more amino acids, or as repeats that are immediately adjacent to each other. Alternatively, one or more modified redox motifs are provided at both the N- and C-termini of the T-cell epitope sequence.

[0135] Other variations envisioned for the peptides of the invention include peptides containing repeats of T cell epitope sequences, where each epitope sequence is preceded and / or followed by a modified redox motif (e.g., "modified redox motif-epitope" repeats or "modified redox motif-epitope-modified redox motif" repeats). Herein, the modified redox motifs all have the same sequence, although this is not required. It is understood that repetitive sequences of peptides containing epitopes that themselves contain modified redox motifs also result in sequences that contain both "epitope" and "modified redox motif." In such peptides, a modified redox motif within one epitope sequence serves as a modified redox motif outside a second epitope sequence.

[0136] Typically, peptides used in the present invention contain a unique T cell epitope. As described below, T cell epitopes in a protein sequence can be identified by functional assays and / or one or more in silica prediction assays. Amino acids in the T cell epitope sequence are numbered according to their position in the binding groove of the MHC protein. T cell epitopes present in a peptide consist of 8 to 25 amino acids, more particularly 8 to 16 amino acids, and most particularly 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids.

[0137] In more particular embodiments, the T cell epitope consists of a sequence of 9 amino acids. In further particular embodiments, the T cell epitope is an epitope presented to T cells by MHC class II molecules (MHC class II-restricted T cell epitope). Typically, T cell epitope sequences refer to octapeptide or, more specifically, nonapeptide sequences that fit into the cleft of MHC II proteins.

[0138] The T cell epitope of the peptide of the present invention can correspond to the native epitope sequence of the protein, or it can be a modified version thereof, provided that the modified T cell epitope retains its ability to bind within the MHC cleft, similar to the native T cell epitope sequence. The modified T cell epitope can have the same binding affinity to the MHC protein as the native epitope, but can also have a lower affinity. In particular, the binding affinity of the modified peptide is at least 10-fold lower, particularly at least 5-fold lower, than that of the original peptide. The peptide of the present invention has a stabilizing effect on the protein complex. Therefore, the stabilizing effect of the peptide-MHC complex compensates for the lower affinity of the modified epitope to the MHC molecule.

[0139] The sequence containing the T cell epitope and reducing compound within the peptide can be further linked to an amino acid sequence (or another organic compound) that promotes uptake of the peptide into late endosomes for processing and presentation among MHC class II determinants. Late endosome targeting is mediated by a signal present in the cytoplasmic tail of the protein and corresponds to a well-defined peptide motif. The late endosome targeting sequence enables processing and efficient presentation of the antigen-derived T cell epitope by MHC class II molecules. Such endosomal targeting sequences are contained, for example, in the gp75 protein (Vijayasaradhi et al. (1995) J. Cell. Biol. 130, 807-820), human CD3 gamma protein, HLA-BM 11 (Copier et al. (1996) J. Immunol. 157, 1017-1027), and the cytoplasmic tail of the DEC205 receptor (Mahnke et al. (2000) J. Cell. Biol. 151, 673-683). Other examples of peptides that function as sorting signals for endosomes are disclosed in the review by Bonifacio and Traub (2003) Annu. Rev. Biochem. 72, 395-447. Alternatively, the sequence may be that of a subdominant or minor T cell epitope from a protein that promotes uptake in late endosomes without overcoming the T cell response to the antigen. The late endosome targeting sequence can be located at the amino- or carboxy-terminus of the antigen-derived peptide for efficient uptake and processing, or can be coupled through a flanking sequence, such as a peptide sequence of up to 10 amino acids. When a recessive T cell epitope is used for targeting purposes, the latter is typically located at the amino-terminus of the antigen-derived peptide.

[0140] Thus, the present invention contemplates the use of peptides of antigenic proteins and their use in eliciting specific immune responses. These peptides can also correspond to fragments of proteins that include a reducing compound and a T-cell epitope within their sequence, i.e., separated by no more than 10, and preferably no more than 7, amino acids. Alternatively, and for most antigenic proteins, peptides of the invention are generated by attaching a reducing compound, particularly a reducing modified redox motif as described herein, to the N- or C-terminus of the T-cell epitope of the antigenic protein (either immediately adjacent thereto or via a linker of no more than 10, and particularly no more than 7, amino acids). Furthermore, the T-cell epitope sequence and / or the modified redox motif of the protein can be modified and / or one or more flanking and / or target sequences can be introduced (or modified) compared to the naturally occurring sequence. Thus, peptides of the invention can include "artificial" or "naturally occurring" sequences, depending on whether the features of the invention can be found in the sequence of the antigenic protein of interest.

[0141] The peptides of the invention can vary substantially in length. The peptides can be as long as 13 or 14 amino acids, i.e., consisting of a 5-amino acid modified redox motif with histidine, an 8-9 amino acid epitope flanked by up to 20, 25, 30, 40, or 50 amino acids. For example, a peptide can contain a 40-amino acid endosomal targeting sequence, approximately 2 amino acids of flanking sequence, a 5-amino acid motif as described herein, a 4-amino acid linker, and a 9-amino acid T cell epitope peptide.

[0142] Thus, in certain embodiments, the complete peptide consists of 13 amino acids up to 20, 25, 30, 40, 50, 75, or 100 amino acids. More particularly, when the reducing compound is a modified redox motif as described herein, the length of the (artificial or natural) sequence comprising the epitope and the modified redox motif, optionally linked to a linker, without the endosomal targeting sequence (herein referred to as the "epitope-modified redox motif" sequence) is important. The "epitope-modified redox motif" more particularly has a length of 13, 14, 15, 16, 17, 18, or 19 amino acids. Such peptides of 13 or 14-19 amino acids can optionally be coupled to an endosomal targeting signal, the size of which is less important.

[0143] As detailed above, in certain embodiments, the peptides of the invention comprise a reduction-modified redox motif as described herein linked to a T-cell epitope sequence.

[0144] In a further particular embodiment, the peptides used in the present invention are peptides comprising T-cell epitopes that do not contain amino acid sequences with redox properties within their native sequence.

[0145] However, in alternative embodiments, a T-cell epitope may comprise any sequence of amino acids that ensures binding of the epitope to the MHC cleft. When the epitope of interest of an antigenic protein comprises within its epitope sequence a modified redox motif as described herein, an immunogenic peptide according to the invention comprises a sequence of the modified redox motif as described herein and / or another redox sequence coupled at the N- or C-terminus to the epitope sequence such that the attached modified redox motif can ensure redox activity (as opposed to a modified redox motif present within an epitope buried within the cleft).

[0146] Thus, T cell epitopes and motifs are either immediately adjacent to each other or separated and non-overlapping. To assess the concepts of "immediately adjacent" or "separate," an 8- or 9-amino acid sequence that fits into the MHC cleft is determined, and the distance between this octapeptide or nonapeptide with a histidine-containing redox tetrapeptide or modified redox motif pentapeptide is determined.

[0147] Generally, the peptides used in the present invention are not naturally occurring (and therefore not themselves fragments of a protein) but are artificial peptides that contain, in addition to a T cell epitope, a modified redox motif as described herein, whereby the modified redox motif is immediately separated from the T cell epitope by a linker of at most 7, more particularly at most 4 or at most 2 amino acids.

[0148] Following administration (i.e., injection) of the peptides disclosed herein (or compositions comprising such peptides) to a mammal, the peptides have been shown to elicit activation of T cells that recognize antigen-derived T cell epitopes and provide further signals to the T cells through the reduction of surface receptors. This upper-limit activation results in T cells acquiring cytolytic properties against cells presenting the T cell epitope and inhibitory properties with respect to bystander T cells. Thus, peptides or compositions comprising peptides described herein that contain antigen-derived T cell epitopes and engineered redox motifs outside the epitope can be used for the direct immunization of mammals, including humans. Accordingly, the present invention provides the use of the peptides disclosed herein and their derivatives for use as pharmaceuticals. Accordingly, the present invention provides methods of treatment comprising administering one or more peptides disclosed herein to a patient in need thereof.

[0149] The present invention provides a method by which antigen-specific T cells with cytolytic properties can be elicited by immunization with small peptides. Peptides containing (i) a sequence encoding a T cell epitope derived from the antigen and (ii) a consensus sequence with redox properties, and optionally a sequence that promotes uptake of the peptide into late endosomes for efficient MHC class II presentation, have been found to elicit suppressor T cells.

[0150] The immunogenic properties of the disclosed peptides make them of particular interest in the treatment and prevention of immune responses.

[0151] The peptides described herein are for use as medicaments, particularly for the manufacture of medicaments for the prevention or treatment of immune disorders in mammals, especially humans.

[0152] The present invention describes a method for treating or preventing an immune disorder in a mammal in need of such treatment or prevention by using a peptide disclosed herein, a homologue or derivative thereof, said method comprising administering a therapeutically effective amount of a peptide disclosed herein, a homologue or derivative thereof to said mammal suffering from or at risk of an immune disorder, e.g., to reduce the symptoms of the immune disorder. Treatment of both humans and animals, such as pets and farm animals, is contemplated. In one embodiment, the mammal to be treated is a human. The immune disorder referred to above is, in certain embodiments, selected from allergic diseases and autoimmune diseases.

[0153] The peptides or pharmaceutical compositions for use in the present invention comprising the peptides as defined herein are preferably administered via subcutaneous or intramuscular administration. Preferably, the peptides or pharmaceutical compositions comprising same can be injected subcutaneously (SC) in the area of ​​the lateral upper arm midway between the elbow and the shoulder. If two or more separate injections are required, they can be administered simultaneously in both arms.

[0154] The peptides for use in the present invention or pharmaceutical compositions comprising same are administered at a therapeutically effective dose. An exemplary, but non-limiting, dosing regimen is 50-1500 μg, preferably 100-1200 μg. More specific dosing schemes may be 50-250 μg, 250-450 μg, or 850-1300 μg, depending on the patient's condition and the severity of the disease. The dosing regimen may include administration of a single dose or two, three, four, five, or more doses, simultaneously or sequentially. An exemplary, non-limiting dosing scheme is as follows: - A low dose scheme involving SC administration of 50 μg of peptide in two separate injections of 25 μg each (100 μL each), followed by three consecutive injections of 25 μg of peptide in two separate injections of 12.5 μg each (50 μL each). - An intermediate dose scheme comprising SC administration of 150 μg of peptide in two separate injections of 75 μg each (300 μL each), followed by three consecutive administrations of 75 μg of peptide in two separate injections of 37.5 μg each (150 μL each). - A high dose scheme involving SC administration of 450 μg of peptide in two separate injections of 225 μg each (900 μL each), followed by three consecutive administrations of 225 μg of peptide in two separate injections of 112.5 μg each (450 μL each).

[0155] For all of the above peptides, further variants are envisaged in which there are one or two amino acids X between the histidine and cysteine. Typically, these extra amino acids are not His, Cys, Ser or Thr.

[0156] The peptides for use in the present invention may also be used in an in vitro diagnostic method for detecting class II-restricted CD4+ T cells in a sample. In this method, the sample is contacted with a complex of an MHC class II molecule and a peptide disclosed herein. CD4+ T cells are detected by measuring binding of the complex to cells in the sample, where binding of the complex to the cells is indicative of the presence of CD4+ T cells in the sample.

[0157] The complex may be a fusion protein of the peptide and the MHC class II molecule. Alternatively, the MHC molecule in the complex is a tetramer. The complex may be provided as a soluble molecule or may be bound to a carrier.

[0158] Thus, in certain embodiments, the treatment and prevention methods of the invention involve the administration of immunogenic peptides as described herein, wherein the peptides comprise T cell epitopes (e.g., as described above) of antigenic proteins that play a role in the disease to be treated. In further specific embodiments, the epitopes used are dominant epitopes in combination with methods of patient stratification or selection that are likely to benefit most from the treatments described above.

[0159] Peptides for use in the present invention can be prepared by peptide synthesis, where the T cell epitope and modified redox motif are separated by 0 to 5 amino acids. In certain embodiments, the modified redox motif can be obtained by introducing one, two, or three mutations outside the epitope sequence to preserve the sequence configuration as present in the protein. Typically, the amino acids at P-2 and P-1, and the amino acids at P+10 and P+11, are conserved in the peptide sequence relative to the nonapeptide that is part of the native sequence. These flanking residues generally stabilize binding to MHC class II. In other embodiments, the sequences at the N- or C-termini of the epitope are unrelated to the sequence of the antigenic peptide containing the T cell epitope sequence.

[0160] Thus, based on the above methods of designing peptides, peptides may be produced by chemical peptide synthesis, recombinant expression methods, or in more exceptional cases, by proteolytic or chemical fragmentation of proteins.

[0161] Peptides such as those produced in the above methods can be tested for the presence of T cell epitopes by in vitro and in vivo methods, and can be tested for their reducing activity in in vitro assays. As a final quality control, peptides can be tested in in vitro assays to verify whether they are capable of generating CD4+ T cells that are cytolytic via the apoptotic pathway in response to antigen-presenting cells that present an antigen containing an epitope sequence that is also present in the peptide with the modified redox motif.

[0162] Peptides for use in the present invention can be produced in bacteria, yeast, insect cells, plant cells, or mammalian cells using recombinant DNA techniques. Given the limited length of peptides, they can be prepared by chemical peptide synthesis, in which peptides are prepared by coupling different amino acids together. Chemical synthesis is particularly suitable for the inclusion of, for example, D-amino acids, amino acids with unnatural side chains, or natural amino acids with modified side chains, such as methylated cysteine.

[0163] Chemical peptide synthesis is well described, and peptides can be ordered from companies such as Applied Biosystems and other companies.

[0164] Peptide synthesis can be carried out either as solid phase peptide synthesis (SPPS) or conversely as solution phase peptide synthesis. The best known SPPS methods are t-Boc and Fmoc solid phase chemistries:

[0165] Several protecting groups are used during peptide synthesis. For example, hydroxyl and carboxyl functional groups are protected by t-butyl groups, lysine and tryptophan are protected by T-Boc, asparagine, glutamine, cysteine, and histidine are protected by trityl groups, and arginine is protected by a pbf group. If appropriate, such protecting groups can be left on the peptide after synthesis. Peptides can be linked together to form longer peptides using a ligation strategy (chemoselective coupling of unprotected peptide fragments) originally described by Kent (Schnelzer & Kent (1992) lnt. J. Pept. Protein Res. 40, pp. 180-193) and outlined, for example, in Tam et al. (2001) Biopolymers 60, pp. 194-205, which offers enormous possibilities for achieving protein synthesis beyond the scope of SPPS. Many proteins ranging in size from 100 to 300 residues have been successfully synthesized by this method. Synthetic peptides continue to play an increasingly important role in the research fields of biochemistry, pharmacology, neurobiology, enzymology, and molecular biology due to the great advances in SPPS.

[0166] Alternatively, peptides can be synthesized by using nucleic acid molecules encoding the peptides of the present invention in a suitable expression vector containing the coding nucleotide sequence. Such DNA molecules can be readily prepared using an automated DNA synthesizer and the well-known codon-amino acid relationships of the genetic code. Such DNA molecules can also be obtained as genomic DNA or cDNA using oligonucleotide probes and conventional hybridization methodologies. Such DNA molecules can be incorporated into expression vectors, including plasmids, adapted for DNA expression and polypeptide production in suitable hosts, such as bacteria, e.g., Escherichia coli, yeast cells, animal cells, or plant cells.

[0167] The physical and chemical properties (e.g., solubility, stability) of the peptide of interest are examined to determine whether the peptide is suitable / will be suitable for use in a therapeutic composition. Typically, this is optimized by adjusting the sequence of the peptide. If necessary, the peptide can be modified after synthesis (chemical modification, e.g., adding / deleting functional groups) using techniques known in the art.

[0168] T cell epitopes themselves are thought to trigger early events at the T helper cell level by binding to appropriate HLA molecules on the surface of antigen-presenting cells and stimulating the relevant T cell subpopulation. These events result in T cell proliferation, lymphokine secretion, a local inflammatory response, recruitment of additional immune cells to the site, and activation of the B cell cascade, leading to the production of antibodies. One of these antibody isotypes, IgE, is fundamentally important in the development of allergic symptoms, and its production is influenced early in the cascade of events at the T helper cell level by the nature of the lymphokines secreted. T cell epitopes are the basic elements or minimal units of recognition by T cell receptors, where an epitope contains amino acid residues essential for receptor recognition, which are contiguous in the amino acid sequence of a protein.

[0169] However, upon administration of a peptide bearing a T cell epitope and a redox motif, the following events are believed to occur: Antigen activation (i) is the activation of specific T cells resulting from a cognate interaction with an antigen-derived peptide presented by an MHC class II molecule. The reductase sequence reduces T cell surface proteins, such as the CD4 molecule, whose second domain contains a constrained disulfide bridge. This transduces a signal to the T cell. Among the consequences associated with increased oxidative pathways, increased calcium influx and nuclear translocation of the NF-κB transcription factor are key events. The latter leads to increased transcription of IFN-gamma and granzymes, which induces the cell to acquire cytolytic properties via an apoptotic mechanism that affects peptide-presenting cells through a mechanism involving granzyme B secretion and Fas-FasL interaction. Because the cell-killing effect is achieved via the apoptotic pathway, cytolytic cells are a more appropriate term for these cells than cytotoxic cells. Destruction of antigen-presenting target cells prevents activation of other T cells specific for epitopes located on the same antigen, or for unrelated antigens processed by the same antigen-presenting cell; a further consequence of T cell activation is the suppression of activation of bystander T cells by cell-to-cell contact-dependent mechanisms. In such cases, when both cytolytic and bystander T cells are in close proximity, i.e., activated on the surface of the same antigen-presenting cell, T cells activated by antigens presented by different antigen-presenting cells are also suppressed.

[0170] The above-hypothesized mechanism of action has been demonstrated using experimental data disclosed in the above-cited PCT application WO2008 / 017517.

[0171] The present invention provides methods for generating antigen-specific cytolytic CD4+ T cells either in vivo or in vitro, and their use in treating patients stratified or selected as likely to benefit from most of the above treatments. Independently, methods can be envisioned for distinguishing cytolytic CD4+ T cells from other cell populations, such as Foxp3+ Tregs, based on characteristic expression data.

[0172] The present invention describes in vivo methods for the generation of antigen-specific CD4+ T cells for use in therapy in light of the present invention. Particular embodiments relate to methods for generating or isolating CD4+ T cells by immunizing an animal (including a human) with a peptide described herein and then isolating the CD4+ T cells from the immunized animal. The present invention describes in vitro methods for the generation of antigen-specific cytolytic CD4+ T cells against APCs. The present application also discloses methods for generating antigen-specific cytolytic CD4+ T cells against APCs.

[0173] In one embodiment, a method is provided that includes isolating peripheral blood cells, stimulating the cell population in vitro with an immunogenic peptide described herein, and expanding the stimulated cell population, particularly in the presence of IL-2. The method according to the invention has the advantage that large numbers of CD4+ T cells are generated, and that CD4+ T cells that are specific for an antigenic protein can be generated (by using peptides that contain antigen-specific epitopes).

[0174] In an alternative embodiment, CD4+ T cells can be generated in vivo, i.e., by injecting an immunogenic peptide described herein into a subject and collecting the cytolytic CD4+ T cells generated in vivo.

[0175] Antigen-specific cytolytic CD4+ T cells against APCs obtainable by the methods disclosed herein are of particular interest for administration to mammals for immunotherapy in the prevention of allergic reactions and the treatment of autoimmune diseases. The use of both allogeneic and autologous cells is envisioned.

[0176] Cytolytic CD4+ T cell populations are obtained as described herein below. Antigen-specific cytolytic CD4+ T cells as described herein can be used as a medicament, more particularly for use in adoptive cell therapy, more particularly in the treatment of acute allergic reactions and relapses of autoimmune diseases such as multiple sclerosis. Isolated cytolytic CD4+ T cells or cell populations, particularly antigen-specific cytolytic CD4+ T cell populations, generated as described are used for the manufacture of a medicament for the prevention or treatment of immune disorders. Methods of treatment using the isolated or generated cytolytic CD4+ T cells are disclosed.

[0177] As explained in WO2008 / 017517, cytolytic CD4+ T cells against APCs can be distinguished from natural Treg cells based on the cellular expression profile. In particular, cytolytic CD4+ T cell populations demonstrate one or more of the following characteristics compared to natural Treg cell populations: After activation, there is increased expression of surface markers including CD103, CTLA-4, Fasl, and ICOS, intermediate expression of CD25, expression of CD4, ICOS, CTLA-4, GITR, and low or no expression of CD127 (IL7-R), no expression of CD27, expression of the transcription factors T-bet and egr-2 (Krox-20) but not the transcriptional repressor Foxp3, high production of IFN-gamma, and no or minimal production of IL-10, IL-4, IL-5, IL-13, or TGF-beta.

[0178] Furthermore, cytolytic T cells express CD45RO and / or CD45RA, but do not express CCR7, CD27, and display high levels of granzyme B and other granzymes as well as Fas ligand.

[0179] The peptides for use in the present invention, after administration to a living animal, generally a human, elicit specific T cells that exert suppressive activity on bystander T cells.

[0180] In specific embodiments, the cytolytic cell populations disclosed herein are characterized by expression of FasL and / or interferon gamma. In specific embodiments, the cytolytic cell populations of the invention are further characterized by expression of granzyme B.

[0181] This mechanism also suggests, and experimental results show, that the peptides of the present invention, which comprise a specific T cell epitope of a particular antigen, can be used to prevent or treat disorders elicited by immune responses to other T cell epitopes of the same antigen, or, in certain circumstances, for the treatment of disorders elicited by immune responses to other T cell epitopes of other, different antigens when those other, different antigens are presented through the same mechanism by MHC class II molecules in the vicinity of the T cells activated by the peptides of the present invention.

[0182] Disclosed are isolated cell populations of cell types that have the characteristics described above and that are further antigen-specific, i.e., capable of suppressing an antigen-specific immune response.

[0183] The present invention provides the use of pharmaceutical compositions comprising one or more peptides according to the present invention, further comprising a pharmaceutically acceptable carrier. As detailed above, the present invention also relates to compositions for medical use or for the manufacture of medicaments for the prevention or treatment of immune disorders, in combination with methods of patient stratification or selection, which are assumed to be beneficial for most of the above treatments. The pharmaceutical composition is, for example, a vaccine suitable for treating or preventing immune disorders, particularly airborne and food-borne allergies, and diseases caused by allergies. As an example of a pharmaceutical composition further described herein, a peptide according to the present invention is adsorbed onto an adjuvant suitable for administration to mammals, such as aluminum hydroxide (alum). Typically, a desired dosage, as described herein, e.g., 50 μg to 1500 μg of peptide adsorbed onto alum, is injected three times at two-week intervals via the subcutaneous route. It should be apparent to those skilled in the art that other routes of administration are possible, including oral, intranasal, or intramuscular. Furthermore, the number of injections and the amount injected may vary depending on the condition being treated. Additionally, other adjuvants besides alum can be used as long as they promote peptide presentation in MHC class II presentation and T cell activation. Therefore, while it is possible to administer the active ingredients alone, they are generally presented as pharmaceutical formulations. Formulations of the present invention for veterinary and human use contain at least one active ingredient described above together with one or more pharmaceutically acceptable carriers. The present disclosure relates to pharmaceutical compositions containing, as an active ingredient, one or more peptides described herein in admixture with a pharmaceutically acceptable carrier. Pharmaceutical compositions should contain a therapeutically effective amount of an active ingredient, such as those indicated hereinafter for methods of treatment or prevention. Optionally, the composition may further contain other therapeutic ingredients. Suitable other therapeutic ingredients, as well as their typical dosages depending on the class to which they belong, are well known to those skilled in the art and can be selected from other known drugs used to treat immune disorders.

[0184] As used herein, the term "pharmaceutically acceptable carrier" refers to any material or substance with which an active ingredient is formulated to facilitate its application or distribution to the site to be treated, for example, by dissolving, dispersing, or diffusing the composition, and / or to facilitate its storage, transportation, or handling without impairing its efficacy. These include total solvents, dispersion media, coatings, antibacterial and antifungal agents (e.g., phenol, sorbic acid, chlorobutanol), isotonic agents (sugars or sodium chloride, etc.), and the like. Additional components may be included to control the duration of action of the immunogenic peptide in the composition. Pharmaceutically acceptable carriers may be solids or liquids or gases compressed to form liquids; i.e., the compositions of the present invention may be suitably used as concentrates, emulsions, solutions, granules, powders, sprays, aerosols, suspensions, ointments, creams, tablets, pellets, or powders. Suitable pharmaceutical carriers for use in pharmaceutical compositions and their formulations are well known to those skilled in the art, and there is no particular limitation on their selection within the present invention. They may also contain additives such as wetting agents, dispersing agents, spreading agents, adhesives, emulsifiers, solvents, coatings, antibacterial and antifungal agents (e.g., phenol, sorbic acid, chlorobutanol), isotonic agents (sugars or sodium chloride, etc.), etc., provided they are consistent with pharmaceutical practice, i.e., carriers and additives that do not cause permanent harm to mammals. The pharmaceutical compositions of the present invention can be prepared by any known method, for example, by homogeneously mixing, coating, and / or grinding the active ingredient together with the selected carrier material and, if appropriate, other additives, such as surfactants, in a single or multi-step procedure. They can also be prepared by micronization, for example, to obtain them in the form of microspheres, usually having a diameter of about 1 to 10 μm, i.e., for the production of microcapsules for controlled or sustained release of the active ingredient.

[0185] Surfactants, also known as expressing agents or emulsifiers, suitable for use in the pharmaceutical compositions of the present invention are nonionic, cationic, and / or anionic materials with excellent emulsifying, dispersing, and / or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surfactants. Suitable soaps include alkali or alkaline earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), such as sodium or potassium salts of oleic acid or stearic acid, or natural fatty acid mixtures obtainable from coconut or tallow oils. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulfonates and sulfates; sulfonated benzimidazole derivatives and alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the form of alkali or alkaline earth metal salts, unsubstituted ammonium salts, or ammonium salts substituted with alkyl or acyl groups having 8 to 22 carbon atoms, such as the sodium or calcium salts of lignosulfonic acid or dodecylsulfonic acid, or mixtures of fatty alcohol sulfates derived from natural fatty acids, sulfates or sulfonates (e.g., sodium lauryl sulfate), and alkali or alkaline earth metal salts of sulfonic acids of fatty alcohol / ethylene oxide adducts. Suitable sulfonated benzimidazole derivatives generally contain 8 to 22 carbon atoms. Examples of alkylarylsulfonic acids are the sodium, calcium, or alkanolamine salts of dodecylbenzenesulfonic acid, dibutyl-naphthalenesulfonic acid, or naphthalenesulfonic acid / formaldehyde condensation products. Corresponding phosphates, such as salts of phosphoric acid esters, and adducts of p-nonylphenol with ethylene and / or propylene oxide, or phospholipids, are also suitable. For example, suitable phospholipids for this purpose are natural (of animal or plant cell origin) or synthetic phospholipids of the cephalin or lecithin type, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerin, lysolecithin, cardiolipin, dioctanylphosphatidylcholine, dipalmitoylphosphatidylcholine and mixtures thereof.

[0186] Suitable nonionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, fatty amines, or amides containing at least 12 carbon atoms in the molecule, alkyl arenesulfonates, and dialkyl sulfosuccinates; for example, polyglycol ether derivatives of aliphatic and alicyclic alcohols, saturated and unsaturated fatty acids, and alkylphenols, which generally contain 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety, and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable nonionic surfactants are polypropylene glycols containing 1 to 10 carbon atoms in the alkyl chain, and water-soluble adducts of polyethylene oxide with ethylenediaminopolypropylene glycol, which adducts contain 20 to 250 ethylene glycol ether groups and / or 10 to 100 propylene glycol ether groups. Such compounds typically contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of nonionic surfactants include nonylphenol-polyethoxyethanol, castor oil polyglycol ether, polypropylene / polyethylene oxide adduct, tributylphenoxypolyethoxyethanol, polyethylene glycol, and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose, and pentaerythritol are also suitable nonionic surfactants. Suitable cationic surfactants include quaternary ammonium salts, particularly halides, having four hydrocarbon groups, optionally substituted with halo, phenyl, substituted phenyl, or hydroxy; for example, quaternary ammonium salts containing at least one C8C22 alkyl group (e.g., cetyl, lauryl, palmityl, myristyl, oleyl, etc.) as an N-substituent and unsubstituted or halogenated lower alkyl, benzyl, and / or hydroxy-lower alkyl groups as further substituents.

[0187] A more detailed description of surfactants suitable for this purpose can be found, for example, in "McCutcheon's Detergents and Emulsifiers Annual" (MC Publishing Crop., Ridgewood, New Jersey, 1981), "Tensid-Taschenbucw", 2nd Edition (Hanser Verlag, Vienna, 1981), and "Encyclopaedia of Surfactants" (Chemical Publishing Co., New York, 1981). The peptides, homologues or derivatives thereof according to the invention (as well as their physiologically acceptable salts or pharmaceutical compositions, all encompassed by the term "active ingredient") can be administered by any route appropriate to the condition to be treated and to the compound, herein the protein and fragment, being administered. Possible routes include regional, systemic, oral (in solid form or by inhalation), rectal, nasal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraarterial, intrathecal and epidural). The preferred route of administration can vary with, for example, the condition of the recipient or the disease to be treated. As used herein, a carrier is optimally "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient. Formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraarterial, intrathecal and epidural) administration.

[0188] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0189] Typical unit dosage formulations contain a daily dose or unit sub-daily dose of the active ingredient, as set forth hereinabove, or an appropriate fraction thereof. It should be understood that in addition to the ingredients specifically noted above, the formulations of the present invention may contain other agents conventionally used in the art pertaining to the formulation type in question; for example, those suitable for oral administration may contain flavoring agents. The peptides, homologs, or derivatives thereof according to the present invention can be used to provide controlled-release pharmaceutical formulations containing one or more compounds of the present invention as active ingredients ("controlled-release formulations"), in which the release of the active ingredient can be controlled and regulated to allow for less frequent dosing or to improve the pharmacokinetic or toxicity profile of a given invention compound. Controlled-release formulations adapted for oral administration, in which individual units contain one or more compounds of the present invention, can be prepared by conventional methods. Additional ingredients may be included to control the duration of action of the active ingredient in the composition. Thus, controlled-release compositions can be achieved by selecting an appropriate polymeric carrier, such as polyesters, polyamino acids, polyvinylpyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate, and the like. The rate and duration of drug release can also be controlled by incorporating the active ingredient into particles, e.g., microcapsules, of polymeric materials such as hydrogels, polylactic acid, hydroxymethylcellulose, poly(dimethyl methacrylate), and other polymers mentioned above. Such methods include colloid drug delivery systems such as liposomes, microspheres, microemulsions, nanoparticles, nanocapsules, and the like. Depending on the route of administration, pharmaceutical compositions may require protective coatings. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for their immediate preparation. Thus, typical carriers for this purpose include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol, and the like, and mixtures thereof.Taking into account the fact that when several active ingredients are used in combination, they do not necessarily simultaneously produce a joint therapeutic effect in the treated mammal, the corresponding composition may also be in the form of a medical kit or package containing the two ingredients in separate but adjacent repositories or compartments. In this latter connection, each active ingredient may therefore be formulated in a way suitable for a route of administration different from that of the other ingredients, for example, one of them may be in the form of an oral or parenteral preparation and the other in the form of an ampule for intravenous injection or aerosol.

[0190] Cytolytic CD4+ T cells, such as those obtained as described herein, induce APC apoptosis after MHC class II-dependent cognate activation, affecting both dendritic cells and B cells, as demonstrated in vitro and in vivo, and (2) suppress bystander T cells through a contact-dependent mechanism in the absence of IL-10 and / or TGF-β. As discussed in detail in WO2008 / 017517, cytolytic CD4+ T cells can be distinguished from natural and adaptive Tregs.

[0191] The present invention will now be illustrated by the following examples, which are not intended to be limiting. Additionally, all references cited herein are expressly incorporated herein by reference. [Example]

[0192] Example 1 Binding of immunogenic peptides containing insulin MHC II T cell epitopes and oxidoreductase motifs to soluble DRB1*0301 or DRB1*0401 recombinant MHC II proteins To examine the binding of peptides containing the MHC class II T cell epitope and oxidoreductase motif from the proinsulin region C20_A1, a soluble phase competition assay was performed in which increasing concentrations of peptides with the sequences HCPYCVRSLQPLALEGSLQKRG (SEQ ID NO: 25) and HCPYCSLQPLALEGSLQKRG (SEQ ID NO: 26) compete with a labeled control peptide (high affinity binder; biotinylated) for binding to soluble DRB1*0301 or DRB1*0401 recombinant human MHC II protein. Once binding approaches equilibrium (18 hours), peptide-MHC II complexes are captured and separated from unbound reagents. The captured peptide-MHC II complexes are visualized by time-resolved fluorescence (Eu 3+ The peptides were quantitatively detected by streptavidin (Figure 1), and the data were processed and plotted to confirm the dose-dependent binding properties of the test peptides and determine IC50 (decrease in fluorescence intensity reflects peptide binding). All tests using these peptides were performed in triplicate, and each test was performed twice. Figure 1 shows the results of one experiment. Peptides with the sequences HCPYCVRSLQPLALEGSLQKRG (SEQ ID NO: 25) and HCPYCSLQPLALEGSLQKRG (SEQ ID NO: 26) are good binders for the DRB1*0301 and DRB1*0401 haplotypes because they can compete with the high-affinity reference epitope binder used in the assay.

[0193] Example 2 Ability of immunogenic peptides containing insulin MHCII T cell epitopes and oxidoreductase motifs to prime and expand CD4+ T cells from various insulin-dependent diabetes mellitus patients. Naive CD4+ T cells from various type 1 diabetes (T1D) patients were tested for their reactivity to a peptide defined by the sequence HCPYCVRSLQPLALEGSLQKRG. HLA DRB1 typing of T1D patients was first tested (see Table 1).

[0194] [Table 1]

[0195] Blood samples were then processed to purify naive CD4+ T cells by magnetic isolation, and the ability of the peptides to prime and expand these naive CD4+ T cells was examined using peptide-preloaded autologous dendritic cells (monocyte-derived DCs differentiated in the presence of GM-CSF and IL-4, followed by maturation with TNF-α) as antigen-presenting cells.

[0196] Figure 2 shows the reactivity measured by the evolution of CD4+ T cell counts after specific peptide stimulation with autologous dendritic cells over successive restimulations (S1, S4, and S6). The data show good responsiveness (cell maintenance and proliferation) for most of the tested patients, except for two patients (T1D02 & T1D06; cells n=0 from S2 to S6) in whom no significant cell reactivity was observed and who were associated with death already after two stimulations. The data also show that patients who do not express the DRB1*04 haplotype did not show a significant response to peptide stimulation under the treatment test conditions described above. More significantly, these non-responsive T1D patients expressed the DRB1*03 haplotype, for which the peptides showed good binding capacity in competitive HLA binding assays.

[0197] Example 3 Phase Ib clinical trial in T1D patients using an immunogenic peptide with the sequence HCPYCSLQPLALEGSLQKRG. The safety, clinical efficacy, and induced immune responses of an immunogenic peptide with the sequence HCPYCSLQPLALEGSLQKRG were evaluated in adult T1D patients with recent-onset disease (within 6 months) in a Phase 1b clinical trial. In this dose-escalating, placebo-controlled study, patients received four biweekly subcutaneous injections of one of three test doses or a matching placebo. The peptide was injected with alum as an adjuvant. Patients were followed for 6 months to assess the safety of the peptide and the induced immune responses.

[0198] The main selection criteria are: - Men and women aged 18 to 30, BMI 17 to 28 kg / m 2 , - Initial diagnosis of type 1 diabetes according to ADA / WHO criteria within the past 6 months, - insulin needs as determined by the researcher, - HLA-DR3 positive and / or HLA-DR4 positive, - the presence of at least one autoantibody (GAD65, IA2 or ZnT8), - Fasting C-peptide ≥ 0.2 nmol / L and / or stimulated C-peptide ≥ 0.4 nmol / L at screening It was.

[0199] Figure 3 shows the scheme of the Phase Ib study design. Patients were divided into three cohorts: The low-dose cohort (Cohort 1) included 8 patients (6 treated and 2 placebo) who received an SC injection of 50 μg of peptide, followed by 3 consecutive injections of 25 μg of peptide. The 4 injections were administered 2 weeks apart. Patients were then followed up for up to 24 weeks. - The medium dose (Cohort 2) included 12 patients (9 treated and 3 placebo) who received an SC injection of 150 μg of peptide followed by three consecutive injections of 75 μg of peptide. The four injections were administered two weeks apart. Patients were then followed up for up to 24 weeks. - The higher dose (Cohort 3) included 21 patients (16 treated and 5 placebo) who received an SC injection of 450 μg of peptide followed by three consecutive injections of 225 μg of peptide. The four injections were administered two weeks apart. Patients were then followed up for up to 24 weeks.

[0200] A data mining analysis using Ariana Pharma's KEM® (Knowledge Extraction and Management) artificial intelligence technology, which performs systematic analysis of all data associations, was performed on the complete data set from the clinical trial. This approach aimed to identify subgroups of patients with a propensity for improvement in clinical parameters. During this analysis, HLA genotype was considered an important factor and was taken into account when assessing future clinical response. Initial data mining results indicated that in Cohort 3 (the higher dose tested), multiple parameters at multiple time points appeared to improve in patients with the HLA-DRA4(+) genotype as well as in patients with the HLA-DR3(-) genotype. Importantly, and reinforcing this observation, patients with the HLA-DR4(-) genotype did not show improvement in the same parameters at different time points under these Phase 1b study conditions. These initial findings are summarized in Table 2 below.

[0201] [Table 2]

[0202] DNA isolation was performed using the Chemagic STAR DNA Blood Kit for Hamilton Robot (Chemagen) according to the analysis plan provided by IMGM, and eluted in 150 μl of Tris-HCl (pH 8.0). Low-resolution HLA typing was performed according to the LABType® SSO Method using sequence-specific oligonucleotide (SSO) probes bound to fluorescently encoded microspheres to identify the alleles encoded by the sample DNA. LABType applied Luminex® technology to the reverse SSO DNA typing method (https: / / www.onelambda.com / en / product / labtype-sso.html) and was evaluated using HLA Fusion™ software. For high-resolution HLA typing, so-called long-range PCR according to SOP AA-1550 was used, which was sequenced using Illumina technology. Evaluation of these sequences was performed using GenDX NGSengine Version 2.13.0 (https: / / www.gendx.com / products / ngsengine).

[0203] Based on this initial, hypothesis-free, driven view, clinical parameters were examined in different subpopulations of the clinical trial according to the expression of HLA genotypes, more precisely HLA-DR3 and HLA-DR4 haplotypes. Table 3 summarizes the distribution of the different genotypes in the clinical trial. As can be observed, the different groups (placebo, cohort 1, cohort 2, and cohort 3) are not balanced with respect to the different genotype combinations. This imbalance is purely due to the small size of the study.

[0204] [Table 3]

[0205] As examples of clinical parameters, the evolution of the C-peptide area under the curve (AUC) during the mixed meal tolerance test (MMTT) and the total daily insulin dose per kg were examined according to the HLA-DR genotype of various patients (Figures 4 and 5, respectively). Notably, patients treated with medium or high doses of the peptide HCPYCSLQPLALEGSLQKRG and expressing HLA-DR4 (DR4(+) or DR3(-)) showed a positive trend for these two endpoints 6 months (Visit 8, V8) after entering the study (Visit 2, V2). This was not observed in the population that did not express HLA-DR4 (DR4(-)) under the test conditions of the phase Ib clinical trial.

[0206] A provisional T1D disease progression model over the first two years after diagnosis has been described by Greenbaum et al. (Diabetes. 2012, 61(8):2066-73) based on a large amount of data accumulated in different clinical trials with newly diagnosed patients in this population aged 7-45 years. 86% of patients in this population were DR3 or DR4 positive. The model used C-peptide secretion as measured through a 2-hour or 4-hour MMTT test. We used this model to compare the progression of our patients with two goals: first, a safety aspect, which confirmed that our treated patients did not show disease progression (i.e., they were less likely to progress faster than the model); and second, an efficacy aspect, with the prediction that treated patients would progress slower than the model. Consistent with the second aspect, we observed that the DR4+ and DR3- subpopulations showed a tendency to improve at 3 months (V6) and 6 months (V8) in cohorts 2 and 3 (median C-peptide decline slower than the model, delta ratio greater than 0), and this was also the case in the placebo group, whereas the HLA-DR4(-) subpopulation had an opposite clinical response under the conditions tested in the Phase Ib clinical trial (Figure 6). Interestingly, in cohort 3 (the highest dose tested), there was a significant difference in the progression of the HLA-DR4(+) and HLA-DR4(-) subpopulations. This significance was not achieved in the other cohorts or in the placebo group.

[0207] The same subgroup difference is also observed over time for the total daily insulin dose per kg. This parameter shows a tendency to decrease for the subpopulation of HLA-DR4+ and DR3(-) patients in Cohort 2 and Cohort 3, which is a positive response to treatment. On the other hand, HLA-DR4(-) patients do not show this positive outcome under the test conditions of the Phase Ib clinical trial (Figure 7). With regard to this parameter, the progress over time in the placebo group is more heterogeneous. The effectiveness of the peptide of SEQ ID NO: 26 in DR3+ and DR4+ individuals will be further investigated in larger studies with larger sample sizes and stratification by HLA type.

Claims

1. 1. An in vitro method for predicting the response of a type 1 diabetes patient to treatment with an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated by 0 to 7 amino acids from said motif, wherein said oxidoreductase motif comprises the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. wherein the method comprises determining the patient's MHC class II HLA haplotype, and patients determined to be HLA-DR4 positive (HLA-DR4+) are predicted to be responsive to the treatment.

2. 2. The method of claim 1, wherein the (pro)insulin MHC class II T cell epitope sequence is defined by the amino acid sequence LALEGSLQK [SEQ ID NO: 3].

3. The method of claim 1 or 2, wherein the responsive patient is homozygous or heterozygous HLA type DR4 positive.

4. 4. The method of any one of claims 1 to 3, wherein the haplotyping in the patient is performed using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis, or through antibody testing.

5. 1. Use of an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated by 0 to 7 amino acids from said motif, for the manufacture of a medicament for use in a method for reducing an immune response to an autoimmune antigen selected from (pro)insulin or C-peptide in a patient, wherein said oxidoreductase motif is selected from the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. wherein the patient is selected based on the presence of a DR4-positive MHC class II HLA haplotype.

6. 1. Use of an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated by 0 to 7 amino acids from the motif, for the manufacture of a medicament for use in a method of treating or preventing type 1 diabetes in a patient selected on the basis of the presence of a DR4-positive MHC class II HLA haplotype, wherein the oxidoreductase motif is selected from the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. Including, use.

7. 7. The use according to claim 6, wherein the medicament is administered to a patient selected based on the presence of DR4 positive (HLA-DR4+) and HLA-DR3 negative (HLA-DR3-) MHC class II HLA haplotypes.

8. 8. The use according to claim 5 or 7, wherein the (pro)insulin MHC class II T cell epitope sequence is defined by the amino acid sequence LALEGSLQK [SEQ ID NO: 3].

9. 9. The use according to any one of claims 5, 7 and 8, wherein the MHC class II HLA haplotype of the patient is determined before or during treatment.

10. The use according to any one of claims 5 and 7 to 9, wherein the haplotyping is carried out using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis or through antibody testing.

11. 11. Use according to any one of claims 5 and 7 to 10, wherein patients who are homozygous for the HLA type DR4+ are considered to be the most responsive, and / or patients who are heterozygous for the HLA type DR4+, e.g. patients who are DR4+ and DR3+, are considered to be intermediate responsive.

12. The use according to any one of claims 5 and 7 to 11, wherein the haplotyping in the patient is carried out using polymerase chain reaction (PCR)-based analysis, sequence analysis, electrophoresis analysis or through antibody testing.

13. The use according to any one of claims 5 and 7 to 12, wherein the peptide is administered in a dosage regimen of 50 to 1500 μg, preferably 100 to 1200 μg.

14. 14. The use according to any one of claims 5 and 7 to 13, wherein the peptide is administered in a single dose or in two, three, four, five or more doses simultaneously or sequentially.

15. The peptide has the following scheme: 1) a first subcutaneous injection of 50 μg of the peptide, followed by three consecutive subcutaneous injections of 25 μg of the peptide, each administered two weeks apart; 2) a first subcutaneous injection of 150 μg of the peptide, followed by three consecutive subcutaneous injections of 75 μg of the peptide, each administered two weeks apart; and 3) A first subcutaneous injection of 450 μg of the peptide, followed by three consecutive subcutaneous injections of 225 μg of the peptide, each administered two weeks apart.

15. The use according to any one of claims 5 and 7 to 14, wherein the compound is administered via four biweekly subcutaneous or intramuscular injections according to any one of the following:

16. The use according to any one of claims 5 and 7 to 15, wherein the patient is further HLA-DR3 negative (HLA-DR3-).

17. The use according to any one of claims 5 and 7 to 16, wherein the peptide is administered as a pharmaceutical composition comprising the peptide and a pharmaceutically acceptable carrier.

18. The use according to any one of claims 5 and 7 to 17, wherein the peptide is administered as a pharmaceutical composition comprising the peptide and an adjuvant.

19. - providing peripheral blood cells; - contacting said cells in vitro with an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from said motif by 0 to 7 amino acids, said oxidoreductase motif comprising the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. a contacting step comprising: - expanding the cells in the presence of IL-2; 1. An in vitro method for predicting the response of a type 1 diabetes patient to treatment with a population of cytolytic CD4+ T cells against APCs presenting an insulin epitope, the population being obtained by a method comprising the steps of:

20. The method of claim 19, wherein the patient is further HLA-DR3 negative (HLA-DR3-).

21. 1. Use of a population of cytolytic CD4+ T cells directed against APCs presenting insulin epitopes for the manufacture of a medicament for use in a method for reducing an immune response to an autoimmune antigen selected from (pro)insulin or C-peptide in a patient, wherein said population of cytolytic CD4+ T cells comprises: - providing peripheral blood cells; - contacting said cells in vitro with an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from said motif by 0 to 7 amino acids, said oxidoreductase motif comprising the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. a contacting step comprising: - expanding the cells in the presence of IL-2; and wherein the patient is selected based on the presence of a DR4-positive MHC class II HLA haplotype.

22. The use according to claim 21, wherein the selected patient is further HLA-DR3 negative (HLA-DR3-).

23. 1. Use of a population of cytolytic CD4+ T cells directed against APCs presenting an insulin epitope for the manufacture of a medicament for use in a method of treating or preventing type 1 diabetes in a patient selected based on the presence of a DR4-positive MHC class II HLA haplotype, wherein the population of cytolytic CD4+ T cells comprises: - providing peripheral blood cells; - contacting said cells in vitro with an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from said motif by 0 to 7 amino acids, said oxidoreductase motif comprising the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. a contacting step comprising: - expanding the cells in the presence of IL-2; The use of an insulin-antibody-specific antibody, which is obtained by an in vitro method for generating a population of cytolytic CD4+ T cells against APCs presenting insulin epitopes, comprising:

24. The use according to claim 23, wherein the patient is further HLA-DR3 negative (HLA-DR3-).

25. The oxidoreductase motif may comprise the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 3, m represents an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents a basic amino acid. The use according to any one of claims 5, 7 to 18 and 21 to 24, comprising:

26. 26. The use according to any one of claims 5, 7 to 18 and 21 to 25, wherein the oxidoreductase motif comprises the tetrapeptide sequence Cxx[CST][SEQ ID NO:1] or [CST]xxC[SEQ ID NO:2].

27. The use according to any one of claims 5, 7 to 18 and 21 to 26, wherein the MHC class II T cell insulin epitope can be defined by the sequence LALEGSLQK [SEQ ID NO: 3].

28. 28. The use according to any one of claims 5, 7 to 18 and 21 to 27, wherein the peptide comprises the sequence Cxx[CST]SLQPLALEGSLQK [SEQ ID NO: 4] or [CST]xxCSLQPLALEGSLQK [SEQ ID NO: 5].

29. 29. The use according to any one of claims 5, 7 to 18 and 21 to 28, wherein the peptide comprises the sequence CxxCSLQPLALEGSLQK [SEQ ID NO: 6].

30. 30. The use according to any one of claims 5, 7 to 18 and 21 to 29, wherein the peptide comprises the sequence HCxx[CST]SLQPLALEGSLQK [SEQ ID NO: 7] or H[CST]xxCSLQPLALEGSLQK [SEQ ID NO: 8].

31. The use according to any one of claims 5, 7 to 18 and 21 to 30, wherein the peptide comprises the sequence HCxxCSLQPLALEGSLQK [SEQ ID NO: 9].

32. 32. The use according to any one of claims 5, 7 to 18 and 21 to 31, wherein the peptide comprises the sequence Cxx[CST][SEQ ID NO:1] or [CST]xxC SEQ ID NO:2] redox motif sequence and the sequence SLQPLALEGSLQKRG [SEQ ID NO:20].

33. 33. The use according to any one of claims 5, 7 to 18 and 21 to 32, wherein the peptide comprises or consists of the amino acid sequence HCPYCSLQPLALEGSLQKRG [SEQ ID NO: 26].

34. 1. An agent for reducing an immune response in a patient against an autoimmune antigen selected from (pro)insulin or C-peptide, comprising an immunogenic peptide having a length of 12 to 50 amino acids, comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated by 0 to 7 amino acids from the motif, wherein the oxidoreductase motif is the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. wherein the patient is selected based on the presence of a DR4-positive MHC class II HLA haplotype.

35. 1. An agent for treating or preventing type 1 diabetes in a patient selected on the basis of the presence of a DR4-positive MHC class II HLA haplotype, comprising an immunogenic peptide having a length of 12 to 50 amino acids, comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated by 0 to 7 amino acids from the motif, wherein the oxidoreductase motif is the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. An agent comprising:

36. 1. An agent for reducing an immune response to an autoimmune antigen selected from (pro)insulin or C-peptide in a patient, the agent comprising a population of cytolytic CD4+ T cells directed against APCs presenting insulin epitopes, wherein the population of cytolytic CD4+ T cells is - providing peripheral blood cells; - contacting said cells in vitro with an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from said motif by 0 to 7 amino acids, said oxidoreductase motif comprising the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. a contacting step comprising: - expanding the cells in the presence of IL-2; wherein the patient is selected based on the presence of a DR4-positive MHC class II HLA haplotype.

37. 1. An agent for treating or preventing type 1 diabetes in a patient selected based on the presence of a DR4-positive MHC class II HLA haplotype comprising a population of cytolytic CD4+ T cells directed against APCs presenting an insulin epitope, wherein the population of cytolytic CD4+ T cells comprises: - providing peripheral blood cells; - contacting said cells in vitro with an immunogenic peptide having a length of 12 to 50 amino acids comprising an oxidoreductase motif and a (pro)insulin MHC class II T cell epitope sequence separated from said motif by 0 to 7 amino acids, said oxidoreductase motif comprising the motif: Zm[CST]XnC or ZmCXn[CST] (wherein n is an integer from 0 to 6, m is an integer from 0 to 2, C represents cysteine, S represents serine, T represents threonine, X represents any amino acid, and Z represents any amino acid, preferably a basic amino acid. a contacting step comprising: - expanding the cells in the presence of IL-2; The agent is obtained by an in vitro method for generating a population of cytolytic CD4+ T cells against APCs presenting insulin epitopes, comprising:

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