WO2025219454A1 - Preproinsulin tolerogenic fragments for treating and monitoring type 1 diabetes - Google Patents

Abstract

The present invention relates to methods for predicting and monitoring of the course of type 1 diabetes with the use of detection of fragments of preproinsulin from sera of the patients, specifically in patients treated with cell therapies with CD4+ Fox P3+ T regulatory cells (in the following Treg cells" or "Tregs"). The present invention relates also to the possibility of the use of the said fragments of preproinsulin in the treatment of patients with type 1 diabetes directly or as agents used during manufacturing of antigen-specific Tregs for the treatment of type 1 diabetes.

Description

Preproinsulin tolerogenic fragments for treating and monitoring type 1 diabetes

FIELD OF THE INVENTION

The present invention relates to the field of diabetes mellitus monitoring and treatment. More precisely, the present invention relates to methods for predicting and monitoring of the course of type 1 diabetes, by detecting specific fragments of preproinsulin from sera of the patients, especially in patients treated with cell therapies with CD4+ Fox P3+ T regulatory cells (in the following “Treg cells” or “Tregs”). The present invention relates also to the use of said fragments of preproinsulin in the treatment of patients with type 1 diabetes, directly (by administration of peptide compositions to the patients) or as agents used during manufacturing of antigen-specific Tregs for the treatment of type 1 diabetes.

BACKGROUND OF THE INVENTION

Type 1 diabetes mellitus (DM1) is a lifelong (chronic) disease in which there are low plasma insulin levels following destruction of p-cells of the pancreas. As part of the natural progression of this disease, some patients regain p-cells activity after DM1 diagnosis and beginning of a treatment. During this period, called DM1 remission, patients manifest transiently improved glycemic control with reduced or no use of insulin or anti-diabetic drugs.

Current treatment of type 1 diabetes involves lifelong injections of insulin. Without this treatment, the disease progresses rapidly and leads to severe complications such as diabetic cardiomyopathy, stroke, kidney failure, diabetic retinopathy, diabetic foot ulcers, ketoacidosis, and diabetic coma, all of which may lead to disability or result in the patient's death.

Both clinical and laboratory data have proved the importance of the immune system in the pathogenesis of type 1 diabetes mellitus (DM1). While the triggering factor may be a viral infection or genetic susceptibility, the disease develops due to an imbalance between exaggerated autoaggressive T cells responses and impaired tolerogenic mechanisms (Budd et al., 2021 ; Raugh et al., 2022). The most promising disease-modifying strategies are therefore created around immunotherapy (Roep, 2023; Roep et al, 2021 ; Herold et al., 2019). Among many ongoing attempts to stop or at least delay DM1 , cell therapy with FoxP3+ T regulatory cells (Treg) seems to be of particular interest. The present inventors and others performed several clinical trials with T-regulatory cells products with promising results (Herold et al., 2019; Dong et al., 2021 ; Zielinski et al., 2022; Bluestone et al., 2015; Marek-Trzonkowska et al., 2014; Marek-Trzonkowska et al., 2012).

There are two major challenges for all these therapies. Firstly, there is a need to find an excellent immune marker to predict the treatment response, which correlates with p-cell destruction leading to the DM1 (Trzonkowski et al., 2015). The tissue available for sampling is usually peripheral blood, which is very distant from local tissue lesions. Thus, the function of p-cells remains the only acceptable endpoint of therapeutic efficacy so far (Roep et al., 2021). Unfortunately, this sort of monitoring allows to detect the disease relatively late, when the destruction of the islets is ongoing. It only shows the progression of DM1 and does not allow to predict the efficacy of the treatment at an early stage, at the time of the therapy administration. A second important need is the improvement in the efficacy of Treg cells involved in imposing the tolerance in type 1 diabetes patients.

The technical problem underlying the present invention is thus the provision of means for predicting the course of type 1 diabetes, specifically in patients treated with cell therapies using Treg cells. Another aim of the invention is to manipulate the immune system of type 1 diabetes patients in order to stop or delay the progression of this disease.

SUMMARY OF THE INVENTION

The present invention is based on the demonstration that, as set out in detail in the experimental part below, certain fragments of preproinsulin are useful in the prediction of the course of type 1 diabetes, specifically in patients administered with cell therapeutic regimen consisting of Tregs or Tregs and a B-cell depleting agent such as rituximab. The biomarkers identified as particularly useful for this purpose are fragments of preproinsulin (used alone or in combination) described in details below.

According to a first aspect, the present invention pertains to an in vitro method of diagnosing type 1 diabetes (DM1) in an individual, comprising assessing the presence of peptides selected from the group consisting of peptides of SEQ ID Nos: 1 , 4, 11, 13, 14, 16, 20, 22, 25, 26, 29, 41 , 48, 49, 52, 53, 58 62, 64, 66, 68, 70, 72 and 74 in a biological sample from said individual, wherein the presence of one or several of these peptides in said sample is indicative that the individual has DM1.

According to another aspect, the present invention relates to an in vitro method of predicting the course of DM1 in an individual diagnosed with DM1 , comprising assessing the presence of peptides selected from the group consisting of

(i) peptides of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 (protolerogenic peptides); and (ii) peptides of SEQ ID Nos: 14, 25, 53 and 58 (disease-accelerating peptides) in a biological sample from said individual, wherein: the presence of one or several protolerogenic peptides and/or the absence diseaseaccelerating peptides are indicative of a good clinical outcome and/or the presence of one or several disease-accelerating peptides and/or the absence of protolerogenic peptides are indicative of unfavorable DM1 evolution.

The invention also pertains to an in vitro method for monitoring a response of an individual having DM1 to a treatment (e.g., a cell therapy treatment with Treg cells, possibly combined to a B-cell depleting treatment such as rituximab), by performing the above method in biological samples obtained from the individual at different time points, wherein the presence of a stable or increasing number of protolerogenic peptides indicate that the individual responds to the treatment.

A pharmaceutical composition comprising at least one protolerogenic peptide as described above or at least one larger region selected from the group consisting of SEQ ID Nos: 72 and 75-79 is also part of the present invention, as well as its use for treating DM1. In such a composition, the peptide can be present as such or be replaced by a nucleic acid encoding it, such as a mRNA, so that it will be expressed in the cells.

According to another aspect, the invention relates to a method for in vitro obtaining antigen-specific Tregs appropriate for the treatment of DM1 , comprising incubating Treg cells with at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 and/or with at least one peptide selected from the group consisting of SEQ ID Nos: 75-79, in conditions enabling preproinsulin-specific Treg cells expansion.

Antigen-specific Treg cells obtainable by the above method and their use for treating DM1 are also part of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Study flow diagram for the clinical trial and healthy control sample acquisition according to Example 1.

Figure 2: Proliferation of Tregs and Tconvs stimulated with monocytes loaded with peptides. Baseline proliferation = proliferation in NonP conditions.

Figure 3: Efficiency of expansion of antigen-specific Tregs

Antigen-specific T regulatory cells (Tregs) and T effector cells (Effs) were expanded with the mixtures of the peptides identified in the patent: MIX1- Protolerogenic (PT) peptides, MIX2 - Disease-accelerating (DA) peptides and MIX3 - R1-R6 peptides. The initial exposure of the cells on monocytes loaded with the mixtures of the peptides resulted in the proliferation Tregs and Effectors, which could be assessed as a percentage of responding cells specific to particular MIXes (A - left chart). The ratio of the percentage of specific Tregs to specific Effs was counted as indexes to assess the prevalent activity of the MIXes towards either Tregs or Effs (A - right chart).

After sorting of Tregs and Effs to the subsets specific (Spec) and unspecific (llnspec) to particular MIXes (Tregs-MIX1 , Tregs-MIX2, Tregs-MIX3, Effs- MIX1 , Effs-MIX2, Effs-MIX3) the cells were expanded for 12 days and the final number of expanded cells was counted (Cells [mln]) (B - upper chart). The ratio of the percentage of specific Tregs to specific Effs and unspecific Tregs to unspecific Effs was counted as indexes to assess the prevalent activity of the MIXes towards either Tregs or Effs (B - lower chart).

Figure 4: Quality of antigen-specific Tregs.

Antigen-specific Tregs were expanded with mixtures of the peptides identified in Example 1 : MIX1- Protolerogenic (PT) peptides, MIX2 - Diseaseaccelerating (DA) peptides and MIX3 - R1-R6 peptides. The following subsets of Tregs were analysed (X axis): MIX1 - specific Tregs (MIX1_Spec), MIX1 - unspecific Tegs (MIX1_unspec), MIX2 - specific Tregs (MIX2_Spec), MIX2 - unspecific Tegs (MIX2_unspec), MIX3 - specific Tregs (MIX3_Spec), MIX3 - unspecific Tegs (MIX3_unspec) and polyclonal Tregs (Poly). The percentage of cells with the expression of FoxP3 was analysed [A] and the percentage of naive CD45RA+CD62L+ Tregs (Tn), T central memory CD45RA+CD62L- Tregs (Tern) and T effector memory CD45RA+CD62L- Tregs (Tern) [B] was assessed in each subset of expanded Tregs.

Figure 5: Suppressive Activity of antigen-specific Tregs

Suppressive Activity of Tregs stimulated with three mixtures of the peptides identified in Example 1 : MIX1- Protolerogenic (PT) peptides, MIX2 - Diseaseaccelerating (DA) peptides and MIX3 - R1-R6 peptides. The Immune suppression tests were performed, where responder cells were PBMC (A), CD4+ T Effector polyspecific cells - EFFs poly (B), CD4+ T Effector antigen-specific cells - EFFs spec (C), CD4+ T Effector antigen-unspecific cells - EFFs unspec (D) where the specificity was towards one of the three mixtures of peptides used. For each test condition, the responders were cocultured with one of the three populations of Tregs (X axis): Tregs unspecific to particular mix of peptides (Tregs_MIXn_unspec), Tregs specific to particular mix of peptides (Tregs_MIXn_spec) and polyclonal Tregs (Tregs_poly). Tregs were cocultured with responders in different Tregs: responders ratios as stated in X axis were the first number is the proportion of Tregs and the second is the proportion of responders with serial increase in the proportion of responders from 1 to 8 [and 1 = 1x10⁵ cells). The positive reference was the culture of responders with antiCD3/antiCD28 beads. All the responders were stained with fluorescent dye V450 prior to commencing the test and the dilution of the dye at the end of the tests due to cell proliferation acquired on flow cytometer was used to assess the percentage of suppression. Each readout was referenced to concomitant readout of the responders cultured with beads using the following formula:

Figure 6: Suppression of immune activation by the peptide mixtures

CD4+ T regulatory cells (Tregs) were stained with VPD450 and CD4+ T effector cells (Effs) with CT Yellow and mixed 1 :1 :1 with autologous PBMC. The cocultured cells were subsequently stimulated with peptide mixtures. The following peptide mixtures were used: MIX1- Protolerogenic (PT) peptides, MIX2 - Disease-accelerating (DA) peptides and MIX3 - R1-R6 peptides. Unstimulated cocultures (unstim) were used as a negative control and the cocultures stimulated with beads (Beads) were used as positive control. The expression of activation markers CD69 and HLA-DR was assessed after 24h, 48h and 72h separately on Tregs and Effectors and presented as a percentage of positive cells: T effectors expressing HLA-DR or CD69 (Effs HLA-DR, Effs CD69, respectively) and T regulatory cells expressing HLA-DR or CD69 (Tregs HLA-DR, Tregs CD69, respectively).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will prevail. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein, the terms “patient,” “subject,” and “individual” can be used interchangeably and refer to a human.

Unless the context clearly dictates otherwise, the singular forms “a”, “an” and “the” include plural referents. For example, reference to “a therapeutic agent” means one or more therapeutic agents.

Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps.

The terms “treatment”, “treating”, and the like, as used herein mean ameliorating or alleviating characteristic symptoms or manifestations of a disease or condition, e.g., DM1. For example, treatment of DM1 can result in the restoration or induction of antigen-specific immune tolerance in the subject. In other examples, treatment means arresting auto-immune diabetes, or reversing autoimmune diabetes. For example, treatment may result in the maintenance of remaining p-cell mass. In other examples, treatment of DM1 involves increasing the frequency or activation of Treg cells. In other examples, treatment may expand antigen-specific Treg cells (e.g., in the thymus), and/or induces migration of Treg cells into peripheral blood. In yet other examples, treatment involves improving at least one of a subject's (a human patient) clinical markers. For example, treatment may raise blood and/or urine C-peptide levels. In other examples, treatment may lower the subject's blood glucose levels (e.g., in response to food ingestion or fasting glucose levels), reduce the amount of injected insulin required to maintain appropriate blood glucose levels in the subject, reduce diabetes-related auto-antibody levels in a subject, and/or increase/preserve C-peptide levels (e.g., following an oral glucose tolerance test). The term “treatment” as used herein also encompasses a delay in the development of DM1 such as a delay of at least about 6 months, preferably at least about 1 year, more preferably at least about 1 ,5 years, still further preferred at least about 2 years. As used herein, “treating” also encompasses, preventing or slowing down the evolution of DM1 or of symptoms associated with DM1. “Preventing” also encompasses preventing the recurrence or relapse of DM1 or of symptoms associated therewith, for instance after a period of remission or improvement. Treatment can mean continuous/chronic treatment, or treatment in which the subject is free of clinical symptoms of the disease or condition for a significant amount of time (e.g., at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years), after the treatment is stopped.

As used herein, the terms “disorder,” “condition,” or “disease” refer to type 1 diabetes (DM1) and associated comorbidities and symptoms.

It is noted that terms like “preferably,” “commonly,” and “advantageously” are not utilized herein to limit the scope of the methods and compositions as described herein nor to imply that certain features are critical or essential to the structure or function of the subject matter recited in the claims.

Peptides are chains of amino acid monomers joined together by amide bonds. The peptides of the present invention comprise or consist essentially of fragment of the naturally occurring human preproinsulin protein. According to a particular embodiment, a peptide according the present invention consists of a fragment of the naturally occurring human preproinsulin protein. The term “peptide” as used herein does not specify or exclude post-expression modifications of peptides, for example, peptides which include covalent attachment of glycosyl groups, acetyl groups, phosphate groups and the like are expressly encompassed by the term peptide. The peptides are generally made from naturally occurring L form amino acids. Peptides according to the present invention may contain one or more D-form. In the context of the present invention, a peptide with a defined sequence also designates its retro-inverso cognate peptide. Also included within the definition are peptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The peptides of the present invention may be generated by any methods known in the art including chemical synthesis, digestion of proteins, or recombinant technology.

Other definitions will be specified below, when necessary.

In the experimental part which follows, the inventors have identified peptide fragments of preproinsulin which are significantly more frequently present in the serum of induviduals having type 1 diabetes mellitus (DM1) than in the serum of individuals not having DM1.

Amongst these peptides more frequently detected in the sera of DM1 patients, the inventors identified preproinsulin fragments correlating with long-term remission of type 1 diabetes, designated in the present text as “protolerogenic peptides”, and preproinsulin fragments correlating with progression of type 1 diabetes of type 1 diabetes, designated herein as “disease-accelerating peptides”. They also identified peptides frequently present in the serum of individuals, regardless of the clinical status of said individuals. Such peptides are herein called “ubiquitous peptides”. These peptides are listed in Table 1 below.

Table 1 : protolerogenic, disease-accelerating and ubiquitous preproinsuling fragments

The presence of protolerogenic peptides and/or disease-accelerating peptides in the serum of an individual diagnosed with DM1 provide information regarding the evolution of DM1 in said individual. According to a first aspect, the present invention thus pertains to an in vitro method of predicting the course of DM1 in an individual diagnosed with DM1 , comprising assessing the presence of peptides selected from the groups consisting of

(i) peptides of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 (protolerogenic peptides); and (ii) peptides of SEQ ID Nos: 14, 25, 53 and 58 (disease-accelerating peptides) in a biological sample from said individual (e.g., serum, plasma or blood sample), wherein the presence of one or several protolerogenic peptides and/or the absence disease-accelerating peptides are indicative of a good clinical outcome.

In the context of DM1 , a “good clinical outcome” means a preservation of the patient’s insulin secretion, possibly leading to long-term remission of DM1.

As mentioned above, “DM1 remission” or “remission” is a period during which patients manifest improved glycemic control with reduced or no use of insulin or anti-diabetic drugs. Almost 80% of children and adolescents show a decline of initial insulin requirements accompanied by an increase of endogenous insulin production. The duration of remission is extremely variable, but usually does not exceed a few months. Various factors seem to influence the remission rates and duration, such as C-peptide level, serum bicarbonate level at the time of diagnosis, duration of DM1 symptoms, haemoglobin A1C (HbA1C) levels at the time of diagnosis, sex, and age of the patient. Mechanism of remission is not clearly understood. Remission seems to be a valuable positive factor for the further DM1 course, as the preservation of p-cell function may decrease the risk of developing vascular complications and the risk of severe hypoglycaemia. In the present text, a “long-term remission” designates a DM1 remission of at least one year, preferably at least two years.

The present invention also pertains to an in vitro method of predicting the course of DM1 in an individual diagnosed with DM1 , comprising assessing the presence of peptides selected from the group consisting of

(i) peptides of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 (protolerogenic peptides); and

(ii) peptides of SEQ ID Nos: 14, 25, 53 and 58 (disease-accelerating peptides) in a biological sample from said individual (e.g., serum, plasma or blood sample), wherein the presence of one or several disease-accelerating peptides and/or the absence of protolerogenic peptides are indicative of unfavorable DM1 evolution.

As used herein, the phrase “unfavorable DM1 evolution” designates a decrease of insulin secretion in said patient. An unfavorable peptides pattern in a patient in remission thus indicates a loss of remission.

Interestingly, in the cohort of DM1 patients studied by the inventors, the presence of protolerogenic peptides and the presence of disease-accelerating peptides were mutually exclusive (/.e., no DM1 patient was found to bear both a protolerogenic peptide and a disease-accelerating peptide). The inventors also observed that the presence of any (even single) protolerogenic peptide indicates a better course of the disease, whereas the presence of any disease-accelerating peptide (even single) indicates a risk of accelerated worsening.

In some embodiments of the above methods, the individual has recent onset DM1. In some embodiments, the individual has long term DM1. The above methods are preferably performed to predict the course of DM1 in a patient having a residual insulin secretion (whatever the duration of DM1).

Residual insulin secretion can originate from native pancreas p-cells which have not (yet) been destroyed, or from transplanted p-cells originating from pancreas transplantation or pancreatic islet transplantation.

Pancreas transplantation (alone or following prior kidney transplantation) may occur in a patient with insulin dependent diabetes. Pancreas retransplantation may be considered after a failed primary pancreas transplant.

Pancreatic islet transplantation is a procedure in which only the islets of Langerhans, which contain the endocrine cells of the pancreas, including the insulin producing p-cells and glucagon producing a-cells, are isolated and transplanted into a patient. Pancreatic islet allotransplantation occurs when islets of Langerhans are isolated from one or more human donor pancreas. Pancreatic islet cells may also be derived from human embryonic stem cells or induced pluripotent stem cells. Pancreatic islet xenotransplantation occurs when islets of Langerhans are isolated from one or more non-human pancreas (e.g., a porcine pancreas or primate pancreas).

Pancreatic islet transplantation can occur in combination with the administration of immunosuppressive agents and/or in the context of an encapsulation device to protect the transplanted pancreatic islet cells from the host autoimmune response, while allowing glucose and nutrients to reach the transplanted pancreatic islet cells.

As outlined above, the inventors observed that the presence of only one protolerogenic or disease-accelerating peptide is enough to provide information on the course of DM1. According to a particular embodiment, the above methods of predicting the course of DM1 are carried out by assessing the presence of at least 15, preferably at least 20 of the recited peptides. According to a particular embodiment, the presence of all the protolerogenic peptides identified above is assessed. According to another particular embodiment, the presence of all the disease-accelerating peptides identified above is assessed. According to yet another particular embodiment, the presence of all the protolerogenic peptides and all the disease-accelerating peptides identified above is assessed.

When performing the above methods, any appropriate technique known by the skilled person can be used to assess the presence of the recited peptides. In Example 1 below, the peptides have been detected using mass spectrometry. However, any other technique, including immunoassays (such as ELISA or radioimmuno assay), can be used to this purpose.

The above methods are particularly useful for monitoring a response of an individual having DM1 to a treatment against DM1. Indeed, the presence of a stable or increasing number of protolerogenic peptides indicates that the individual responds to the treatment. Another aspect of the present invention is thus an in vitro method for monitoring a response of an individual having DM1 to a treatment, comprising assessing the presence of protolerogenic peptides and disease-accelerating peptides as defined above in biological samples obtained from the individual at different time points, wherein the presence of a stable or increasing number of protolerogenic peptides indicates that the individual responds to the treatment.

According to a particular embodiment, the above method is carried out by assessing the presence of protolerogenic peptides and disease-accelerating peptides in samples obtained at regular intervals. For continuously monitoring the patient’s response to the treatment, the method is carried out in a continuous fashion (e.g., every 2 weeks, every month, every two months, every 6 months or every year). The frequency of the detection of the peptides can be adjusted by the skilled person depending on the patient’s profile (for example, a patient in remission since several years will be tested less frequently than a patient who just started a treatment after DM1 diagnosis).

The monitoring method of the invention is particularly useful for monitoring a cell therapy based on Treg cells in a patient having or developing an autoimmune disease. Autoimmune disease in the context of the invention is type 1 diabetes mellitus (DM1). According to a particular embodiment, the above method is used for monitoring a cell therapy in a patient treated with CD4+ FoxP3+ Treg cells (with exogenous insulin when needed).

According to a particular embodiment, the patient’s response to the Treg cells treatment is assessed by the above method at least once, e.g. at least 2 weeks, preferably 1 month, more preferably 2 months, even more preferably 3 months after the administration of the cell therapy.

As already mentioned, the monitoring can be performed at regular or irregular intervals, preferably regular intervals (at least at the beginning of the treatment). According to a particular embodiment, the monitoring method is carried out in a continuous fashion for continuously monitoring the cell therapy of the patient, if possible as long as the cell therapeutic treatment of the patient continues.

The Treg cell therapy may be combined with a further medication useful in the treatment of DM1. According to a particular embodiment, the patient diagnosed with DM1 is treated with Treg cells combined with a B-cell depleting agent.

A “B cell depleting agent” is a molecule which depletes or destroys B cells in a patient and/or interferes with one or more B cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell. The B cell depleting agent preferably binds to a B cell surface marker. The B cell depleting agent preferably is able to deplete B cells (/.e., reduce circulating B cell levels) in a patient treated therewith. Such depletion may be achieved via various mechanisms such as antibody-dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death (e.g. via apoptosis). B cell depleting agents include but are not limited to antibodies, synthetic or native sequence peptides and small molecule antagonists which preferably bind to the B cell surface marker, optionally conjugated with or fused to a cytotoxic agent. The preferred B cell depleting agent comprises an antibody, more preferably a B cell depleting antibody, for example an anti-CD20, anti-CD19 or anti-CD22 antibody.

An example of anti-CD20 antibody that can be administered in combination with Treg cells is rituximab (designated as “RTX” in the experimental part below). Rituximab is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of immune system B cells. When it binds to this protein it triggers cell death. Other examples of such antibodies include:

• ocrelizumab, humanized (90%-95% human)

• ofatumumab (HuMax-CD20), a fully human B cell-depleting agent

• third-generation anti-CD20s such as obinutuzumab, having a glycoengineered Fc fragment (Fc) with enhanced binding to Fc gamma receptors to increase ADCC.

The present invention also relates to a cell therapy method of a patient diagnosed with DM1 , comprising the administration of Treg cells as outlined above, and carrying out the monitoring and/or assessment method of the invention, preferably further comprising the administration of a B-cell depleting agent such as rituximab.

According to another aspect of the present invention, the fragments of preproinsulin (alone or in combination) described as protolerogenic biomarkers as outlined above can be also used to deliberately manipulate the immune system toward tolerance. More specifically, they can be used in the treatment of patients with type 1 diabetes directly as a constituent(s) of a drug inducing tolerance in DM1 patients. The present invention thus also pertains to a pharmaceutical composition comprising at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 52, 62, 64, 66, 68, 70, 72, 74 and optionally, a peptide SEQ ID No:1 and/or a peptide SEQ ID No:49.

As explained in the experimental part which follows, the identified protolerogenic peptides were found in 6 separated regions which are shown in Table 2 Delow.

Table 2: regions encompassing the protolerogenic peptides

As shown in Example 3 below, the inventors surprisingly found that these larger peptides are particularly efficient for generating Tregs with a high suppressive activity. The present invention thus also pertains to a pharmaceutical peptide composition comprising at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 72 and 75-79.

By “at least one peptide selected from the group consisting of SEQ ID Nos: 1, 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74” and “at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 72 and 75-79” is herein meant that the composition comprises at least one isolated peptide having one of the recited sequences. The term "isolated" with respect to a peptide herein refers to a peptide that is not associated with naturally associated components that accompany it in its native state. An isolated peptide of SEQ ID No: X according to the invention can be fused to a second peptide moiety provided this second moiety is not a sequence flanking SEQ ID No: X in natural preproinsulin. For example, two or more of the above peptides can be concatenated in a single polypeptide, possibly separated by linkers not originating from the sequence of preproinsulin. The peptide composition can also comprise other non-peptidic components and even further peptide components, provided that it comprises at least one isolated peptide as above-described. According to a preferred embodiment, the composition comprises at least one peptide consisting of one of the above-listed sequences. In other words, a composition comprising only larger insulin or preproinsulin fragments is not in the scope of the invention, even if one or these fragments encompasses one or several of the recited peptides. The open wording “comprising” indeed applies to the pharmaceutical composition, not to the preproinsulin fragment(s) comprised in said composition. According to a particular embodiment, the composition does not comprise full-length insulin nor full-length preproinsulin. According to another particular embodiment, the composition does not comprise either fragments of preproinsulin larger than the above described peptides.

The skilled person knows that peptides and proteins can be administered either as such or through administration of a nucleic acid encoding them. According to a particular embodiment of the present invention, the pharmaceutical composition is a peptide composition. According to another particular embodiment, the pharmaceutical composition comprises a nucleic acid (DNA or RNA) encoding the peptides to be expressed in the body. For example, the pharmaceutical composition can advantageously comprise one or several mRNA(s) for expression of one or several of the protolerogenic peptides recited above. The technology of mRNA vaccines is now well known by persons skilled in the art of vaccination (Jackson et al. npj Vaccines 2020; Gote et al., Int J Mol Sci. 2023) and is particularly advantageous for expressing the protolerogenic peptides and peptides mixtures according to the invention. All the features described above regarding the sequences of the peptides apply whatever the way the peptides are vectorized, /.e., as such or through the delivery or a nucleic acid such as DNA and mRNA(s).

The peptides and nucleic acids described herein may be administered in pure form, combined with other active ingredient(s), and/or combined with pharmaceutically acceptable (/.e., nontoxic) excipients or carriers. The composition can comprise other pharmaceutically acceptable ingredients such as a carrier, salts, buffers and adjuvants. The term “pharmaceutically acceptable” is used herein in accordance with its art-recognized meaning and refers to components that are compatible with the other ingredients of a pharmaceutical composition, and are not deleterious to the recipient thereof.

The compositions described herein can be prepared in any known or otherwise effective dosage or product form suitable for use in providing delivery of the peptides in such a way that it activates Treg cells. The compositions according to the invention can be administered by any appropriate route, such as parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; vaginally; intramuscular route, or as inhalants.

According to a particular embodiment, the composition is formulated as a peptide composition comprising a carrier such as albumin and is administered through intradermal or subcutaneous route so that the peptides are then presented by the patient’s dendritic cells. According to another particular embodiment, the composition is formulated as a mRNA composition and is combined with a nucleic-acid delivery agent suitable for delivery of mRNA into mammalian host cells. Such mRNA delivery agents are well-known in the art and include a polymeric carrier, polycationic protein or peptide, lipid nanoparticle or other. For example, the mRNA (non-replicating or self-amplifying) may be delivered into cells using a cationic lipid nanoparticle (LNP). For example the formulation may comprise 20-60% cationic lipid; 5-25% non-cationic lipid, 25-55% sterol and 0.5-15% PEG-modified lipid as disclosed WO 2015/164674. The mRNA may also be formulated in RNA decorated particles such as RNA decorated lipid particles, preferably RNA decorated liposomes as disclosed in WO 2015/043613.

According to a particular embodiment, the pharmaceutical composition according to the invention comprises a mixture of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 protolerogenic peptides selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74. When the peptides are vectorized as mRNAs, the composition can comprise one or several mRNA sequences and each mRNA can encode one or several peptides, or a longer polypeptide encompassing several of the recited peptides, separated by cleavable linkers. The mRNA can also be designed to express the aboverecited peptides fused to another peptidic moiety which is not present in proinsulin. For example, the peptides can be expressed as a fusion with a signal peptide favoring the trafficking, especially the secretion of the molecule (such as, but not limited to the Ig VH peptide illustrated in Example 4). Such functional moieties, exogenous to proinsulin, can be linked in N-ter and/or in C-ter positions, possibly via linkers that can themselves be cleavable, self-cleavable or not cleavable.

According to a particular embodiment, the above pharmaceutical peptide or nucleic acid (DNA or mRNA) composition is for use in the treatment of DM1.

In particular, the pharmaceutical peptide or nucleic acid composition according to the invention can be used in the treatment of DM1 in an individual treated with T regs cells, possibly further combined with a B-cell depleting agent as recited above.

As an alternative or complement to the above-described use of the protolerogenic peptides according to the present invention, the same peptides can also be used during manufacturing of antigen-specific Tregs for the treatment of type 1 diabetes. Indeed, as explained in WO2021/034208, using antigen-specific Tregs in cell therapy of autoimmune diseases, rather than a polyspecific set of lymphocytes with specificity against many different antigens, allow for a more precise treatment and a reduction of the Treg dose, leading to an increase in the effectiveness of the Treg treatment and a reduction of possible side effects. The inventors also showed that tolerance is more associated with the presentation of the longer peptides via HLA class II to CD4+ T cells, so that the regions R1-R6 (SEQ ID Nos: 75-79 and 72) of the preproinsulin can also be used to expand antigen-specific Tregs.

According to another aspect, the present invention thus relates to a method for in vitro obtaining antigen-specific T regs appropriate for the treatment of DM 1 , comprising incubating Treg cells with at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 and/or with at least one peptide selected from the group consisting of SEQ ID Nos: 75-79 and 72, in conditions enabling preproinsulin-specific Treg cells expansion.

Antigen-specific Tregs can be obtained in vitro by incubating Treg cells with antigen presenting cells loaded with fragments of the targeted antigen, so that Treg cells specific for this antigen are expanded. The expanded antigen-specific Treg cells can then be isolated from the polyspecific Treg cells population.

Examples of conditions enabling antigen-specific Treg cells expansion are described by Iwaszkiewicz-Grzes et al. (Cytotherapy, 2020) and in WQ2021/034208.

In particular, WQ2021/034208 discloses a process for manufacturing antigen-specific T lymphocytes marked with monoclonal antibodies and sorted, wherein the lymphocytes: a) are generated by the use of autologous monocytes loaded with the antigen; b) T regulatory or T effector lymphocytes to be generated are suspended in PBS and stained intracellularly with a fluorescent dye; c) the lymphocytes are subsequently incubated in the dark; d) the lymphocyte cells are subsequently washed intensively several times with culture medium; e) T regulatory or T effector lymphocytes stained with intracellular fluorescent dye are suspended in the culture medium with gammairradiated autologous CD 14+ monocytes loaded with antigen; f) the co-culture of T regulatory or T effector lymphocytes with CD 14+ monocytes is coincubated with anti-CD 154 and anti-CD28 antibodies; g) the co-culture is incubated in culture medium; and h) antigen-specific T lymphocytes after incubation are sorted based on the low intensity of intracellular dye where the low intensity of fluorescence is a marker of antigen-specificity in that a loss of fluorescence correlated with the intensity of proliferation. The protolerogenic peptides and the regions R1 to R6 described herein can be used for expanding antigen-specific Treg through the above process or any other appropriate method.

The method for in vitro obtaining antigen-specific Tregs appropriate for the treatment of DM1 according to the invention can be performed using any source of Treg cells, such as natural Tregs (nTregs), induced Tregs (iTregs), genetically modified Tregs, etc. According to a particular embodiment, the starting material for obtaining such antigen-specific Tregs is a population of Tregs sorted from blood using a cell sorter, as described in W02013/184011.

When performing the above method, the Tregs can be incubated with only one peptide selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74-79, or with a mixture of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15 of these (possibly with all of them).

After sufficient expansion of the antigen-specific Treg cells, the antigenspecific Treg cells are isolated (for example using a cell sorter) and prepared in a cell formulation appropriate for administration to a patient.

In the above method, the Tregs which are incubated with the recited peptides are preferably CD4⁺ FoxP3⁺ CD25^high CD127' T cells.

The present invention also pertains to antigen-specific Treg cells obtainable by the above method, /.e., preproinsulin-specific Treg cells.

In a particular embodiment of the invention, Tregs manufactured with the use of protolerogenic fragments of preproinsulin (single or in combination) and/or regions R1 to R6 of the preproinsulin as defined herein are CD4⁺ FoxP3⁺ T regulatory cells. In a preferred embodiment of the invention, Tregs manufactured according to the invention are of the phenotype CD4⁺ FoxP3⁺ CD25^high CD127' doublets'.

The present invention also pertains to the use of preproinsulin-specific Treg cells as described above in the treatment of DM1 , either alone or combined with a B-cell depleting agent as defined above.

In such a treatment, the antigen-specific Treg cells can be derived from cells obtained from the patient to be treated (autologous cell therapy), or can be derived from cells obtained from a donor (allogenic cell therapy).

The present invention is also directed to the use of antigen-specific Treg cells as described herein for treatment of a patient having or developing DM1 , by cell therapy, comprising the administration of said Treg cells, and optionally a B-cell depleting agent as defined above (e.g. rituximab) to the patient, and further comprising carrying out the assessment method and/or the monitoring method of the invention. The methods of the present invention can also be used to prevent or reduce destruction of p cells following pancreas transplantation or pancreatic islet transplantation. Thus, other aspects of the present disclosure relate to methods of preventing destruction of pancreatic beta cells in a transplant patient. Such methods involve administering to a transplant patient a peptide composition and/or antigenspecific Tregs as above-described, wherein said administering is carried out under conditions effective to prevent or reverse loss of pancreatic p-cells in the subject.

Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.

EXAMPLES

Example 1 : Identification of serum insulin and proinsulin fragments associated with good clinical outcome of the immune intervention in DM1

The study described herein aimed to identify and validate serum insulin and proinsulin fragments associated with good clinical outcome of the immune intervention in DM1 in the clinical trial TregVAC2.0 (clinical trial registration ISRCTN37116985).

The inventors assessed the evolution of the insulin and proinsulin fragments present in the sera of DM1 patients treated with combined therapy of autologous polyclonal CD3⁺CD4⁺CD25^highCD127' T regulatory cells and anti-CD20 antibody (Tregs+RTX group) as compared to the patients treated with polyclonal Treg administration only (Tregs only group) or standard-of-care treatment with insulin (standard-of-care group).

As a final result, we expected to find a peptide or peptides, which are insulin or proinsulin fragments, which correlate with p-cell function and can be used as a immunotherapeutic directly or indirectly, for example as a stimulant of cells prepared for immunotherapy of DM1. Materials and Methods

• Study design

The present inventors have performed a phase 1/2, prospective, multicenter clinical trial to study efficacy and impact on selected immune parameters of combined therapy with autologous Treg administration and anti-CD20 monoclonal antibodies in children and adolescents with recently diagnosed DM1. The study was a prospective, open-label and randomised 24-months-lasting clinical trial registered as ISRCTN37116985 and EudraCT 2014-004319-35. In this three-arm trial, the follow-up was performed in a standard-of-care group (standard-of-care; 11 patients), the group treated with autologous polyclonal Treg only (Tregs only; 12 patients), and the group treated with combined therapy with autologous polyclonal Treg and anti CD20 antibodies (Tregs+RTX; 13 patients). Interventional groups, which consisted of Tregs+RTX and Tregs only patients, received two doses of Treg in an open-label manner, 30x10⁶ cells/kg b.w. per dose, three months apart starting from day 0. The administration of the anti- CD20 antibody was blinded and placebo-controlled. The patients were randomly assigned to an anti-CD20 antibody or placebo by the element of chance (coin) to receive four dosages of rituximab (Tregs+RTX group) or placebo (Tregs only group) on +14, +22, +29, and +36 days of the trial. All the participants were followed-up for two years posttherapy and assessed at administration, three, six, twelve, and twenty-four months after, as given in the study flow diagram shown in Figure 1.

The report on the efficacy and safety of combined therapy made in this clinical trial has already been published. We have proved that the combined autologous Treg administration and anti-CD20 antibodies therapy was superior to the Treg administration-only treatment in DM 1 , as assessed by the AUG of C-peptide mixed meal tolerance test and the percentage of patients in clinical remission at 24 months of the trial. The therapy was safe, despite the 80% adverse events rate in the Tregs+RTX patients, as no adverse event led to the withdrawal of the intervention or the patient's death (Zielinski et al., 2022).

The study described herein aimed to find a peptide or peptides, which are insulin or proinsulin fragments, which correlate with p-cell function and can be used as marker of DM1 progression or as a immunotherapeutic directly or indirectly, for example as a stimulant of cells prepared for immunotherapy of DM1.

In a two-years-lasting follow-up, we analysed sera of patients and healthy volunteers (healthy control group) in order to find insulin or proinsulin fragments and tried to correlate the presence of particular sequences with the clinical outcomes of DM1 patients. The study was approved by an independent institutional review board (NKBBN/374/2012-NKBBN/374-7/2014) and all participants signed an informed consent form.

• Patients All the participants were recruited based on the detailed inclusion and exclusion criteria for both the clinical trial and healthy volunteer cohort (Table 3, 4 and 5). By the power analysis for sample numbers, thirteen patients in the randomised treatment arms were satisfactory to find a 20% difference in the geometric mean ratio of AUC (0-240 min) of C-peptide (alpha 5%), a significant outcome of the clinical experiment. For healthy volunteers, 12 healthy controls were enrolled selected from age- adjusted patients, who were under diagnostic procedures and found healthy. The samples of sera from DM1 patients for the analysis were taken at times: 0, +12 months and +24 months. The samples from healthy controls were taken during diagnostic procedures.

Table 3: Clinical trial participant's patient's baseline characteristics. P-values are based on one-way ANOVA F-statistics for continuous and Kruskal-Wallis statistics for multilevel categorical data; adapted from Zielinski et al., 2022. Table 4: Healthy control patient's baseline characteristics. Healthy controls were anonymous blood donors, all characteristics assumed to be within normal range.

Table 5: Clinical trial and in-vitro model participant’s inclusion and exclusion criteria. Adapted from Zielinski et al., 2022.

Methods • Samples collection and preparation

Venous blood was collected during the trial visits into clot activatorcontaining tubes and allowed to clot for 30 min followed by centrifugation for 15 min at 1000g. Aliquoted serum samples were then stored at - 80 °C for further analyses. Due to the quality of stored sera or technical errors, the samples of sera from one patient from standard-of-care group, one patient from Tregs only group and three patients from Tregs+RTX group were excluded from final analysis. • Detection of the peptides

The data from collected sera were acquired using mass spectrometry (Orbitrap Exploris 480, ThermoFisher Scientific). The library of spectra from samples were acquired separately for each sample. There were separate DDA acquisitions for 2- 3 times charged peptides and 4-6 times charged peptides. The samples were prepared in duplicates and intrinsic acquisition was performed with DIA method using Evosep One- Exploris 480 system in 88min gradient. For the library samples and for specific peptides, iRT standards (Biognosys) were added in order to correct retention times and obtain better resolution of the peaks.

The acquired data were analysed with MaxQuant software with MaxDIA algorithm (MaxQuant library).

• The choice of the sequences of preproinsulin fragments

The staff responsible for acquisition of the data and search for the sequences was blinded to the diagnosis of the patients and the timepoints of the follow up. The search focused on the fragments of preproinsulin. The sequences present in less than three samples were excluded.

The peptides detected in the sera were mapped to the sequence of preproinsulin. This way the most frequent regions of preproinsulin translated to the peptides and present in the sera were identified.

The presence of each peptide was then analysed in the samples of particular groups of patients, such as healthy controls [n=12], ‘time 0’ [n=31] (samples from all three groups from clinical trial at recruitment), standard-of-care [n=20] (samples from +12months and +24 months timepoints of the follow up), Tregs only [n=22] (samples from +12months and +24 months timepoints of the follow up), and Tregs+RTX [n=20] (samples from +12months and +24 months timepoints of the follow up), and according to the clinical outcomes within clinical trial groups [good outcome/ bad outcome]. The good clinical outcome/ ‘in remission’ status was defined as a preservation of c-peptide secretion in MMTT test and clinical remission defined as daily dose of insulin below 0.5 units per kg b.w. and glycated haemoglobin (HbA1C) levels below 6,5% (TDD <0.5 Ul/kg/day and HbA1c <6.5%) (Zielinski et al., 2022). Three categories of the peptides were distinguished according to the clinical outcomes:

1. Protolerogenic/ possibly associated with immune tolerance

2. Ubiquitous/ presented frequently and regardless of the clinical status of the patients

3. Dangerous/ potentially accelerating disease Criteria to include the peptides to those called protolerogenic were the following:

1. When the peptide was detected in any of the trial groups (control, Tregs only and Tregs+RTX) at +12 months or/and +24 months of the follow up, 50% or more of all of the counts must be detected in patients with good clinical outcome/ ‘in remission’ (the peptides were taken into account for further analysis, when they were present in sera of 3 or more patients),

And

2a. The peptide was detected in less than 11 of sera of patients at the time of diagnosis (‘time O’) [the number of sera equal to the number of patients in any single group at this time point]

Or

2b. The peptide was not detected in the sera of patients at the time of diagnosis (‘time O’) [n=31 consists of the sera of patients from all the trial groups]

Or

2c. The peptide was not detected in the sera of patients form control (standard-of- care) group

And

3. The number of sera with the detected peptide associated with the good clinical outcome was higher than or equal to the number of sera with the detected peptide at the time of diagnosis (‘time O’)

Criteria to include the peptides to those called ubiquitous were the following:

1. When the peptide was detected in any of the trial groups (control, Tregs only and Tregs+RTX) at +12 months or/and +24 months of the follow up, 50% or more of all of the counts must be detected in patients with good clinical outcome,

And

2. The peptide was detected in more than 11 of sera of patients at the time of diagnosis (‘time O’) [the number of sera equal to the number of patients in any single group at this time point] Criteria to include the peptides to those called dangerous were the following:

1. When the peptide was detected in any of the trial groups (control, Tregs only and Tregs+RTX) at +12 months or/and +24 months of the follow up, less than 50% of all the counts were detected in patients with good clinical outcome (the peptides were taken into account for further analysis when they were present in sera of 3 or more patients).

Results

• The most common regions of preproinsulin in the detected peptides There were 121 peptides detected 1251 times in total in the sera according to the criteria from the methods. The peptides detected in the sera were then mapped to the sequence of preproinsulin. This way the most frequent regions of preproinsulin translated to peptides and present in the sera were identified. There were 1172 counts, out of the total 1251 counts, containing the sequences from these frequent regions. The following regions of preproinsulin were identified as the most frequently included in the peptides: 8-30 (141 counts), 21-36 (102 counts), 39-55 (141 counts), 58-89 (434 counts), 71-96 (283 counts), and 98-108 (71 counts) (Table 6). In fact, while we define the whole regions, what mostly differentiates the regions is where the peptides start, i.e. 8-30 (starts 8-18), 21-36 (starts 21-25), 39-55 (starts 39-49), 58- 89 (starts 58-68), 71-96 (starts 71-87), and 98-108 (starts 98-101). The peptides found in the sera of healthy controls as well as the peptides classified as protolerogenic, ubiquitous and dangerous were mainly from these regions.

PT: protolerogenic peptide; UB: ubiquitous peptide; DA: disease accelerating peptide.

• Frequency of protolerogenic/ ubiquitous/ dangerous peptides

The region 8-30 contained 2 protolerogenic and 1 ubiquitous peptides, the region 21-36 contained 3 protolerogenic and 1 dangerous peptides, the region 39-55 contained 2 protolerogenic, 1 ubiquitous and 1 dangerous peptides, the region 58-89 contained 4 protolerogenic and 3 ubiquitous peptides, the region 71-96 contained 7 protolerogenic, 2 ubiquitous and 2 dangerous peptides, and the region 98-108 contained 2 protolerogenic peptides (Table 6).

Discussion

The present study attempted to identify the most accurate immune biomarkers predicting the outcome of type 1 diabetes. We used sera from TregVAC2.0 trial (clinical trial ISRCTN37116985) where we followed both standard-of-care DM1 patients and those treated with Tregs monotherapy or combined treatment with Tregs and rituximab, and therefore the biomarkers are of special interest in the assessment of the efficacy of the therapy with Tregs in DM1. Importantly, the preproinsulin fragment peptides identified in the study as protolerogenic might be also considered as therapeutic agents. It would be possible to administer them directly to DM1 patients as a peptide- based medicinal product (separately single peptides or as a combination of several or all peptides in the product) in order to increase the level of protolerogenic peptides in the body. It might also be possible to use them in vitro, in the manufacturing of antigenspecific Tregs, which could then be administered in the treatment of DM1 similarly to the currently used polyclonal Tregs.

Current research and findings are the natural consequence of the success of TregVAC2.0 clinical trial (Zielinski et al., 2022). We confirmed there that combined treatment with Tregs and rituximab is superior to monotherapies with these agents. The hypothesis behind this strategy was that the depletion of B cells, the most probably presenting autoantigens, in patients with relatively advanced disease should significantly reduce the epitope spread. This, in turn, would significantly down regulate/ reduce the activation of the immune system and facilitate immune regulation provided by administered ex vivo expanded Tregs. It is very important that in this line of thinking about autoimmunity, B cells are not regarded as autoantibody producers but mainly as autoantigen presenting cells accelerating the epitope spread. And the epitope spread existed in our DM1 patients as suggested by more than 100 various insulin fragments, including abundant ‘ubiquitous’ fragments, identified in the sera in all groups of the study. If this hypothesis is true, the early events in the progression of DM1 should be dependent on the autoantigens presented to the lymphocytes. The primary autoantigen candidates are insulin or/and insulin-like peptides as islet p-cells producing insulin are the first targets for autoaggression in DM1. Indeed, in our previous works we found that the progression of DM1 depended on the presence of insulin-specific T conventional cells (Tconvs), while insulin-specific Tregs could delay this process (Gliwihski et al., 2020). Interestingly, similar families of clones of Tconvs and Tregs cells were increasing their proportions with the progression of DM1 , which suggested that similar epitopes were activating both subsets (Gliwihski et al., 2020). The onset of DM1 could therefore be related to relatively faster and more abundant activation of Tconvs over Tregs of similar specificity. Hence, it is highly probable that the detection of disease-specific epitopes recognizable by T cells has diagnostic and predictive value and the manipulation with the number and content of such epitopes may be a powerful immunotherapeutic tool.

Indeed, using the algorithm of the insulin fragment screening in the sera of TregVAC2.0 patients developed in the present Example we have found fragments associated with long-term remission of the disease as well as peptides associated with fast progression of the disease. They were defined as ‘protolerogenic’ and ‘disease accelerating’, respectively. The predictive role of these peptides in the patients treated with Tregs seems to be obvious. Nevertheless, as we could observe the remission and good outcomes not only in the interventional arms, but also in patients treated under the standard of care, the predictive/diagnostic role of these peptides seems to be wider and attributable to all DM1 patients.

Therapeutic maneuvers aiming at increasing the level of protolerogenic peptides or/and decreasing the level of disease accelerating peptides could also be utilized in the immunotherapy of DM1. The fact that only some fragments and not the complete sequence of insulin are associated with the course of the disease may explain failures of clinical trials with native insulin used as tolerogenic agent, which have been reported in the literature (Pozzilli et al., 2000). Protolerogenic peptides identified in the present Example can be considered as a tool to expand antigen-specific Tregs in vivo, when administered directly into the body. It is also possible to use such peptides in vitro in the expansion cultures of Tregs in order to select antigen-specific Tregs for cellular therapy, like in our previously described method of the manufacturing (Iwaszkiewicz- Grzes et al., 2020).

In addition, a striking difference in the peptide length between protolerogenic and disease accelerating insulin fragments should be emphasized. While the former are usually longer than 10 amino acids, the latter do not exceed 10 amino acids. Taking into account a paradigm of antigen presentation, this phenomenon suggests that the autoaggression is more related to the presentation of the shorter peptides, possibly via HLA class I to CD8+ T cells, while tolerance is more associated with the presentation of the longer peptides via HLA class II to CD4+ T cells. The regions of the preproinsulin which sequences were most frequently represented in the detected peptides can also be a tool to expand antigen-specific Tregs, as they are even longer than the detected protolerogenic peptides.

DM1 is a classical autoimmune disease in which pathologic mechanisms of epitope spread and failed peripheral regulation are known and confirmed. Nevertheless, there are many other mechanisms of disturbed tolerance which may constitute a background of the discovered variety of insulin fragments or other peptides and their association with the course of DM1 (Lernmark et al., 2023). This should be determined in future studies. In addition, at least some of the fragments of insulin detected in this Example are definitely associated with the processing of exogenous insulin or its analogues administered as a substitutive treatment to the patients. On a one side, it is an artifact, while on the other, it might be a hint for future research on the routes of administration of protolerogenic insulin derivatives in patients with DM1.

In summary, we have developed an algorithm of screening of the peptides with diagnostic value in DM1 and identified such peptides. Some of these peptides defined as protolerogenic might constitute a content of medicinal products inducing tolerance in DM1 patients and therefore slow progression of the disease. The peptides might be administered directly to the patients for in vivo activities or used in vitro as a selection agent in the manufacturing of insulin-specific Tregs used subsequently in the therapy of DM1.

Example 2: in vitro proliferation of Treg and Tconv cells in cocultures with es loaded with preproinsulin peptides

Peripheral blood mononuclear cells from healthy volunteers were sorted to monocytes, T regulatory cells (Tregs) and T conventional (Tconv) cells. Monocytes were loaded with the peptides and cocultures of peptide-loaded monocytes with Tregs or Tconv were performed for 6 days.

The following cocultures were performed:

Controls:

Beads - Tregs or Tconv stimulated with antiCD3/antiCD28 beads (positive control)

UnStim - Tregs or Tconv not stimulated (negative control) NonP - Tregs or Tconv stimulated with monocytes not loaded with peptides (baseline proliferation)

PX - Tregs or Tconv stimulated with monocytes loaded with peptides.

The following cultures of Tregs or Tconv with peptide-loaded monocytes were used:

P1 : monocytes loaded with LPLLALLALWGPD (SEQ ID No: 1)

P2: monocytes loaded with PAAAFVNQHL (SEQ ID No: 11)

P3: monocytes loaded with FVNQHLCGSH (SEQ ID No: 16)

P4: monocytes loaded with YLVCGERG (SEQ ID No: 20)

P5: monocytes loaded with VCGERGFFYTPKTR (SEQ ID No: 22)

P6: monocytes loaded with FYTPK (SEQ ID No: 25)

P7: monocytes loaded with LQVGQVELGGGP (SEQ ID No: 33)

P8: monocytes loaded with GQVELGGGPGAGSL (SEQ ID No: 41)

P9: monocytes loaded with LGGGPGAGSLQPLA (SEQ ID No: 48)

P10: monocytes loaded with LQKRGIVEQ (SEQ ID No: 68)

P11 : monocytes loaded with SLYQLENY (SEQ ID No: 74)

P12: monocytes loaded with a MIX of all peptides P1 to P11

Tregs and Tconvs in the cocultures were stained with fluorescent proliferation cell-tracker Violet. Proliferating cells divide the dye into the cytoplasm of daughter cells, 50% of the dye to each, and therefore the offspring cells can be detected due to fluorescence dilution.

After 6 days, the percentage of proliferating cells was measured in each culture and the results are presented in Figure 2.

It seems that Tregs proliferate above baseline proliferation with the majority of the peptides (also with P6 and P11 , which were identified as disease accelerating and ubiquitous peptides, respectively) which is not the case of Tconvs. The highest values are for the MIX of all peptides (and peptide P1).

Example 3: Expansion and activity of antigen-specific Tregs

We expanded antigen-specific Tregs with mixtures of peptides disclosed in Examples 1 and 2 and verified the activity of obtained peptide-specific Treg with in vitro functional tests using previously described methods from our lab (Iwaszkiewicz- Grzes D et al. Cytotherapy. 2020 Nov;22(11):629-641). METHODS

Generation of antigen-specific T regulatory and T effector cells

Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy volunteers. A proportion of PBMC was used to obtain autologous CD14+ monocytes which were subsequently incubated for 24 hours with three mixtures of peptides identified in Example 1 :

MIX1- Protolerogenic (PT) peptides (SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74),

MIX2 - Disease-accelerating (DA) peptides (SEQ ID Nos: 14, 25, 53 and 58) and MIX3 - R1-R6 peptides (SEQ ID Nos: 72 and 75-79).

The remaining PBMC were used to sort CD4+CD25highCD127- T regulatory cells (Tregs) and CD4+CD127+ T effector cells (Teff) using a cell sorter. These cells were subsequently stained with VPD450 proliferation dye.

After 24h incubation, monocytes loaded with mixtures of peptides (MIX1 , MIX2 or MIX3) were added to VPD450-labelled Tregs and Teff and cocultured for 7 days. Separate sets of Tregs and Teff were incubated with antiCD3/antiCD28 beads in order to receive polyclonal Tregs and Teff. A proportion of Tregs and Teff was left cultured without stimulation.

At day 7, the cocultures were sorted gating on Tregs or Teff and sorted based on the dilution of proliferation dye into antigen-specific and antigen-unspecific cells (Figure 3A). The cells recognizing the mixtures of peptides proliferated and diluted the dye and were therefore classified as antigen-specific, while those non-proliferating with high fluorescence of the dye were treated as unspecific. The following subsets were achieved for Tregs: MIX1 - specific Tregs (MIX1_Spec), MIX1 - unspecific Tegs (MIX1_unspec), MIX2 - specific Tregs (MIX2_Spec), MIX2 - unspecific Tegs (MIX2_unspec), MIX3 - specific Tregs (MIX3_Spec), MIX3 - unspecific Tegs (MIX3_unspec) and polyclonal Tregs (Poly). Similar subsets were acquired for T effectors: MIX1 - specific Effs (Effs_MIX1_Spec), MIX1 - unspecific Effs (Effs_MIX1_unspec), MIX2 - specific Effs (Effs_MIX2_Spec), MIX2 - unspecific Effs (Effs_MIX2_unspec), MIX3 - specific Effs (Effs_MIX3_Spec), MIX3 - unspecific Effs (Effs_MIX3_unspec) and polyclonal Effs (Effs_poly) All acquired subsets were further expanded with antiCD3/antiCD28 beads and IL2 for 12 days (Figure 3B).

At day 19, the cells were analysed in flow cytometer to assess the level of FoxP3 and the proportion of naive/memory subsets in the expanded Tregs (Figure 4). Suppression assay

Also, at day 19, the cultures of Tregs and Teffs were used for the preparation of suppression assays in order to assess functional properties of the expanded Tregs (Figure 5). For the suppression assay, autologous PBMC and different subsets of effectors, such as CD4+ T Effector polyspecific cells (EFFs poly) agnostic to the antigen, CD4+ T Effector antigen-specific cells (EFFs spec), CD4+ T Effector antigen-unspecific cells (EFFs unspec) were used as responders. All subsets of responders were restained with proliferation dye VPD450 and cocultured in different proportions with Tregs from the cultures expanded with one of the mixtures of the peptides: MIX1 , MIX2 or MIX3. Each set of the experiments was prepared with co-cultures where both responders and Tregs were specific/unspecific to the particular mixture of peptides. For example, T regs specific to MIX1 were tested against the responders specific to MIX1 or unspecific to MIX1 , Tregs unspecific to MIX1 were tested against the responders specific to MIX1 or unspecific to MIX1. In addition, both Tregs specific to MIX1 and unspecific to MIX1 as well as polyclonal Tregs were tested against PBMC and polyclonal Effectors. Similar sets of the co-cultures were performed for MIX2 and MIX3. The co-cultures were stimulated with antiCD3/antiCD28 beads for 3 days.

For each culture condition, the reference culture with the corresponding responders stimulated with antiCD3/antiCD28 beads without Tregs in the culture was performed.

After 3 days of co-culture, the fluorescence of the responders was acquired as a Mean Fluorescence Intensity (MFI) using flow cytometer. The intensity of the suppression was assed using the following formula: where

- “proliferation in coculture” was MFI from the analysed coculture of responders with Tregs

- “proliferation of responders with beads” was MFI from the reference culture with the corresponding responders stimulated only with antiCD3/antiCD28 beads without Tregs in the culture.

Suppression of immune activation by the peptide mixtures

Another functional assessment of the presented peptides was prepared in the cocultures of Tregs, T effectors and PBMC stimulated with the mixtures of peptides to assess the expression of activation markers CD69 and HLA-DR. (Figure 6). Autologous CD4+ T regulatory cells (Tregs) were stained with VPD450 dye and CD4+ T effector cells (Effs) with CT Yellow and mixed 1 : 1:1 with autologous PBMC. The cocultures were subsequently stimulated with peptide mixtures MIX1 , MIX2 and MIX3. Unstimulated cocultures (unstim) were used as a negative control and the cocultures stimulated with beads (Beads) were used as positive control. The expression of activation markers CD69 and HLA-DR was assessed after 24h, 48h and 72h separately on Tregs and Effectors as a percentage of positive cells.

RESULTS AND DISCUSSION

Generation of antigen-specific T regulatory and T effector cells

All three mixtures of peptides allowed proliferation of Tregs and Teffs. But MIX1 and noticeably MIX3 were preferentially expanding Tregs, while MIX2 was preferentially expanding Eff as revealed by Treg/Effs index (Figure 3A). In addition, final numbers of antigen-specific cells expanded after selection of antigen-specific cells revealed that although the yield of Tregs were comparable between the conditions, the proliferation of effectors specific to MIX2 (Eff-MIX2) was exceptionally intense (Figure 3B). Treg/Effs index from the number of cells expanded after selection confirmed that the lowest expansion of Tregs at the expense of Effectors was in the cells treated with MIX2 and the index confirmed preferential expansion of Tregs treated with MIX3.

The quality of all antigen-specific Tregs measured as the expression of FoxP3 and the proportion of naive/memory cells (Figure 4) confirmed high quality of Tregs expanded with all MIXes and this quality was comparable to polyclonal Tregs. The percentage of FoxP3+ Tregs was above 95% and the proportion of naive CD45RA+CD62L+ Tregs was above 80% in all expanded conditions.

Suppressive Activity of antigen-specific Tregs

Suppressive Activity of Tregs generated with the mixtures of the peptides from the patent (MIX, MIX2, MIX3) and polyclonal Tregs were tested against different autologous responders: PBMC, CD4+ T Effector polyspecific cells (EFFs poly), CD4+ T Effector antigen-specific cells (EFFs spec) and CD4+ T Effector antigen-unspecific cells (EFFs unspec) (Figure 5). All populations of Tregs were able to suppress proliferative responses of PBMC (Figure 5A). The level of suppression significantly decreased, when responders were polyclonal T effectors (Effs_poly) - especially in Tregs generated with MIX1 and MIX2. This is possibly due to a higher concentration of lymphocytes to be suppressed in the responder population. Importantly, Tregs generated with MIX3 (Tregs_MIX3_spec) still kept high suppressive potential against Effs_poly Figure 5B). The suppressive abilities of Tregs_MIX3_spec were also sustained against CD4+ T Effector MIX3-specific cells (EFFs_MIX3_spec). Although weaker, the suppressive potential of Tregs_MIX1_spec was also preserved when tested against EFFs_MIX1_spec, whereas Tregs_MIX2_spec did not reveal any suppressive potential against EFFs_MIX3_spec responders (Figure 5C). The suppressive potentials similar to those against specific responders were also noted when unspecific responders (Effs_unspec) were applied (Figure 5D). These results clearly show antigen-specific suppressive abilities of Tregs generated with MIX3 (Tregs_MIX3_spec), to a lesser extent Tregs generated with MIX1 (Tregs_MIX1_spec) and no to little suppressive activity of Tregs generated with MIX2 (Tregs_MIX2_spec). All the cells regulated PBMC (all subsets are Tregs, anyway) but increasing concentration of specific cells to be regulated (from polyclonal Effectors to MIX-specific Effectors) revealed differences in the suppressive activity of the generated subsets of antigen-specific Tregs. These results also highlight the specificity of the used peptide MIXes in the generation of efficient Tregs.

Suppression of immune activation by the peptide mixtures

The suppression of immune activation with the peptide mixtures (MIX1 , MIX2 and MIX3) was assessed in the stimulated cocultures of PBMC enriched with Tregs and T effectors (Effs). Compared to the cocultures stimulated with antiCD3/antiCD28 beads, all three MIXes were able to control the activation and kept the level of activation markers CD69 and/or HLA-DR (the percentage of Tregs and Effectors expressing the markers) at levels similar to those noted in unstimulated cultures (Figure 6).

Example 4: Increased stability of peptides expression when administered as mRNA

The peptides disclosed in the Examples 1 to 3 were rewritten to mRNA sequences and prepared to stabilize their expression, when taken up by cells. The sequences can thus be administered as a drug, either as peptide or mRNA sequences which will be translated into peptides in the body.

Vector Design

In vitro transcription (IVT) constructs were based on the pmRVac vector from VectorBuilder® containing a T7 promotor, a 5’ and 3’-globin UTR, a 110-bp poly(A) tail and the kanamycin-resistance gene.

The vectors were designed to express the peptides coupled to a human Ig VH signal peptide (MDWTWRILFLVAAATGAHS, SEQ ID No: 105) for peptide secretion, in N-ter, separated by a self-cleavable AD linker, and the C-terminal V5 tag (GKPIPNPLLGLDST, SEQ ID No: 106) for expression detection, separated by the G4S linker. The encoded peptides are shown in Table 7 below. Table 7: peptides expressed in vitro. The number at the end of the name corresponds to the SEQ ID No of the preproinsulin peptide (as described in Example 1)

In vitro Transcription

In vitro transcription from plasmid DNA templates linearized with restriction enzyme Sapl was completed using T7 RNA polymerase (Invitrogen), CleanCap® AG and methylpseudouridine reagents (TriLink Biotechnologies). The resulting modRNAs contain a 5’ Cap structure with pseudouridine incorporated instead of uridine. The size and integrity of transcripts were assessed on TBE-urea polyacrylimide gels (BioRad) stained with Sybr Gold Nucleic Acid Stain (Invitrogen). In vitro Expression

Expression of peptides following transfection of the modRNAs with Lipofectamine MessengerMAX (Invitrogen) was evaluated in HEK293T cells. Total cellular and secreted peptide expression was assessed by Western blotting with a V5- HRP (Invitrogen).

REFERENCES

Bluestone JA, Buckner JH, Fitch M, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sei Transl Med. 2015;7(315):315ra189. doi : 10.1126/scitranslmed.aad4134

Budd MA, Monajemi M, Colpitts SJ, Crome SQ, Verchere CB, Levings MK. Interactions between islets and regulatory immune cells in health and type 1 diabetes. Diabetologia. 2021 ;64(11):2378-2388. doi:10.1007/S00125-021-05565-6

Dong S, Hiam-Galvez KJ, Mowery CT, et al. The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes. JCI insight. 2021 ;6(18). doi:10.1172/JCI. INSIGHT.147474

Gliwihski M, Iwaszkiewicz-Grzes D, Woloszyn-Durkiewicz A, et al. Proinsulinspecific T regulatory cells may control immune responses in type 1 diabetes: implications for adoptive therapy. BMJ Open Diabetes Res Care. 2020; 8(1):e000873. doi: 10.1136/bmjdrc-2019-000873.

Gote V, Bolla PK, Kommineni N, Butreddy A, Nukala PK, Palakurthi SS, Khan W. A Comprehensive Review of mRNA Vaccines. Int J Mol Sci. 2023 Jan 31 ;24(3):2700. doi: 10.3390/ijms24032700. PMID: 36769023; PMCID: PMC9917162.

Herold KC, Bundy BN, Long SA, et al. An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes. N Engl J Med. 2019;381 (7):603-613. doi:10.1056/NEJMOA1902226

Iwaszkiewicz-Grzes D, Gliwinski M, Eugster A, et al. Antigen-reactive regulatory T cells can be expanded in vitro with monocytes and anti-CD28 and anti-CD154 antibodies. Cytotherapy. 2020; 22(11):629-641. doi: 10.1016/j.jcyt.2020.07.001

Jackson, N.A.C., Kester, K.E., Casimiro, D. et al. The promise of mRNA vaccines: a biotech and industrial perspective, npj Vaccines 5, 11 (2020).

Lernmark A, Akolkar B, Hagopian W et al. Possible heterogeneity of initial pancreatic islet beta-cell autoimmunity heralding type 1 diabetes. J Intern Med. 2023; 294(2):145-158. doi: 10.1111/joim.13648.

Marek-Trzonkowska N, Wujtewicz MA, Mysliwiec M, et al. Administration of CD4+CD25highCD127- regulatory T cells preserves p-cell function in type 1 diabetes in children. Diabetes Care. 2012;35(9):1817-1820. doi:10.2337/DC12-0038

Marek-Trzonkowska N, Mysliwiec M, Dobyszuk A, et al. Therapy of type 1 diabetes with CD4(+)CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets - results of one year follow-up. Clin Immunol. 2014;153(1):23-30. doi:10.1016/j.clim.2014.03.016 Pozzilli P, Pitocco D, Visalli N, et al. No effect of oral insulin on residual betacell function in recent-onset type I diabetes (the IMDIAB VII). IMDIAB Group. Diabetologia. 2000;43(8): 1000-4. doi: 10.1007/s001250051482

Raugh A, Allard D, Bettini M. Nature vs. nurture: FOXP3, genetics, and tissue environment shape Treg function. Front Immunol. 2022; 13:4199. doi:10.3389/FIMMU.2022.911151/BIBTEX

Roep BO, Montero E, van Tienhoven R, Atkinson MA, Schatz DA, Mathieu C. Defining a cure for type 1 diabetes: a call to action, lancet Diabetes Endocrinol. 2021 ;9(9):553-555. doi: 10.1016/S2213-8587(21 )00181-9

Roep BO. The need and benefit of immune monitoring to define patient and disease heterogeneity, mechanisms of therapeutic action and efficacy of intervention therapy for precision medicine in type 1 diabetes. Front Immunol. 2023; 14. doi : 10.3389/FIM M U .2023.1112858

Trzonkowski P, Bacchetta R, Battaglia M, et al. Hurdles in therapy with regulatory T cells. Sci Transl Med. 2015;7(304). doi:10.1126/SCITRANSLMED.AAA7721

Zielinski M, Zalihska M, Iwaszkiewicz-Grzes D, et al. Combined therapy with CD4+CD25highCD127- T regulatory cells and anti-CD20 antibody in recent-onset type 1 diabetes is superior to monotherapy: Randomised phase l/ll trial. Diabetes, Obes Metab. 2022 ;24(8): 1534- 1543. doi: 10.1111/DOM.14723

Claims

1. A pharmaceutical composition comprising at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 52, 62, 64, 66, 68, 70, 72, 74 and 75-79, or a mRNA encoding the same.

2. The pharmaceutical composition of claim 1 , comprising a mixture of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 protolerogenic peptides selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72, 74 and 75-79.

3. The pharmaceutical composition of claim 1 , comprising one or several mRNA sequences for expressing at Ieast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 protolerogenic peptides selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72, 74 and 75-79.

4. The pharmaceutical composition according to any one of claims 1 to 3, which comprises at least one, preferably 2, 3, 4, 5 or 6 protolerogenic peptide selected from the group consisting of SEQ ID Nos: 72 and 75-79, or one or several mRNA sequence(s) encoding the same.

5. The pharmaceutical composition of any one of claims 1 to 4, for use in the treatment of DM 1.

6. The pharmaceutical composition for use according to claim 5, for use in the treatment of DM1 in an individual treated with Tregs cells.

7. The pharmaceutical composition for use according to claim 6, wherein the individual also receives a B-cell depleting agent.

8. A method for in vitro obtaining antigen-specific Tregs appropriate for the treatment of DM1, comprising incubating Treg cells with at least one protolerogenic peptide selected from the group consisting of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 and/or with at least one peptide selected from the group consisting of SEQ ID Nos: 75- 79, in conditions enabling preproinsulin-specific Treg cells expansion.

9. The method of claim 8, further comprising the steps of isolating antigen-specific CD4+ FoxP3+ Treg cells and including these in a cell formulation appropriate for administration to a patient.

10. Antigen-specific Treg cells obtainable by the method of claim 8 or claim 9.

11. The antigen-specific Treg cells of claim 10, for use in the treatment of DM1.

12. The antigen-specific Treg cells for use according to claim 11 , for treating DM1 in an individual who also receives a B-cell depleting agent.

13. An in vitro method of predicting the course of DM1 in an individual diagnosed with DM1 , comprising assessing the presence of peptides selected from the group consisting of

(i) peptides of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 (protolerogenic peptides); and

(ii) peptides of SEQ ID Nos: 14, 25, 53 and 58 (disease-accelerating peptides) in a biological sample from said individual, wherein the presence of one or several protolerogenic peptides and/or the absence disease-accelerating peptides are indicative of a good clinical outcome.

14. An in vitro method of predicting the course of DM1 in an individual diagnosed with DM1 , comprising assessing the presence of peptides selected from the group consisting of

(i) peptides of SEQ ID Nos: 1 , 4, 11 , 13, 16, 20, 22, 26, 29, 41 , 48, 49, 52, 62, 64, 66, 68, 70, 72 and 74 (protolerogenic peptides); and

(ii) peptides of SEQ ID Nos: 14, 25, 53 and 58 (disease-accelerating peptides) in a biological sample from said individual, wherein the presence of one or several disease-accelerating peptides and/or the absence of protolerogenic peptides are indicative of unfavorable DM1 evolution.

15. An in vitro method for monitoring a response of an individual having DM1 to a treatment, comprising performing the method of claim 13 or claim 14 in biological samples obtained from the individual at different time points, wherein the presence of a stable or increasing number of protolerogenic peptides indicates that the individual responds to the treatment.

16. The method of any one of claims 13 to 15, wherein the individual has a DM1 with residual insulin secretion.

17. The method of any one of claim 13 to 16, wherein the individual is treated with Treg cells.

18. The method of claim 17, wherein the individual is treated with Treg cells combined with a B-cell depleting agent.