RU2824503C2 - Novel combinations for antigen therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method for preventing and/or treating autoimmune diabetes. Method of preventing and/or treating autoimmune diabetes involves administering to a patient a composition, wherein said composition contains at least one beta-cell autoantigen, which is an insulinoma antigen-2, by injection directly into a lymph node.

EFFECT: invention provides effective treatment of autoimmune diabetes.

12 cl, 2 tbl, 7 ex

Description

Cross-reference to related applications

This international application claims priority from Swedish Patent Application No. 1450678-6, filed June 4, 2014, and Swedish Patent Application No. 1451315-4, filed November 4, 2014, which are expressly incorporated herein by reference in their entirety.

Field of technology to which the invention relates

The present invention generally relates to the field of immunology, and in particular relates to the field of immunotherapy. More particularly, the present invention relates to the prevention and/or treatment of autoimmune diseases, such as type 1 diabetes or autoimmune diabetes. The present invention relates to compositions and combinations thereof that are particularly suitable for the prevention and/or treatment of such diseases. The invention also relates to methods of combination therapy for the treatment and/or prevention of autoimmune diseases.

State of the art

The immune defense system, like many other defense systems, consists of multiple and largely interactive parts. There is the innate system, which is thought to respond primarily via pattern recognition receptors such as Toll-like receptors to well-known and common microbial antigens (Ag); and there is the acquired system, which can adapt to respond to a wide variety of foreign organisms and also creates and maintains T cells that respond rapidly when new contact with the same Ag occurs.

In the case of an autoimmune disease, the adaptive immune system typically reacts with at least one self-antigen (autoantigen, aag). Although not fully understood, autoimmunity can be triggered in several ways. Examples of possible triggers discussed include, but are not limited to, innate responses to viruses, diets, vitamins, stress, microbiomes, vaccines, antibiotics, and other drugs. An additional important factor is genetic predisposition.

In the case of endogenously produced self-antigens, they are processed intracellularly and their peptides are transported to the cell surface together with MHC-I molecules and presented to a variety of cells, including naive CD8+ T cells; mature cytotoxic CD8+ T cells; and/or CD8+ effector memory T cells. To activate naive CD8+ T cells to mature cytotoxic T cells, a secondary costimulatory signal associated with CD28 is required.

Self-antigen, which is not located inside the cell, is typically taken up by an antigen-presenting cell (APC) such as a dendritic cell (DC) or macrophage, and the processed peptides are transported in association with MHC-II molecules and presented to naive T cells or CD4+ memory T cells. Here, a secondary costimulatory signal involving CD28 is also required to activate the naive CD4+ T cells into the T helper (Th) cell form. Two forms of Th cells are commonly discussed: Th1; Th2.

The type of Th cell generated upon contact between the APC-MHCII-aag complex and the CD4+ T cell receptor (TCR) depends on several factors, including DC maturity and the environment, including, but not limited to, the availability of IL-10 at the time of presentation.

While Th1 cells secrete inflammatory cytokines including IFNg and stimulate an inflammatory cell-mediated response, Th2 cells typically secrete IL-4 and stimulate a humoral response where BCs produce non-cytolytic and neutralizing antibodies. Antibodies for "coating" and complement fixation (antibody-dependent cellular toxicity) are typically stimulated by the innate system.

Each B lymphocyte (BC) expresses a distinct and specific B cell receptor (BCR) in the form of an immobilized antibody molecule. While T cells recognize cognate Ag as a processed peptide surrounded by an MHC molecule, B cells recognize antigens in their native form (such unprocessed antigens usually do not respond to T cell activation) and, primarily via Th2 signaling, differentiate into short-lived antibody-producing plasma cells, 10% of which develop into long-lived antigen-specific memory B lymphocytes.

It is generally accepted in the art that insulin and insulin-associated molecules are early molecules that are involved in the process leading to autoimmune diabetes, including type 1 diabetes and latent autoimmune diabetes in adults. Extensive clinical trials have been and are currently being conducted using various insulin-based drugs to induce tolerance and stop or prevent disease progression. Many other beta-cell antigen molecules and their peptides have been tested for the same purpose, including, but not limited to, GAD65, and other molecules not limited to the B-chain of insulin and proinsulin, as well as their derivatives, are being prepared for clinical trials.

During the development of the human immune system, self-reactive lymphocytes are thought to be clonogenically excluded in order to distinguish self from non-self. Non-self-active T cells persist and continue to differentiate into naive CD4+ or CD8+ T cells that can be activated by contact with their specific antigen. Some self-reactive lymphocytes, however, manage to escape the elimination process. This is seen to a significant extent if the self-antigen is sequenced, for example, within the cell. However, in the case of insulin, an example of a common molecule, relatively few insulin-reactive lymphocytes manage to escape the elimination process and, as a consequence, there are relatively few naive insulin-reactive CD4+ or CD8+ T cells.

Molecules that are abundant in nature, such as insulin, are generally not adsorbed and presented by dendritic cells unless this process is initiated by activated CD8+ T cells and/or TLR signaling. Thus, and what may not have been noted in the art, the autoimmune process that leads to diabetes may in many cases be initiated by naive CD8+ insulin-reactive lymphocytes that have escaped the clearance process and that recognize aag directly linked to MHC class 1 on target organ cells. In genetically susceptible patients, the insulin-reactive "hidden" naive CD8+ thymocyte may appropriately initiate an inflammatory process, including the generation of cytotoxic CD8+ cells, which in turn may induce cytokines that alert the innate system, including excitation that causes B cells to produce insulin antibodies. Some beta cells fail and release sequenced antigens, such as GAD65, for which there are large numbers of naive CD4+ and CD8+ T cells. GAD65 may also share features with certain viral structures that further stimulate TLR-like signaling.

As the autoimmune reaction progresses, anti-inflammatory mechanisms develop, such as expression of CTLA-4 molecules and secretion of IL-10. Since in the case of insulin the contribution of ADC-MHCII to the resulting autoimmune disease is weak, such compensatory mechanisms induce insulin tolerance in an autoantigen-specific manner. However, GAD molecules are taken up, processed DCs and GAD peptides are presented to naive CD4+ T cells, activating them into T helper cells. Thus, the transient immune response to insulin is replaced by a more robust response to GAD. This explains why in many genetically predisposed young children the first autoantibody is often an insulin antibody and why such antibodies often disappear as the disease progresses.

Mocellin, Cytokine Growth Factor Rev. 2004, The multifaceted relationship between IL-10 and adaptive immunity: Putting the pieces of the puzzle together describes how interleukin-10 (IL-10) acts as a pleiotropic cytokine regulating the function of multiple adaptive immune cells. Although generally considered an immunosuppressant molecule, IL-10 has immunostimulatory properties in in vivo models. As an example, IL-10 produced locally by pancreatic islet cells promotes autoimmunity in NOD mice, while systemic administration can exert beta-cell-preserving and anti-inflammatory effects. The relationship between IL-10 and infectious diseases, autoimmune diseases, allergies, cancers, and transplantation involving adaptive immunity is reviewed.

Russel et al, Islets, 2014, The impact of anti-inflammatory cytokines on the pancreatic β-cell, have recently shown that anti-inflammatory molecules such as interleukins IL-4, IL-10 and IL-13 can have a direct impact on beta-cell function and viability and that circulating levels of these cytokines may be reduced in type 1 diabetes, and it has been suggested that targeting anti-inflammatory pathways may have therapeutic potential in type 1 diabetes.

Calcinaro et al, Diabetologia, 2005, Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse, concluded that early oral administration of probiotic bacteria prevents the development of diabetes in NOD mice and is associated with increased IL-10 expression in the pancreas, where IL-10-positive mononuclear cells were found infiltrating into the islets.

Van Dongen et al, Int. J. Cancer. 2010 Aug 15;127(4):899-909. doi: 10.1002/ijc.25113. Anti-inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor-treated gastrointestinal stromal tumors (GIST), report that tumors treated with tyrosine kinase inhibitors imatinib and sunitinib induced secretion of anti-inflammatory IL-10 in macrophage cultures, suggesting that treatment with these inhibitors may contribute to the immunosuppressive microenvironment of GIST. Overall, these data implicate GIST as an active site of tumor-immune interaction in which suppressive mechanisms override potential antitumor responses. Tyrosine kinase inhibitors may contribute to this negative balance.

Louvet et al, Proc. Natl. Acad. Sci. U S A. 2008, Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice, report how tyrosine kinase (TK) inhibitors offer potential for the treatment of autoimmune diseases. Treatment with imatinib (Gleevec) prevents and reverses T1D in NOD mice. Similar results were observed with sunitinib (Sutent). Another TK inhibitor, PLX647, showed only modest efficacy, while a soluble form of the platelet-derived growth factor receptor (PDGFR), PDGFRbetalg, rapidly reversed diabetes. Imatinib treatment resulted in durable remission and long-term efficacy, and tolerance appears to be dependent on inhibition of a combination of tyrosine kinases, supporting the use of selective kinase inhibitors for the treatment of T1D.

Agostinoet al, J. Oncol. Pharm. Pract. Epub 2010, Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice, describe how tyrosine kinase inhibitors (TKIs) affect changes in blood glucose (BG) concentrations in patients treated with dasatinib, imatinib, sorafenib, and sunitinib. All showed significant reductions in BG. Although the mechanism of the hypoglycemic effect of these drugs is unclear, common kinases targeted include c-kit and PDGFRβ.

Adorini, Ann. N. Y. Acad. Sci. 2003, Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting autoimmune diabetes, report that 1,25-dihydroxyvitamin D(3) induces dendritic cells with a tolerogenic phenotype characterized by decreased expression of costimulatory molecules CD40, CD80, CD86 and low levels of IL-12 and increased secretion of IL-10. Treatment with 1,25-(OH)(2)D(3) induced tolerance in murine islet allografts associated with impaired CD4(+) and CD8(+) type 1 cell development and increased percentage of CD4(+)CD25(+) regulatory cells, and suppressed diabetes development at non-hypercalcemic doses, suggesting that vitamin D is an approach for the treatment of type 1 diabetes.

Heine et al, Eur. J. Immunol. 2008, 1,25-dihydroxyvitamin D(3) promotes IL-10 production in human B cells, report that 1,25-dihydroxyvitamin D(3) (calcitriol) inhibits IgE expression by B cells and enhances IL-10 expression by dendritic cells and T cells. The presence of a molecular link in activated B cells between the activation of the vitamin D signaling pathway, IL-10 expression, and their ability to produce calcitriol from its precursor suggests that 25-hydroxyvitamin D(3) may be used as a regulator of immune responses.

Niiro et al, Biochem. Biophys. Res. Commun. 1998, MAP kinase pathways as a route for regulatory mechanisms of IL-10 and IL-4 which inhibit COX-2 expression in human monocytes, describe how mitogen-activated protein kinases (MAPKs) are activated and play an important role in regulating the expression of proinflammatory molecules in monocytes/macrophages. Lipopolysaccharide (LPS)-stimulated human monocytes induce the expression of COX-2 protein and COX-2 mRNA, as well as signal-regulated protein kinase (ERK)2 and p38 MAPK in monocytes. The induction of COX-2 mRNA, COX-2 protein and prostaglandin (PG)E2 by LPS is inhibited by specific inhibitors of ERK and p38 MAPK. Similarly, IL-10 inhibits COX-2 expression. LPS-induced phosphorylation and activation of ERK2 and p38 MAPK are significantly suppressed by IL-10, suggesting that the inhibition of LPS-induced expression of proinflammatory molecules by IL-10 may be attributed to the regulatory effects of both cytokines on MAPK activation.

Obermajer et al, Blood. 2011, Positive feedback between PGE2 and COX2 redirects the differentiation of human dendritic cells toward stable myeloid-derived suppressor cells, describe how dendritic cells (CDs) and myeloid-derived suppressor cells (MDSCs) have opposing effects on the immune system.

Cyclooxygenase 2 (COX2) is a key regulator of PGE(2) synthesis and a determinant in the redirection of CD1a(+) dendritic cells into monocytic CD14(+)CD33(+)CD34(+) MDSCs. Exogenous PGE(2) and similar diverse COX2 activators such as lipopolysaccharide, IL-1β, and IFNγ all induce COX2 expression by monocytes, blocking their differentiation into CD1a(+) DCs and inducing endogenous PGE(2), IDO1, IL-4Rα, NOS2, and IL-10, typical MDSC-associated suppressive factors. Disruption of COX2-PGE(2) feedback by COX2 inhibitors or EP2 and EP4 antagonists suppresses the production of MDSC-associated suppressive factors and CTL inhibitory function of fully developed MDSCs in cancer patients. The central role of COX2-PGE(2) feedback in the induction and maintenance of MDSCs highlights the potential for its application to enhance or suppress immune responses in cancer, autoimmunity, or transplantation.

Lieb, Med. Hypotheses, 2007, Antidepressants, prostaglandins and the prevention and treatment of cancer, report that the proposed mechanism of cancer development is increased cyclooxygenase levels, oncogene synthesis and expression, viral activation, signal propagation abnormalities, failed apoptosis, tumor initiation and progression, angiogenesis, metastasis, immunosuppression, telomerase activity and autoimmunity. All of these are regulated by prostaglandins. Observational and radiographic regression of cancer has been reported in patients taking nonsteroidal anti-inflammatory drugs containing prostaglandins, such as indomethacin and ibuprofen.

Kim, et al, Immune Netw. 2010, Cyclooxygenase Inhibitors, Aspirin and Ibuprofen, Inhibit MHC-restricted Antigen Presentation in Dendritic Cells, report that nonsteroidal anti-inflammatory drugs (NSAIDs) have immunoregulatory effects on T and B cells and that ibuprofen inhibits MHC class I and class II-restricted antigen presentation in dendritic cells (DCs). Ibuprofen does not inhibit phagocytic activity of dendritic cells, nor the expression of all MHC molecules and costimulatory molecules on dendritic cells. Rather, ibuprofen increases the expression of all MHC molecules and costimulatory molecules on dendritic cells. The results show that ibuprofen inhibits intracellular processing of phagocytosed antigen, and suggest that chronic administration of high doses of NSAIDs may impair the ability of dendritic cells to present antigens in association with MHC molecules.

Gribben et al, Immunology 1994, CTLA4 mediates antigen-specific apoptosis of human T cells, describe how CTLA4 is a T cell-restricted molecule that is induced by TCR or CD28 activation. During the immune response, there is a balance between proinflammatory and non-proinflammatory signals. Cross-linking of CD28 or the common CD28/CTLA4 binding region by monoclonal antibodies or the natural ligands B7-1 and 2 provides a positive costimulatory signal, resulting in increased levels of proinflammatory IL-2. In fact, CTLA4 cross-linking provides a weak costimulatory signal to CD28. Following T cell activation, CD28 expression is downregulated and CTLA4 expression is upregulated. At such times, CTLA4 cross-linking in the absence of CD28 costimulation can induce deletion of previously activated T cells, suggesting that CTLA4 can both costimulate and induce deletion depending on the activation state of the T cell.

Posadas et al, Clinical Immunology 2009, Abatacept in the treatment of rheumatoid arthritis, describe that in rheumatoid arthritis (RA), T cells and several other cells, including dendritic cells, macrophages, and fibroblasts, express activation markers such as CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4). In the peripheral blood, naive T cells require two signals to be activated to their full functional potential: i) antigen flanked by an MHC molecule on the antigen-presenting cell (APC), presented to the corresponding antigen-specific T cell receptor (TCR) on the T cell; and ii) ligation of CD28 on the T cell to CD80/86 on the APC. Stimulation of naive T cells with cognate antigen without costimulation results in T cell anergy, whereas ligation of costimulatory molecules onto T cells in the absence of cognate antigen has no effect on the T cell.

One of the surface molecules that is upregulated on an activated T cell after successful stimulation by both signals is CTLA4. It binds to CD80/80 with higher avidity than CD28. Not only does this prevent CD28 from binding, but CTLA4 also induces inhibitory signals in the newly activated T cell.

Abatacept (Fc-modified immunoglobulin CTLA4) is a T-cell-depleting immunomodulatory fusion protein that consists of the extracellular portion of human CTLA4 and the heavy chain of human IgG1. It blocks a costimulatory signal that is involved in the activation of naive T cells. Its ligation to CD80/86 on APCs can also interfere with and reduce IL-6, which induces CD80/86, which can suppress inflammatory cytokines such as IL-1β, IFNγ, and IL-17. Further ligation of abatacept to CD80/86 can induce indoleamine dioxygenase (IDO) in APCs, which in turn can cause anergy in T cells, as well as suppress paracrine activation of naive T cells by activated T cells.

Controversy has been expressed regarding the ability of abatacept to influence the secondary immune response, restimulation of CD4+ memory T cells. However, expression of CD80/86 on B cells may represent another pathway where abatacept may exert an immune regulatory function.

Patakas et al, abstract #723 ACR/ARHP, 2013, Abatacept is Highly Effective At Inhibiting T cell Priming and Induces a Unique Transcriptional Profile In CD4+ T Cells, indicate that subcutaneous priming with ovalbumin in the presence of abatacept produces large amounts of IL-2, resembling naive T cells newly exposed to antigen. These T cells include fewer Tregs and CD4+ T cells than other (non-primed or naive) T cell populations, and their condition is accompanied by transcriptional suppression of dendritic cell activation. Patakas et al. conclude that while abatacept significantly regulates T-DC communication that occurs upon priming of the defective cell, this is distinct from anergic tolerance.

Orban et al, Lancet 2011, Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled trial, report that abatacept significantly preserves endogenous insulin secretion as measured by stimulated C-peptide during the first 6 months compared with placebo and then declines in parallel with placebo. The early beneficial effect is thought to be due to abatacept blocking the B80/86-CD28 co-stimulatory signal, preventing the activation of naive T cells. The consistent decline in beta-cell function after 6 months is thought to be due to continued T-cell activation, which declines as the disease progresses.

Although Orban et al. indicate that two signals are required to activate naive T cells, one originating from antigen presented in the context of an MHC molecule on the APC and the other being a stimulatory signal involving CD28 molecules on the T cell, no specific antigens were used in the trials. This may suggest that while abatacept initially and successfully prevents the activation of naive T cells into cytotoxic T cells, its action may eventually be neutralized and/or compensated for by other parts of the immune system, such as increased IL-2 secretion, and when this occurs, the opportunity to induce tolerance to a particular antigen is lost.

Orban et al, Diabetes Care 2013, Costimulation Modulation With Abatacept in Patients With Recent-Onset Type 1 Diabetes: Follow-up 1 Year After Cessation of Treatment, further report a sustained beneficial effect beyond one year after stopping the drug, although the differences between the active-agent and placebo groups had diminished. Thus, 35% of patients in the active-agent group had a peak stimulated C-peptide > 0.2 nmol/L, compared with 30% for patients in the placebo group at 36 months after starting treatment. There was no effect of treatment with abatacept in DR3-negative patients.

Both of the Orban et al. studies mentioned above indicate that immune regulation by abatacept as a stand-alone treatment in patients with recent onset of type 1 diabetes is not sufficient to achieve a beta-cell function-preserving effect.

Verhagen et al, PLOS 2014, CTLA-4 Modulates the Differentiation of Inducible Foxp3+ Treg Cells but IL-10 Mediates Their Function in Experimental Autoimmune Encephalomyelitis, concluded that the presence of IL-10 is an important factor in enhancing the suppressive effects of Tregs.

GAD65 (65 kD isoform of glutamic acid decarboxylase) is one of the major beta-cell autoantigens, as its production in islet cells with increased secretion is observed in response to beta-cell stimulation. It has been shown that this protein has a profound effect on the autoimmune immunological process. Numerous studies have shown that GAD can prevent the development of diabetes mellitus in experimental animals. The similarity of GAD to viral proteins may be important for the therapeutic effect. The observed effect, even after the onset of the immune process, suggests that the same effect could be expected in humans. Recombinant GAD has been formulated with aluminum hydroxide (alum), and in a phase II study in LADA patients, administration of a single low dose of 20 μg of the diamide did not improve beta-cell function for up to 2 years, compared with the placebo group, without any adverse effects. Other doses were also tried: 4 mcg had no effect, 100 mcg showed the same effect as 20 mcg, while 500 mcg showed no effect. An association with a change in the CD4+CD25+/CD4-CD25- cell ratio was found, suggesting a mechanism for the effect. Against this background, a phase II study was conducted in patients aged 10-18 years with recent onset of type 1 diabetes. Patients were randomly assigned to either 20 mcg GAD-alum (diamide), administered subcutaneously on Day 1 and Day 30, or placebo. After 30 months, the effect was still visible and was clearly statistically and clinically significant, with about half the C-peptide decreased in the GAD group compared with the placebo group. In patients with diabetes duration <3 months, a surprisingly good effect was observed with no or minimal decline in beta-cell function over the next 15 months. And after 48 months, patients treated for <6 months maintained significant C-peptide levels and had no adverse effects.

Subsequently, phase III trials were initiated in Europe and in the USA. The European study enrolled 334 patients and divided them into three groups, one group with GAD-alum (diamide) 20 mcg on Day 1, 30, 90 and 270, another group with GAD-alum 20 mcg on Day 1 and 30 and placebo on Day 90 and 270 and a third group with placebo on Day 1, 30, 90 and 270. Despite positive trends (16% efficacy, p=0.1), the primary endpoint, the area under the curve of serum C-peptide after the food tolerance test (FTT), was not met after 15 months, leading to early termination of the phase III trials. However, the European phase III trials did show statistically significant efficacy in some prespecified subgroups. Furthermore, 45 Swedish patients were seen by their physicians at 30 months when the study was stopped, and the 15 patients who received two doses of 20 mcg GAD-alum (diamide) showed significant preservation of C-peptide at 30 months compared to placebo. It was noted that these Swedish patients were the ones who did not show efficacy after 15 months, while efficacy was seen at 15 months in the non-Scandinavian patients. It was concluded that although partially effective, GAD-alum may need to be combined with other compounds to become part of a clinically effective treatment regimen for type 1 diabetes.

Denes et al, Diabetes Technol. Ther. 2010, Autoantigens plus interleukin-10 suppress diabetes autoimmunity, report that while a recombinant vaccinia virus (rVV) strain that expresses the immunomodulatory cholera toxin B subunit (CTB) fused to a fragment of the autoantigen glutamic acid decarboxylase (GAD65) or the immunosuppressive cytokine interleukin-10 (IL-10) were independently able to generate only low levels of immune suppression of type 1 diabetes mellitus (T1DM) in NOD mice, a vaccinia virus (VV)-mediated combination of CTB::GAD and IL-10 fusion proteins proved to be a more effective and durable immunotherapeutic strategy for T1DM.

Robert et al, Diabetes, 2014, Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice, recently showed that oral administration of live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen peptide GAD65370-575 and the anti-inflammatory cytokine interleukin-10, in combination with a short course of low-dose anti-CD3, preserves functional β-cell mass in recent-onset NOD mice.

Skyler et al, Diabetes 2011, Stopping Type 1 Diabetes, Attempts to Prevent or Cure Type 1 Diabetes in Man (which is incorporated herein by reference in its entirety) indicate that combination therapy regimens using an immunomodulator and antigen-specific therapy provide a rationale for longer-term treatment of T1D.

Staeva et al, DIABETES, VOL. 62, JANUARY 2013, Recent Lessons Learned from Prevention and Recent-Onset Type 1 Diabetes Immunotherapy Trials (which is incorporated herein by reference in its entirety) provides a review of current concepts in the art and provides recommendations for evaluating combination therapy using autoantigens and anti-inflammatory compounds.

Skyler, DIABETES TECHNOLOGY & THERAPEUTICS, Volume 16, Supplement 1, 2014, Immune Intervention for Type 1 Diabetes, 2012-2013 (which is incorporated herein by reference in its entirety), provides a review of recent trials in real-world settings.

Rigby et al, Current Opinion Endocrinol. Diabetes Obes. 2014, 21:271-278, Targeted immune interventions for type 1 diabetes: not as easy as it looks! (which is incorporated by reference in its entirety) review numerous attempts to combat autoimmune diabetes and conclude that despite nearly a dozen trials involving many hundreds of participants, no monotherapy has been offered and that: a) different subpopulations of T1D patients appear to respond differently to immune interventions, suggesting significant heterogeneity; b) effective therapy must combine Teff inhibition (by depletion, increased suppression, or both) and Tregs stimulation (by increasing frequency or function, including by sequestering the proinflammatory milieu); and this may require combinations including a Teff-depleting agent, a Treg-enhancing agent, and an antigen.

As stated above, several monotherapy and combination regimens of drug administration including various compounds have shown promising effects in preventing and treating autoimmune diabetes in a mouse model. However, all attempts to transfer such drug administration regimens to humans have failed to provide a clinically significant effect. Thus, there remains a great need to save society and patients from the devastating disease of autoimmune diabetes and its long-term complications. The object of the present invention is to develop methods and compositions for preventing and treating the autoimmune component of T1D and LADA, which opens the way for endogenous or otherwise achievable enhancement of the functional mass of beta cells.

The essence of the invention

According to one aspect, the present invention relates to a method for preventing and/or treating an autoimmune disease, which comprises administering a composition, wherein said composition comprises at least one beta cell autoantigen, to a patient having a serum vitamin D level above 50 nanomol/liter.

The present invention discloses a method of using anti-inflammatory compounds and autoantigens in combination with methods of imparting greater "tolerizing capacity" to dendritic antigen-presenting cells (APCs) when presenting said autoantigens to the immune system.

The present invention discloses methods wherein combination regimens of drug administration including anti-inflammatory compounds, autoantigens and vitamin D can be effectively used as methods for the prevention and treatment of T1D and other autoimmune diseases.

According to another aspect, the present invention relates to a method for the prevention and/or treatment of an autoimmune disease, which comprises administering to a patient a composition, wherein said composition comprises at least one beta cell autoantigen, by intralymphatic injection or injection directly into a lymph node.

According to another aspect, the present invention relates to a method for the prevention and/or treatment of an autoimmune disease, which comprises administering to a patient at least one beta cell autoantigen in increasing doses over several weeks, months or years.

According to another aspect, the present invention relates to a composition comprising a plurality of particles, each of which comprises at least one first and at least one second antigen immobilized on its surface, wherein the first antigen is a beta cell autoantigen and the second antigen is either a tolerogenic factor or a beta cell autoantigen, wherein said composition optionally further comprises pharmaceutically acceptable auxiliary substances, excipients, solvents and/or buffering agents.

According to another aspect, the present invention relates to a composition comprising:

i) at least one beta cell autoantigen and at least one of the following

iia) a compound that causes IL-10 induction, which is selected from the group consisting of vitamin D, vitamin D analogues, tyrosine kinase inhibitors, gamma-aminobutyric acid and gamma-aminobutyric acid analogues; and

iib) a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor;

and optionally pharmaceutically acceptable auxiliary substances, excipients, solvents and/or buffering agents.

According to another aspect, the present invention relates to a pharmaceutical kit that comprises:

i) a composition comprising a beta cell autoantigen and at least one of the following:

iia) a composition comprising a compound that induces IL-10, which is selected from the group consisting of vitamin D, vitamin D analogues, tyrosine kinase inhibitors, gamma-aminobutyric acid, gamma-aminobutyric acid analogues; and

iib) a composition comprising a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor.

According to another aspect, the present invention relates to a beta cell autoantigen for use in the method of the present invention.

Preferred embodiments of the present invention are set forth in the dependent claims.

Definitions

All terms and expressions used in this specification are intended to have the meaning assigned to them by those skilled in the art on the filing date of this application, unless a different meaning is not obvious from the context of this specification. However, for clarity, some terms and expressions are specifically defined below.

An "autoantigen" or "self-antigen" is an endogenous tissue component that has the ability to interact with an autoantibody and elicit an immune response. A "beta-cell autoantigen" is an autoantigen produced in the beta cells of the pancreas.

An "autoantibody" is an antibody that reacts with the self-antigens of the organism that produced them.

The term "vitamin D" includes vitamin _D2 and vitamin _D3 . "Vitamin D analogs" include, without limitation, ergocalciferol, dihydrotachysterol, alfacalcidol, calcitriol, cholecalciferol, and calcifediol and combinations thereof, and any other vitamin D analog classified in group A11CC of the Anatomical Therapeutic Chemical Classification System.

The term "cyclooxygenase inhibitor" or "COX inhibitor" refers to compounds that combine with cyclooxygenase and thereby prevent its substrate-enzyme combinations with arachidonic acid and the formation of eicosanoids, prostaglandins, and thromboxanes. A subgroup of cyclooxygenase inhibitors are the cyclooxygenase-2 inhibitors, which have specificity for cyclooxygenase-2.

The term "TNF alpha inhibitor" refers to compounds that inhibit the action of tumor necrosis factor alpha (TNF-alpha) and includes adalimumab, sertolizumab, etanercept, golimumab, infliximab, and any other compound classified in group L04AB of the Anatomical Therapeutic Chemical Classification System.

An "epitope" is a portion of the surface of an antigen that is capable of eliciting an immune response and binding to an antibody produced to counteract that response or to a T-cell receptor.

The term "gamma-aminobutyric acid analogs" includes vigabatrin and baclofen.

Expressions using the singular, etc., should be considered as including the plural.

As used herein, the term "co-administration" refers to the administration of the compounds according to the dosage regimens of the present invention such that their dosing regimens overlap. They do not necessarily have to be administered simultaneously.

The abbreviation "T1D" stands for "type 1 diabetes."

Detailed description of the invention

At the point where the first insulin autoantibody appears in a genetically susceptible individual, such as, but not limited to, a DR3 haplotype individual, and no other autoantibodies are detected, this may indicate that GAD-reactive CD8+ and CD4+ T cells have not yet been activated, suggesting a window of opportunity when the disease can be completely reversed by anti-inflammatory agents and/or lymphocyte differentiation inhibitors alone, without the use of beta cell antigen, since insulin autoimmunity is likely to be self-limited by a counter-measure mechanism. In this situation, when few, if any, GAD-reactive activated CD4+ T helper cells are still being produced, it is important to direct activation away from the pro-inflammatory Th1 type. In GAD antibody-positive individuals and/or in diabetic patients, GAD and other beta cell antigens formulated with aluminum hydroxide, commonly considered a Th2 activating adjuvant, can be administered to provide antigen-specific tolerance-inducing agents. Although this may result in more Th2 cells and Tregs than proinflammatory cells, several factors, including intrinsic environmental factors, may offset the tolerance-inducing effect. Therefore, it may be insufficient to use Th2 activating autoantigen compositions as monotherapy, and the present invention provides compositions and combination regimens for enhancing beta cell tolerance-inducing methods.

Previous attempts to perform immune regulation in autoimmune diseases by administering monotherapeutic drugs such as GAD and insulin antigens; anti-inflammatory compounds such as CD3, CD20 antibodies; thymoglobulin, abatacept, alefacept or TNF alpha inhibitors such as etanercept have shown some efficacy, however, the duration of these effects or their effectiveness after disease onset limits the application of these approaches in clinical settings. The present invention discloses a new strategy for the treatment and prevention of autoimmune diseases in dendritic cells enhanced by vitamin D, which acts synergistically with autoantigens and anti-inflammatory agents, demonstrating varying degrees of effect.

Thus, the present invention relates to means (eg, methods, compositions and combinations thereof) for the prevention and/or treatment of autoimmune diseases, such as autoimmune diabetes.

The present invention utilizes the knowledge that vitamin D (Vit D) enhances immature dendritic cells (DCs) in a manner that renders them more tolerogenic, which has not yet been shown to be sufficient to alter the course of any autoimmune disease, and combines the timely administration of Vit D with the administration of an autoantigen associated with said autoimmune disease, thereby enhancing the tolerogenic immunoregulation of the administered autoantigen.

To further enhance the tolerogenic environment in which dendritic cells present autoantigens to the immune system of a patient with sufficient serum Vit D concentration, the present invention discloses that combination therapy regimens using a conventional anti-inflammatory compound together with an autoantigen are useful for the treatment and prevention of T1D.

In addition to its potential synergistic effects, another advantage of co-administration of the compounds of the present invention concerns the problem that autoimmune disorders often involve autoimmune reactions against several autoantigens. It may be difficult to attempt to induce anergy or remove all autoaggressive lymphocytes using direct antigen-specific tolerance induction to all their cognate antigens, since all cognate antigens may be unknown. For example, type 1 diabetes mellitus (T1D) is thought to be caused by autoaggressive lymphocytes that enter the islets of Langerhans, where they destroy beta cells. Activation of such cells is likely multifactorial, involving genetic predisposition, environmental activators such as viruses, and may cause pancreatic (islet cell) damage caused by, for example, a local proinflammatory response.

Because autoaggressive processes are usually somewhat accelerated when pre-diabetic individuals are identified by islet cell antibody screening, it is reasonable to assume that aggressive responses to more than one islet cell antigen will continue at this stage of the disease. Furthermore, chronic non-specific systemic immune suppression is not considered an option because diabetes often affects young individuals and lifelong immune suppression is associated with adverse effects that are unacceptable, even when compared with insulin therapy alone.

Thus, a therapeutic immune intervention with specificity and low systemic side effects is highly desirable. The methods of the present invention circumvent the problem of multiple autoantigen targets, since co-administration of the compounds of the present invention can be sufficient to restore tolerance to multiple autoantigens that are targets in an autoimmune disorder.

The methods of the present invention also circumvent the problem of chronic non-specific systemic immune suppression because co-administration of the compounds of the present invention can restore long-term tolerance without the need for continuous lifelong dosing.

Without wishing to be bound by any theory, the present inventors believe that co-administration of the compounds of the present invention has the potential to synergistically establish long-term tolerance, in part by inducing activation/expansion of regulatory T cells (as well as regulatory antigen-presenting cells (APCs)).

A number of different phenotypes of regulatory T cells have been described. They can arise after thymectomy and can be induced after systemic immune regulation. Their effector functions are not fully known. They probably form part of the intrinsic balance of the immune system and their loss leads to severe immune dysregulation and autoimmune diseases. Th2-like regulators with defined antigen specificity have been described. They are thought to suppress the immune response as an unrelated antigen and arise after antigen-specific immunization. Homann et al., J. Immunol. (1999) 163:1833-8. Based on their effector function, they have been named Th3 (TGF-beta producers). These cells are antigen-specific lymphocytes with specialized effector functions and their behavior differs from that of Th2 cells. Therefore, applying the so-called Th1/Th2 paradigm to these cells may lead down the wrong path.

Suppression of the immune response by unrelated antigens refers to the phenomenon of antigenic spreading. Antigenic spreading is considered to be an important component of the progression of local autoimmune processes. It can therefore be assumed that when patients have multiple autoantibodies, the autoaggressive response may involve multiple self-antigens (or "autoantigens"). Since most autoantigens cannot be defined for a specific autoimmune disorder, it is not possible to induce tolerance to each autoaggressive specificity using therapeutic drug regimens that include knowledge of the corresponding restriction element and MHC peptide. In this situation, inducing regulatory cells by the methods of the present invention offers a number of advantages. It is known, for example, that regulatory T cells in T1D can act locally in lymph nodes and islets as suppressors of the immune response as an unrelated antigen, which means that they can suppress aggressive lymphocytes with a different autoantigen specificity. This may occur by modulating antigen-presenting cells (APCs), such as by secreting cytokines with immune modulating function. Thus, such T-regulatory cells as an unrelated antigen in suppressing the immune response can weaken autoaggression to several other autoantigens, without knowing their specific specificity.

In the presence of a first autoantibody to a common 1st antigen or autoantigen, such as insulin, in a genetically susceptible individual, while no antibodies to at least one other 2nd sequenced (less common) autoantigen are detected, a time window may arise with the possibility of stopping the potential disease process at its inception with anti-inflammatory agents and/or lymphocyte differentiation inhibitors alone. In fact, since the inflammatory response to such a weak 1st autoantigen can be caused by a relatively small number of insulin-specific autoreactive CD8+ and CD4+ T cells (TCs) that have escaped the clonal deletion process in the thymus, the inflammation can disappear and said 1st antigen in some embodiments of the present invention can be used as a tolerogen inducing tolerance to other, possibly more sequenced or unusual autoantigens for which a larger number of naive autoreactive CD8+ and CD4+ T cells (TCs) that have escaped the deletion process are available for activation.

In situations where autoimmunity activates more potent autoantigens, such as, for example, GAD65 in the case of autoimmune diabetes, and where it is able to control the disease process, it may be insufficient to use anti-inflammatory agents and/or inhibitors of lymphocyte differentiation alone without the use of these potent autoantigens to induce active tolerization. On the other hand, the use of potent autoantigens alone, for example, GAD65 in admixture with an adjuvant such as aluminum hydroxide (herein referred to as "alum"), which is usually considered a Th2-activating adjuvant agent, may lead to the activation of not only regulatory components but also proinflammatory and/or cytotoxic molecules and cells, not limited to molecules belonging to the innate immune system, macrophages, dendritic cells, B cells (BCs), T cells (TCs) or other factors, including environmental factors, that may contribute to counteract tolerization. In addition, other less Th2-activating auxiliary compounds such as physiological saline or human serum albumin can be used. Therefore, it may be insufficient to use compositions of individual autoantigens as monotherapy, and the present invention discloses compositions and methods that use at least two autoantigens to enhance the effect of specific tolerization of the administered autoantigens, using which formulations are prepared that include aluminum hydroxide, physiological saline or human serum albumin.

According to one aspect, the present invention provides at least one pharmaceutical composition comprising at least one antigen. The at least one pharmaceutical composition of the present invention may, in some embodiments, comprise at least two antigens. According to some other embodiments, the at least one composition comprises at least three autoantigens. According to some other embodiments, the at least one composition comprises at least four autoantigens. According to some other embodiments, the antigens may be prepared into separate compositions. Most preferably, all of the antigens of the present invention may be included in the same formulation. In some embodiments, all of the antigens are autoantigens, in some other embodiments, some or all of the antigens are antigens (tolerogens) to which the immune system has developed a regulatory response, and is thus able to tolerantly influence the response to other autoantigens.

The subject of the present invention are disclosed compositions, drug administration regimens and methods that allow inducing tolerance of self-structures that are attacked by an autoimmune disease. In one specific embodiment of the present invention, at least two antigens are bound to one carrier particle and thereby expose one immune cell of the adaptive immune system to the action of at least two antigens that have a different effect on said immune cell, which leads to a modified response of the patient's self-structure to an autoimmune attack.

By adsorbing, binding, or encapsulating an antigen protein or peptide onto carrier particles with which the immune system reacts and introducing the particles into human tissues by, for example, subcutaneous injection, the protein or peptide can be targeted for entry into the immune system. This entry is most likely accomplished by dendritic cells or macrophages (antigen-presenting cells, APCs). Some of the proteins or peptides entered into the body by this process will be presented to the adaptive immune system by HLA proteins (MHC), and the adaptive immune response will proceed. The adaptive response will depend on the level of innate immune activity and the immune response reactions that exist in relation to any antigens that are presented by the HLA proteins during this process. These influencing proteins may be peptide- or protein-linked particles, or native proteins, autoantigens, or tolerogens (antigens to which the immune system reacts in a tolerogenic manner) that enter the body and are presented in the same process. If these proteins are taken up by the same immune cell, they will be presented together with the HLA proteins on the cell surface.

The presentation of a foreign peptide to the immune system can be influenced by signals from the innate immune system in response to tissue destruction, which has a Th2 character. Endogenous antigens, including autoantibodies, RNA nucleotides, etc., can do the same. A native protein or a protein to which a response already exists will elicit signaling by adaptive immune cells that influences the overall immune response and may activate tolerogenic signals or a pre-existing Th1 response. If the signaling induced by injection and by the interaction of naive immune cells and antigen-presenting immune cells is stronger than the pre-existing response, a new immune response will be formed, including memory.

If a combination of proteins present on the same carrier particles is administered and is taken up and presented by the same APC, the effects will be additive due to the expansion of the number of possible adaptive immune cells with which interaction occurs, leading to an increased likelihood of the formation of a new immune response, despite a possible pre-existing response to one of the proteins.

If the autoantigen protein is administered on the same carrier particle together with another protein (native or foreign) to which an immune response already exists, the immune response to the antigen will largely depend on the reaction that the immune system has already generated in relation to the accompanying antigens. If the accompanying antigens are native proteins for which central regulatory T cells exist (such as, for example, IL-10, insulin, human serum albumin, hemoglobin), the reaction will be directed towards tolerance. If the said proteins are antigens to which the person has already been immunized, for example, as is often the case with diphtheria or tetanus toxoid, the immune response will depend on the existing reactions (which are mainly inflammatory reactions).

The present invention includes methods for treating autoimmunity and/or establishing or inducing tolerance by co-administering the compounds of the present invention. In addition to autoimmune diseases, the methods of the present invention can also be used to establish tolerance to allergens, wherein allergenic peptides or proteins are co-administered with the compounds of the present invention, wherein the antigens, instead of self-antigens, are allergens (antigens) specific for an allergic disease.

It should be understood that the details provided herein for one aspect, in particular the details regarding autoantigens, influence antigens, a compound that causes IL-10 induction, and a compound that reduces the ability of the immune system to activate naive TCs and BCs and to elicit a secondary immune response from activated and memory lymphocytes, the time and route of administration, apply mutatis mutandis to all other aspects of the present invention.

Methods

According to one aspect, the present invention relates to a method for preventing and/or treating an autoimmune disease, which comprises administering a composition, wherein said composition comprises at least one beta cell autoantigen, to a patient having a serum vitamin D level above 50 nmol/liter. Each of the at least two molecules can thus influence the response of adaptive immune cells to which the antigens are presented.

A patient may have a serum vitamin D level in the range of 50-150 nmol/L, such as 60-100 nmol/L, 75-100 nmol/L, or 100-150 nmol/L.

The method may include pre-treatment of the patient in order to regulate the serum vitamin D level, and such premedication may include administration of vitamin D and/or vitamin D analogues and/or exposure to UVB radiation, preferably for 7 to 90 days prior to administration to said patient of a composition comprising at least one beta cell autoantigen.

The method may further include the administration of vitamin D and/or vitamin D analogues in an amount of 7,000-70,000 international units per week for 3-48 months.

The beta cell autoantigen may be the beta cell autoantigen discussed below in the Autoantigens section.

The method may further comprise administering a cyclooxygenase inhibitor, as discussed below under "Cyclooxygenase Inhibitors."

The method may further comprise administering a CTLA4 compound, as discussed below under "CTLA4 Compounds."

The method may further include administering a TNF alpha inhibitor, as discussed below under "TNF alpha inhibitors."

The present invention provides a method for the prevention and/or treatment of an autoimmune disease in a patient, comprising administering to said patient:

a) for the purpose of tolerizing specific antigens of at least one autoantigen or fragments thereof; or nucleic acids, plasmids or vectors encoding such molecules associated with at least one of the above autoimmune and inflammatory diseases. In one embodiment of the present invention, the autoantigen is administered when the serum vitamin D level is from 50 to 150 nM/L, more preferably from 75 to 100 nM/L and most preferably from 100 to 150 nM/L; and

b) in order to hinder the maturation of APCs, administering to said patient at least one IL-10 inducing compound selected from the group consisting of vitamin D, vitamin D analogs, gamma-aminobutyric acid, gamma-aminobutyric acid analogs, and tyrosine kinase inhibitors; as defined above. In one embodiment of the present invention, IL-10 induction is enhanced or achieved by exposure to UVB radiation; and

c) in order to interfere with the ability of the immune system to activate naive TCs and BCs and elicit a secondary immune response from activated and memory lymphocytes, administering a compound such as an NSAID compound; a CTLA-4 compound; or a TNF alpha inhibitor; as described above.

According to some aspects, the present invention relates to methods for preventing and/or treating autoimmune diseases, such as type 1 diabetes (T1D) and autoimmune diabetes.

The present invention discloses a method for treating autoimmune diseases such as T1D and autoimmune diabetes, which comprises administering to a patient suffering from said disease:

a) a course of Vit D to enhance the ability of antigen-presenting dendritic cells to present antigen peptides to the immune system in a tolerizing manner;

b) an autoantigen, such as GAD65, formulated with a pharmaceutical carrier that is administered in an amount sufficient to restore or induce tolerance to the autoantigen; and optionally,

c) a therapeutic dose of an anti-inflammatory compound, such as a cyclooxygenase inhibitor such as ibuprofen, or a more potent cox-2 or cox-1 inhibitor.

The Vit D course is preferably started 15-90 days before the autoantigen administration, or 7 to 90 days before the autoantigen administration, and Vit D is administered in liquid form or as tablets at doses corresponding to 7,000 to 70,000 international units per week for 3 to 48 months.

Premedication with vitamin D is intended to raise the patient's serum vitamin D level above approximately 50 nmol/L, or above 60, 75, or 100 nmol/L. Premedication may be omitted if the patient's serum vitamin D level already meets these levels.

In another embodiment of the present invention, the serum Vit D concentration can be increased by phototherapy. In this case, patients are exposed to ultraviolet B radiation, preferably in the range of 10-120 minutes daily for 15-90 days before the administration of the autoantigen. Phototherapy should continue for 3 to 48 months.

Preferred doses of the autoantigen are two to four administrations, at least two weeks apart, more preferably one month apart, each in an amount of 10 to 200 μg of antigen if administered by injection. When administered orally, preferred doses are in the range of 500 mg to 5 g daily for three months to 48 months. Ibuprofen is preferably administered in daily doses of 100 to 800 mg for 60 to 150 days, and during this period the autoantigen is administered.

The autoantigen may be administered by intralymphatic injection, injection directly into a lymph node, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, intranasal, transmucosal or sublingual; or orally, including administration in the form of tablets, pills, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays or in dry powder form that is reconstituted with a liquid medium. The anti-inflammatory compound and the autoantigen may be administered together with a pharmaceutically acceptable carrier, excipient or diluent.

In one embodiment of the present invention, and in the case of treatment or prevention of T1D, injections of autoantigen are performed subcutaneously on one side or both sides of the stomach area to increase the trafficking of APC cells presenting autoantigen peptides to the pancreatic lymph nodes.

According to some other embodiments of the present invention, at least one autoantigen is administered subcutaneously, for example, into the stomach, in the vicinity of the draining lymph nodes of the pancreas. In some embodiments of the present invention, the volume of subcutaneous injection of antigens is from 0.2 to 2 ml, more preferably from 0.4 to 0.6 ml.

According to some other embodiments of the present invention, at least one autoantigen is administered by intradermal injection.

According to some embodiments of the present invention, when at least one antigen is administered by subcutaneous injection in a "vaccine-like" manner, a dose of at least 4-250 international units, more preferably 10-100 international units, most preferably 10-50 international units, is administered at each administration during treatment of the at least one antigen.

According to some embodiments of the present invention, at least one antigen is administered by subcutaneous injection of "allergic type", wherein various schedules of administration of increasing medicinal doses of each used autoantigen can be used. Such a method can include administration of beta-cell autoantigen at increasing doses for several weeks, months or years.

In one embodiment of the present invention, the composition comprising a beta cell autoantigen is administered 1-4 weeks apart, such as 2-4 weeks apart or 2 weeks apart, in an initial treatment period of 3 to 4 months, and optionally 2-3 months apart, in a long-term treatment period lasting 6-9 months.

In one embodiment of the present invention, the amount of beta cell autoantigen is increased from 1-5 mcg per administration during the initial treatment period to approximately 40-100 mcg per administration for the final drug administrations.

In one embodiment of the present invention, the method comprises subcutaneous injections of a beta cell autoantigen (e.g., GAD65) at escalating doses starting from baseline (Month 1) as follows: 0.4; 0.8; 2; 3.2; 4; 6.4; 8; 12; 16; 20; 24, 32, 40 mcg of autoantigen weekly, where starting with 40 mcg, the autoantigen is administered for weeks 15 to 27 at intervals of 2, 4, and 8 weeks. 40 mcg of autoantigen is then administered every 8 to 12 weeks for 1 year. Vitamin D can be administered as described above (starting on Day 1).

The preferred dosing regimen includes escalating doses of autoantigen 1 mcg, 5 mcg, 20 mcg, 50 mcg, two injections of each dose 4 weeks apart, resulting in 28 weeks of treatment with GAD-alum. Alternatively, the preferred dosing regimen includes escalating doses such as 4 mcg, 8 mcg, 16 mcg, 20 mcg, 40 mcg on Days 0, 15, 30, 45, 60, and then 40 mcg on Days 120, 180, 270.

In one embodiment of the present invention, the autoantigen is administered directly to the lymph nodes or to the lymphatic system in order to allow resident APCs to present the antigen peptides to the immune system. If the autoantigens are administered directly to the lymph node or to the lymphatic system, the dose is preferably 1 to 15 μg based on the autoantigen, more preferably 2 to 10 μg based on the autoantigen or 2 to 5 μg based on the autoantigen. The composition together with aluminum hydroxide is preferred.

According to some embodiments of the present invention, at least one autoantigen is administered intrainguinal, intralymph node or intralymphatic system. In some embodiments of the present invention, the volume of intrainguinal injection of antigens is from 0.05 to 0.2 ml, more preferably from 0.05 to 0.15 ml.

According to some embodiments of the present invention, when at least one antigen is administered intralymph node or as an intralymphatic injection, the preferred dose is 1-15 μg, more preferably in the range of 2-10 and most preferably in the range of 2-5 μg per injection and the autoantigen used, wherein such administrations are carried out at least 2 times, more preferably at least 3 times and most preferably at least 4 times with an interval of at least 14 days from each other, more preferably at least 30 days from each other.

According to some embodiments of the present invention, when at least one antigen is administered intravenously, at least 100-10,000 μg of each antigen is administered at one time, at least twice, at least one week apart.

According to some embodiments of the present invention, when at least one antigen is administered orally, at least 0.5-5 g of each antigen is administered at one time at least once a week.

According to some embodiments of the present invention, at least one antigen is formulated, alone or together with other antigens, as appropriate, in aluminum hydroxide, saline, or human serum albumin.

According to some embodiments of the present invention, at least one pharmaceutical composition comprises at least two antigens on the same carrier particle. According to some other embodiments of the present invention, at least one pharmaceutical composition comprises at least two antigens on different carrier particles. According to some embodiments of the present invention, the at least two antigens are administered separately, at different times and frequencies, with different schedules and in different compositions that are suitable for the specific antigens. More preferably, the at least two antigens are formulated as the same pharmaceutical composition and are therefore administered simultaneously.

In some embodiments of the present invention, at least one antigen is formulated together with an auxiliary compound, such as aluminum hydroxide. In more specific embodiments of the present invention, at least one antigen is formulated in saline or human serum albumin.

According to some embodiments of the present invention, at least one antigen and at least one IL-10 inducing compound are administered simultaneously. According to some embodiments of the present invention, at least one antigen and at least one IL-10 inducing compound are administered separately.

According to some embodiments of the present invention, at least one IL-10 inducing compound is administered simultaneously with at least one antigen. According to some embodiments of the present invention, the administration of at least one IL-10 inducing compound is started within 1-14 days before the first administration of at least one antigen. According to some embodiments of the present invention, the administration of at least one IL-10 inducing compound is started at least 2 weeks, preferably at least 10 weeks before the start of the first administration of at least one autoantigen.

According to some embodiments of the present invention that include periods of treatment using vitamin D, a range of 500 to 10,000 international units, more preferably 1,000 to 3,000 international units of vitamin D, such as vitamin D3, is administered daily.

According to some embodiments of the present invention that include premedication using vitamin D, a range of 7,000-100,000 international units of vitamin D, such as vitamin _D3 , is administered daily, administered once a week prior to the first administration of at least one antigen, and then as a maintenance dose of 500-2,000 international units per day thereafter.

In some embodiments of the present invention, the period of treatment using vitamin D is from 60 to 420 days after the 1st administration of the antigen.

A compound that reduces the ability of the immune system to activate naive TCs and BCs and to elicit a secondary immune response from activated and memory lymphocytes, such as an NSAID compound; a CTLA-4 compound; or a TNFα inhibitor, can be administered simultaneously or separately with at least one additional antigen and/or at least one IL-10 inducing compound.

In some embodiments of the present invention, a compound that reduces the ability of the immune system to activate naive TCs and BCs and elicit a secondary immune response from activated and memory lymphocytes is an NSAID compound, such as a COX inhibitor.

According to some embodiments of the present invention, the treatment period using an NSAID begins at least 2 weeks prior to the first administration of at least one antigen.

According to some embodiments of the present invention, when the COX inhibitor is ibuprofen, at least one dose in the range of 400 to 1000 mg is administered per day during the treatment period using the NSAID.

The treatment period with NSAIDs is at least 4 to 14 weeks, more preferably 4 to 8 weeks.

In the methods of the present invention, the anti-inflammatory compound is to be administered orally or by injection.

In some embodiments of the present invention, a compound that reduces the ability of the immune system to activate naive TCs and BCs and elicit a secondary immune response from activated and memory lymphocytes is a TNFα inhibitor.

Blockade of TNF alpha reduces the state of immune activation and decreases the activity of DCs and other immune cells. In addition, because TNF alpha disrupts the structure of FSCs and GCs in lymphoid tissue and causes impairment of B cell functions, activation of antigen-specific effector T cells and autoantibody production are reduced. Recent preclinical data in diabetes indicate that beta cell antigen delivered by inactive DCs can induce peripheral T cell unresponsiveness, attenuate ongoing beta cell destruction, and delay beta cell destruction. Thus, with autoantigen-TNF alpha inhibitor combination therapy, autoantibody levels and effector T cell activity and numbers will be somewhat reduced, while the number and function of autoantigen-specific Tregs will at least be preserved. It takes months to reverse these immunological processes and characterize these effects, but in that time, because of its clear beta-cell protective and metabolic effects, inhibiting TNF alpha will directly preserve beta cells. Since GAD65 is known to be a major autoantigen in T1DM, this approach biases the diabetic autoimmune response toward a regulatory phenotype with sufficient critical mass to result in a significant and lasting effect on beta cell preservation.

Thus, according to one aspect of the present invention, a TNFα inhibitor, which is not limited to, for example, infliximab, adalimumab, golimumab and etanercept, is used as an anti-inflammatory compound. The preferred dose when using etanercept is 0.2 to 1 mg/kg subcutaneously, once or twice a week for 2 to 9 months.

According to some embodiments of the present invention, when the TNFα inhibitor is etanercept, the FDA-approved dosage of 5 mg/kg at Weeks 0, 2, and 6 is preferred. In another embodiment of the present invention, the dosage is the same as that used in a phase I study of monotherapy using a TNFα inhibitor (0.4 mg/kg (not more than 25 mg) subcutaneously twice a week for 26 weeks). In another most preferred embodiment of the present invention, only two doses of not more than 25 mg/dose are used in a combination therapy regimen including vitamin D and autoantigens.

According to some embodiments of the present invention, the TNFα inhibitor is administered prior to the first administration of at least one antigen.

In some embodiments of the present invention, a compound that reduces the ability of the immune system to activate naive TCs and BCs and to elicit a secondary immune response from activated and memory lymphocytes is a CTLA-4 compound such as abatacept. According to certain embodiments of the present invention, when the compound is abatacept, the doses of abatacept are at least 2-20 mg/kg and are administered at one time, starting within +/- 7 days from the time of the first administration of at least one antigen.

According to some embodiments of the present invention, the CTLA-4 compound is administered concurrently with a first administration of at least one antigen.

According to some embodiments of the present invention, the administration of at least one antigen and a compound that reduces the ability of the immune system to activate naive TCs and BCs and to elicit a secondary immune response from activated and memory lymphocytes is repeated on Day 14, 28, and 45 +/- one week as appropriate to ensure blockade of CD28 on TCs, where Day 1 represents the first administration of at least one compound that causes IL-10 induction.

According to one aspect, the present invention provides a method for treating one or more symptoms associated with T1D. Symptoms associated with T1D include, but are not limited to, decreased insulin production, decreased insulin sensitivity, high blood glucose levels, destruction of insulin-producing cells, and abnormal C-peptide levels.

The methods of the present invention can be directed to the treatment and prevention of not only T1D, but autoimmune diseases and disorders in general. For example, patients can suffer from Graves' disease, Hashimoto's thyroiditis, hypoglycemia, multiple sclerosis, mixed essential cryoglobulinemia, systemic lupus erythematosus, rheumatoid arthritis (RA), celiac disease, T1D or any combination thereof. In such cases, disease-relevant autoantigens should be included as autoantigens in the treatment methods. According to one aspect of the present invention, patients can suffer from autoimmune reactions involving T cells or B cells having antigen specificity, or a T cell receptor (TCR) and/or B cells that have the antigen specificity of a T cell receptor (TCR) or a B cell receptor (BCR) for an autoantigen.

According to some embodiments of the present invention, the individual to be treated according to the present invention is a mammal. According to particular embodiments of the present invention, the individual to be treated according to the present invention is a human. According to some embodiments of the present invention, the individual to be treated according to the present invention is an infant. According to particular embodiments of the present invention, the individual to be treated according to the present invention is an adolescent. According to particular embodiments of the present invention, the individual to be treated according to the present invention is an adult.

In some embodiments of the present invention, the human being treated is over 4 years of age.

In other embodiments of the present invention, the human being treated is 8 years of age or older.

In other embodiments of the present invention, the human being treated is 10 years of age or older.

In some embodiments of the present invention, the human being treated is 18 years of age or younger.

In some embodiments of the present invention, the age of the human being treated is 4-10, or 4-18, or 8-18, or 10-18 years.

In other embodiments of the present invention, the human being treated is 18 years of age or older.

In some embodiments of the present invention, the age of the human being treated is 18-30 years.

Compositions

The present invention also discloses a composition comprising a plurality of particles, each of which comprises at least one first and at least one second antigen immobilized on the surface, wherein the first antigen is a beta cell autoantigen and the second antigen is either a tolerogen or a beta cell autoantigen, wherein the composition further optionally comprises pharmaceutically acceptable auxiliary compounds, excipients, solvents and/or buffering agents.

According to some embodiments of the present invention, at least one autoantigen is formulated with an auxiliary compound. According to particular embodiments of the present invention, the auxiliary compound is aluminum hydroxide. In other particular embodiments of the present invention, at least one antigen is formulated in saline or in human serum albumin. In more particular embodiments of the present invention, the autoantigen may be administered as plasmids or encoded by a viral vector, such as adeno-associated viral vectors or herpes simplex virus vectors.

According to some embodiments of the present invention, at least one self-antigen is an self-antigen, which is discussed below.

The compositions of the present invention may comprise more than one autoantigen. Thus, according to some embodiments of the present invention, said composition comprises at least two autoantigens. According to some other embodiments of the present invention, said composition comprises at least three autoantigens. According to some other embodiments of the present invention, said composition comprises at least four autoantigens.

Thus, according to specific embodiments of the present invention, said composition comprises at least a GAD, such as GAD-65, and insulin. According to other specific embodiments of the present invention, said composition comprises at least insulinoma antigen-2 and insulin. According to other specific embodiments of the present invention, said composition comprises at least ZnT8 and insulin. According to other specific embodiments of the present invention, said composition comprises at least IGRP and insulin. According to other specific embodiments of the present invention, said composition comprises at least chromogranin A and insulin.

According to other specific embodiments of the present invention, said composition comprises at least a GAD, such as GAD-65, and an insulin B-chain. According to other specific embodiments of the present invention, said composition comprises at least insulinoma antigen-2 and an insulin B-chain. According to other specific embodiments of the present invention, said composition comprises at least ZnT8 and an insulin B-chain. According to other specific embodiments of the present invention, said composition comprises at least IGRP and an insulin B-chain. According to other specific embodiments of the present invention, said composition comprises at least chromogranin A and an insulin B-chain.

According to other specific embodiments of the present invention, said composition comprises at least a GAD, such as GAD-65, and proinsulin. According to other specific embodiments of the present invention, said composition comprises at least insulinoma antigen-2 and proinsulin. According to other specific embodiments of the present invention, said composition comprises at least ZnT8 and proinsulin. According to other specific embodiments of the present invention, said composition comprises at least IGRP and proinsulin. According to other specific embodiments of the present invention, said composition comprises at least chromogranin A and proinsulin.

According to other specific embodiments of the present invention, said composition comprises at least insulin and an insulin B-chain. According to other specific embodiments of the present invention, said composition comprises at least insulin and proinsulin. According to other specific embodiments of the present invention, said composition comprises at least an insulin B-chain and proinsulin.

According to specific embodiments of the present invention, said composition comprises at least a GAD, such as GAD-65, insulin and an insulin B-chain. According to another specific embodiment of the present invention, said composition comprises at least a GAD, such as GAD-65, insulin and proinsulin. According to other specific embodiments of the present invention, said composition comprises at least a GAD, such as GAD-65, an insulin B-chain and proinsulin. According to other specific embodiments of the present invention, said composition contains at least insulin, an insulin B-chain and proinsulin.

According to other specific embodiments of the present invention, said composition comprises GAD, such as GAD-65, insulin, insulin B-chain and insulin B-chain.

According to some embodiments of the present invention, a composition of at least one autoantigen, such as at least two autoantigens or at least three autoantigens, is prepared together with an excipient.

Autoantigens can be prepared in the form of compositions with such auxiliary substances as aluminum hydroxide, MAS-1, human serum albumin, lipid emulsions.

According to specific embodiments of the present invention, the excipient is aluminum hydroxide.

According to one embodiment, the present invention relates to a composition that comprises:

i) at least one beta cell autoantigen and at least one of the following

iia) a compound that causes IL-10 induction, which is selected from the group consisting of vitamin D, vitamin D analogues, tyrosine kinase inhibitors, gamma-aminobutyric acid and gamma-aminobutyric acid analogues; and

iib) a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor;

and optionally pharmaceutically acceptable auxiliary substances, excipients, solvents and/or buffering agents.

The present invention also relates to a composition (e.g., a pharmaceutical composition) comprising i) at least one autoantigen and ii) at least one compound that causes the induction of IL-10.

More particularly, the present invention relates to a composition (e.g., a pharmaceutical composition) comprising i) at least one autoantigen selected from the group consisting of insulin, insulin B chain, proinsulin, and beta cell autoantigen, and ii) at least one IL-10 inducing compound selected from the group consisting of vitamin D, vitamin D analogs, and tyrosine kinase inhibitors.

The composition of the present invention may comprise more than one IL-10 inducing compound. Thus, in accordance with some embodiments of the present invention, the composition comprises at least two IL-10 inducing compounds. In accordance with some other embodiments of the present invention, the composition comprises at least three IL-10 inducing compounds. In accordance with some other embodiments of the present invention, the composition comprises at least four IL-10 inducing compounds.

Thus, in accordance with specific embodiments of the present invention, the composition comprises at least vitamin D and a tyrosine kinase inhibitor. In accordance with other specific embodiments of the present invention, the composition comprises at least a vitamin D analogue and a tyrosine kinase inhibitor.

According to more specific embodiments of the present invention, the composition comprises at least vitamin D and dasatinib. According to other more specific embodiments of the present invention, the composition comprises at least vitamin D and bosutinib. According to other more specific embodiments of the present invention, the composition comprises at least vitamin D and saracatinib. According to other more specific embodiments of the present invention, the composition comprises at least vitamin D and imatinib. According to other more specific embodiments of the present invention, the composition comprises at least vitamin D and sunitinib.

According to more specific embodiments of the present invention, the composition comprises at least a vitamin D analog and dasatinib. According to other more specific embodiments of the present invention, the composition comprises at least a vitamin D analog and bosutinib. According to other more specific embodiments of the present invention, the composition comprises at least a vitamin D analog and saracatinib. According to other more specific embodiments of the present invention, the composition comprises at least a vitamin D analog and imatinib. According to other more specific embodiments of the present invention, the composition comprises at least a vitamin D analog and sunitinib.

According to some embodiments of the present invention, the composition further comprises iii) a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor.

According to specific embodiments of the present invention, the compound is a cyclooxygenase inhibitor, such as a non-steroidal anti-inflammatory drug (NSAID). According to more specific embodiments of the present invention, the NSAID is selected from the group consisting of ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, salsalate (disalcid), piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib and nimesulide.

In one embodiment of the present invention, at least one composition comprising a protein/peptide antigen or a combination of proteins/peptides antigens is prepared by mixing together individual GMP-produced protein/peptide solutions in a formulation buffer. The combined protein solution is then sterilized by filtration into a closed composition storage vessel with constant stirring, then a sterile solution of an excipient is added to the composition storage vessel. The protein-containing particles are then aseptically placed in sterile and pyrogen-free glass injection vials or pre-filled syringes and sealed. The articles with adsorbed proteins can be removed aseptically from each injection vial and injected into the patient's tissue.

The proportions of the combined proteins in the composition may vary, although equal proportions in grams of weight are most preferred. If used, the proportion of the influencing antigen should be at least equal to the combined weight of the other proteins or peptides of the antigens. The proportion of the influencing antigen may be greater than the combined weight of the other antigens. The buffer of the composition may be an isotonic solution of phosphate-buffered mannitol. The excipient may be aluminum hydroxide (alum), liposome, poly(lactide-co-glycolide) microparticles, or saline.

The medicinal products, pharmaceutical compositions or therapeutic combinations of the present invention can be prepared in any form suitable for use in humans and/or animals, preferably in humans, including infants, children and adults, and can be prepared using standard procedures known to those skilled in the art. The medicinal product, (pharmaceutical) composition or therapeutic combination can be prepared using standard procedures known to those skilled in the art, for example, from the monographs “Pharmaceutics: The Science of Dosage Forms”, Second Edition, Aulton, M.E. (ED. Churchill Livingstone, Edinburgh (2002); “Encyclopedia of Pharmaceutical Technology”, Second Edition, Swarbrick, J. and Boylan 15 J.C. (Eds.), Marcel Dekker, Inc. New York (2002); “Modern Pharmaceutics”, Fourth Edition, Banker G.S. and Rhodes C.T. (Eds.) Marcel Dekker, Inc. New York 2002 “The Theory and Practice of Industrial Pharmacy”, Lachman L., Lieberman H. and Kanig J. (Eds.), Lea & Febiger, Philadelphia (1986). The corresponding descriptions are incorporated herein by reference and are an integral part of the description of the present application. The terms “drug”, “pharmaceutical composition” and “pharmaceutical preparation” may be used interchangeably.

The drug, pharmaceutical composition or therapeutic combination of the present invention may further comprise one or more pharmaceutically acceptable excipients. Carriers, diluents and excipients that are suitable for preparing the drug, pharmaceutical composition or therapeutic combination of the present invention are well known to those skilled in the art, for example, from the Handbook of Pharmaceutical Excipients, Sixth Edition, Raymond C. Rowe, Paul J. Sheskey and Marian E Quinn (Eds.), American Pharmaceutical Association (July 2009), which is incorporated herein by reference and forms part of the present description.

Autoantigens

Autoantigens suitable for use in the methods, compositions and reagent kits of the present invention are beta cell autoantigens.

These include: glutamic acid decarboxylase (GAD65, or GAD67, or GAD32) (Baekkeskov et al., Nature (1990) 347: 151)); insulin (Palmer et al., Science (1983) 222: 1337); including peptide B9-23, containing amino acids 9-23 of the B-chain of insulin (Daniel et al., Proc. Natl. Acad. Sci. USA (1995) 93: 956-960; Wong et al., Nat. Med. (1999) 5: 1026-1031), proinsulin; including the B24-C36 peptide, which contains amino acids 24-36, encompassing the junction of the B-chain of proinsulin and C-peptide (Chen et al., J. Immunol. (2001) 167: 4926-4935; Rudy et al., Mol. Med., (1995) 1: 625-633); HSP60 (heat shock protein 60, Raz et al., Lancet (2001), 358:1749-53); ICA512/IA-2 (islet cell antigen 512; Rabin et al., J. Immunol. (1994) 152: 3183), insulinoma antigen-2, ZnT8, islet-specific glucose-6-phosphate catalytic subunit protein (IGRP), chromogranin A, insulin B chain, preproinsulin, proinsulin II, proinsulin peptide without cytotoxic T-lymphocyte epitope, insulin peptide C13-A5, islet cell antigen p69, or any peptide derivative, including citrullinated forms of iDS and corresponding nucleotides of the above compounds.

For alignment of insulin sequences between species, see Table I in Homann et al., J. Immunol. (1999) 63: 1833–1838.

According to specific embodiments of the present invention, the beta cell autoantigen is GAD, such as GAD-65 or GAD-67, including fragments thereof, derivatives thereof, or nucleic acid encoding it.

According to more specific embodiments of the present invention, the beta cell autoantigen is GAD-65, a fragment thereof, a derivative thereof, or a nucleic acid encoding it. According to more specific embodiments of the present invention, the beta cell autoantigen is GAD-65. According to other specific embodiments of the present invention, the beta cell autoantigen is a fragment derived from GAD-65 (i.e., a GAD65 fragment).

According to other specific embodiments of the present invention, the beta cell autoantigen is insulinoma antigen-2.

According to other specific embodiments of the present invention, the beta cell autoantigen is ZnT8.

According to other specific embodiments of the present invention, the beta cell autoantigen is islet-specific glucose-6-phosphate catalytic subunit protein (IGRP).

According to other specific embodiments of the present invention, the beta cell autoantigen is chromogranin A.

According to some other embodiments of the present invention, the beta cell autoantigen is insulin.

According to some other embodiments of the present invention, the beta cell autoantigen is the insulin B chain.

According to some other embodiments of the present invention, the beta cell autoantigen is proinsulin.

According to some other embodiments of the present invention, the beta cell autoantigen is preproinsulin.

According to some aspects of the present invention, a tolerogen is administered to a patient being treated. A tolerogen is an antigen that induces a state of specific immunological unresponsiveness to subsequent increasing doses of the antigen. Tolerogens suitable for use in the present invention are native endogenous human proteins and other molecules that are widely distributed and exposed to the immune system and are generally recognized as self. Examples of tolerogens include IL-10, human serum albumin or hemoglobin, or gamma-aminobutyric acid.

Autoantigens or tolerogens for use in the present invention may be administered as full-length proteins, or they may alternatively be administered as fragments or variants of such full-length proteins, provided that the fragments or variants of the autoantigens have at least one epitope conserved with respect to the original autoantigen and are effective in the methods of the present invention.

The fragment of the protein of the autoantigen or tolerogen has the same amino acid sequence as the original autoantigen or tolerogen, but lacks at least one N-terminal or C-terminal amino acid residue. The fragment of the autoantigen should include at least one corresponding epitope of the original autoantigen. The fragments of the autoantigen and tolerogen preferably have a length of at least 8 amino acids, such as at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids.

One variant of an autoantigen or tolerogen protein may have an amino acid sequence that is less than 100% identical to the original autoantigen or tolerogen, such as 99%, 95%, 90%, 85%, 80%, 70%, 60% or 50% identical (when compared using a protein sequence aligner such as Clustal Omega, available from the European Bioinformatics Institute, Hinxton, UK), while the autoantigens at the same time have at least one corresponding epitope that is conserved with respect to the original autoantigen. Variants may also have shorter (i.e., be a fragment) or longer amino acid sequences, compared to the original antigen or tolerogen.

In the present invention, the administration of an autoantigen or tolerogen may include an autoantigen or tolerogen that contains a protein or a peptide fragment of the original protein. It may also include the administration of a variant of an autoantigen or tolerogen.

In addition, the protein or peptide can be administered to the patient as a protein or peptide in a pharmaceutically acceptable carrier, or the protein or peptide can be encoded by an expression vector, wherein the expression vector is administered (see, in particular, examples where the self-antigen is expressed in the patient using a pCMV vector). Such an expression vector can be a nucleic acid, such as DNA or RNA, and delivery can also be accomplished by needle injection, gene guns, jet injection, or by cytofectin, as is known in the art. Nucleic acids can be prepared in saline, on gold beads, in liposomes, or in lipid formulations. For further description regarding the administration of self-antigens via their expression vectors (and for specific antigens that can be administered), see US Patent Publication No. 2002/0107210, which is incorporated herein by reference.

Compounds that induce IL-10

In some aspects, the methods, compositions, and kits of the present invention utilize compounds that induce IL-10.

According to some embodiments of the present invention, at least one IL-10 inducing compound is vitamin D, such as 1,25-dihydroxyvitamin D.

According to some other embodiments of the present invention, at least one IL-10 inducing compound is a vitamin D analog, such as TX527.

According to some other embodiments of the present invention, at least one IL-10 inducing compound comprises increasing serum vitamin D levels using UVB radiation.

According to some other embodiments of the present invention, at least one IL-10 inducing compound is a tyrosine kinase inhibitor, such as dasatinib, bosutinib, saracatinib, imatinib, sunitinib, or combinations thereof.

According to specific embodiments of the present invention, the tyrosine kinase inhibitor is dasatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is bosutinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is saracatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is imatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is sunitinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of at least two compounds selected from dasatinib, bosutinib, saracatinib, imatinib and sunitinib. For example, the tyrosine kinase inhibitor can be a combination of dasatinib and bosutinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and saracatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and imatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and saracatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and imatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of imatinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib, bosutinib and saracatinib.

The composition of the present invention may comprise more than one IL-10 inducing compound. Thus, according to some embodiments of the present invention, said composition comprises at least two IL-10 inducing compounds. According to some other embodiments of the present invention, said composition comprises at least three IL-10 inducing compounds. According to some other embodiments of the present invention, said composition comprises at least four IL-10 inducing compounds.

Cyclooxygenase inhibitors

In some aspects, the methods, compositions, and kits of the present invention utilize one or more cyclooxygenase inhibitors.

The cyclooxygenase inhibitors listed may be nonsteroidal anti-inflammatory drugs (NSAIDs). According to more specific embodiments of the present invention, the NSAID is selected from the group consisting of ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, salsalate (disalcid), piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib and nimesulide.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a propionic acid derivative such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin or loxoprofen.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an acetic acid derivative such as indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac or nabumetone.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a salicylate, such as aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, or salsalate.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an enolic acid derivative (oxicam), such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam or isoxicam.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an anthranilic acid derivative such as mefenamic acid, meclofenamic acid, flufenamic acid or tolfenamic acid.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a selective COX-2 inhibitor, such as celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, or etoricoxib.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is ibuprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is dexibuprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is naproxen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is fenoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is ketoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is dexketoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is flurbiprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is oxaprozin.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is loxoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is indomethacin.

Ibuprofen primarily blocks cox-2, but also cox-1 to some extent. Ibuprofen has a somewhat broader effect on the immune system than a narrowly targeted IL-1 blocker, and has very pronounced anti-inflammatory effects without the serious risks. Ibuprofen use dampens beta cell inflammation and allows Vit D to enhance DCs in a way that induces tolerance to peptides from autoantigens presented to T cells, thereby protecting the patient's beta cells.

CTLA4 compounds

In some aspects, the methods, compositions, and kits of the present invention utilize a CTLA-4 compound, such as cytotoxic T lymphocyte-associated immunoglobulin 4 antigen.

According to more specific embodiments of the present invention, the CTLA-4 compound is abatacept.

Abatacept (Fc modified immunoglobulin CTLA4) is a T cell-depleting, immunomodulatory fusion protein composed of the extracellular portion of human CTLA4 and the heavy chain of human IgG1. It blocks a costimulatory signal involved in the activation of naive T cells. Its ligation to CD80/86 on APCs can also be interfered with by IL-6, which induces CD80/86, which is capable of suppressing inflammatory cytokines such as IL-1 beta, IFN-gamma, and IL-17. Further ligation of abatacept to CD80/86 can induce indoleamine dioxygenase (IDO) on APCs, which in turn can induce T cell anergy and also suppress paracrine activation of naive T cells by activated T cells. CD80/86 expressed on B cells may be another pathway where abatacept may function as an immunomodulator.

TNF alpha inhibitors

In some aspects, the methods, compositions, and kits of the present invention utilize TNF alpha inhibitors such as adalimumab, sertolizumab, etanercept, golimumab, or infliximab. In more specific embodiments, the TNF alpha inhibitor is adalimumab. In other more specific embodiments, the TNF alpha inhibitor is sertolizumab. In other more specific embodiments, the TNF alpha inhibitor is etanercept. In other more specific embodiments, the TNF alpha inhibitor is golimumab. In other more specific embodiments, the TNF alpha inhibitor is infliximab.

Reagent kits

The present invention also provides kits of reagents corresponding to the methods of the present invention. For example, according to one aspect, a kit of reagents may include: (a) an anti-inflammatory compound; (b) an autoantigen; (c) vitamin D, and optionally (d) instructions for using the dosage regimens of the present invention.

The present invention also discloses a pharmaceutical kit that includes:

i) a composition comprising a beta cell autoantigen and at least one of the following:

iia) a composition comprising a compound that induces IL-10, selected from the group consisting of vitamin D, vitamin D analogues, tyrosine kinase inhibitors, gamma-aminobutyric acid and gamma-aminobutyric acid analogues; and

iib) a composition comprising a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor.

The present invention also discloses a pharmaceutical kit that includes:

i) a composition comprising a beta cell autoantigen and at least one of the following:

iia) a composition comprising a compound that induces IL-10, selected from the group consisting of vitamin D, vitamin D analogues, tyrosine kinase inhibitors, gamma-aminobutyric acid and gamma-aminobutyric acid analogues; and

iib) a composition comprising a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor.

The present invention also discloses the use of compositions comprising at least two of the following:

i) at least one antigen or fragments thereof; or nucleic acids encoding such molecules associated with at least one of the autoimmune and inflammatory diseases that are included in the group specified in the present description; and

ii) at least one IL-10 inducing compound selected from the group consisting of vitamin D, such as 1,25-dihydroxyvitamin D. According to some other embodiments of the present invention, the at least one IL-10 inducing compound is a vitamin D analog, such as TX527. According to some other embodiments of the present invention, the at least one IL-10 inducing compound is a tyrosine kinase inhibitor, such as dasatinib, bosutinib, saracatinib, imatinib, sunitinib, or combinations thereof; and

iii) at least one compound that reduces the ability of the immune system to activate naive TCs and BCs and elicit a secondary immune response from activated and memory lymphocytes, such as a COX inhibitor; a CTLA-4 compound; or a TNF alpha inhibitor; as described herein;

to obtain a set of reagents comprising at least two pharmaceutical compositions (i.e. medicinal products).

At least one of the two pharmaceutical compositions may include at least one antigen prepared as a composition in aluminum hydroxide, saline or human serum albumin and in the form of injections in a pre-filled vial or syringe, and the other of said at least two pharmaceutical compositions may include a compound that causes IL-10 induction in the form of tablets for oral administration.

In some embodiments, the present invention relates to a kit of reagents comprising at least two pharmaceutical compositions that comprise i) at least one antigen selected from the group provided herein, and ii) at least one compound that reduces the ability of dendritic cells to activate naive lymphocytes or to receive a secondary immune response from activated lymphocytes and memory lymphocytes, selected from the group: COX inhibitors; CTLA-4 compounds; and TNF alpha inhibitors. As an example, at least one of the two pharmaceutical compositions can comprise at least one antigen prepared as a formulation in aluminum hydroxide, saline, or human serum albumin and as an injection in a prefilled vial or syringe, and the other of said at least two pharmaceutical compositions can comprise one compound selected from the group of COX inhibitors; CTLA-4 compounds; and TNFα inhibitors.

In some embodiments, the present invention relates to a kit comprising at least three pharmaceutical compositions that comprise i) at least one antigen selected from the group provided herein; ii) at least one IL-10 inducing compound selected from the group consisting of vitamin D, vitamin D analogs, and tyrosine kinase inhibitors, and iii) at least one compound that reduces the ability of dendritic cells to activate naive lymphocytes or to receive a secondary immune response from activated lymphocytes and memory lymphocytes, selected from the group: COX inhibitors; CTLA-4 compounds; and TNF alpha inhibitors. As an example, at least one of the three pharmaceutical compositions may comprise at least one antigen prepared as a formulation in aluminum hydroxide, saline or human serum albumin and as injections in a pre-filled vial or syringe, and another of the at least three pharmaceutical compositions may comprise at least one IL-10 inducing compound selected from the group consisting of vitamin D, vitamin D analogs, gamma-aminobutyric acid, gamma-aminobutyric acid analogs and tyrosine kinase inhibitors, and the third of the at least three pharmaceutical compositions may contain one compound selected from the group of COX inhibitors; CTLA-4 compounds; and TNFα inhibitors.

Application in medicine

The composition of the present invention can be used in therapy, in particular for the purpose of preventing and/or treating an autoimmune disease. Accordingly, the composition of the present invention can be a pharmaceutical composition.

According to some embodiments of the present invention, the composition is intended for use as a medicine, for example for use in the prevention and/or treatment of an autoimmune disease.

According to specific embodiments of the present invention, the composition is intended for use in the prevention and/or treatment of type 1 diabetes mellitus, such as type 1.

According to other specific embodiments of the present invention, the composition is intended for use in the prevention and/or treatment of autoimmune diabetes.

According to other specific embodiments of the present invention, the composition is intended for use in the prevention and/or treatment of latent autoimmune diabetes.

According to other specific embodiments of the present invention, the composition is intended for use in the prevention and/or treatment of relapse of autoimmune diabetes, such as relapse of autoimmune diabetes in an individual (eg, a human) with autoimmune diabetes who has undergone islet cell transplantation or other types of cell therapy, including stem cell therapy.

According to another aspect, the present invention provides an autoantigen in combination with at least one IL-10 inducing compound for use in the prevention and/or treatment of an autoimmune disease.

More particularly, the present invention relates to an autoantigen selected from the group consisting of insulin, insulin B chain, proinsulin and beta cell autoantigens, in combination with at least one IL-10 inducing compound, such as at least one IL-10 inducing compound selected from the group consisting of vitamin D, vitamin D analogues and tyrosine kinase inhibitors, for use in the prevention and/or treatment of an autoimmune disease.

According to some embodiments of the present invention, the autoantigen is a beta cell autoantigen, such as glutamic acid decarboxylase (GAD), insulinoma antigen-2, ZnT8, islet-specific glucose-6-phosphate catalytic subunit protein (IGRP), or chromogranin A.

According to specific embodiments of the present invention, the beta cell autoantigen is GAD, such as GAD-65, including fragments thereof, derivatives thereof, or the nucleotide encoding it.

According to more specific embodiments of the present invention, the beta cell autoantigen is GAD-65, a fragment thereof, a derivative thereof, or a nucleotide encoding it. According to more specific embodiments of the present invention, the beta cell autoantigen is GAD-65. According to other specific embodiments of the present invention, the beta cell autoantigen is a fragment derived from GAD-65 (i.e., a GAD65 fragment).

According to other specific embodiments of the present invention, the beta cell autoantigen is insulinoma antigen-2.

According to other specific embodiments of the present invention, the beta cell autoantigen is ZnT8.

According to other specific embodiments of the present invention, the beta cell autoantigen is islet-specific glucose-6-phosphate catalytic subunit protein (IGRP).

According to other specific embodiments of the present invention, the beta cell autoantigen is chromogranin A.

According to some embodiments of the present invention, the autoantigen is insulin.

According to some other embodiments of the present invention, the autoantigen is the B chain of insulin.

According to certain other embodiments of the present invention, the autoantigen is proinsulin.

According to some embodiments of the present invention, the autoantigen is prepared as a composition together with an excipient.

According to specific embodiments of the present invention, the excipient is aluminum hydroxide.

According to some embodiments of the present invention, at least one IL-10 inducing compound is vitamin D, such as 1,25-dihydroxyvitamin D.

According to some other embodiments of the present invention, at least one IL-10 inducing compound is a vitamin D analog, such as TX527.

According to some other embodiments of the present invention, at least one IL-10 inducing compound is a tyrosine kinase inhibitor, such as dasatinib, bosutinib, saracatinib, imatinib, sunitinib, or combinations thereof.

According to specific embodiments of the present invention, the tyrosine kinase inhibitor is dasatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is bosutinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is saracatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is imatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is sunitinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of at least two compounds selected from dasatinib, bosutinib, saracatinib, imatinib and sunitinib. For example, the tyrosine kinase inhibitor can be a combination of dasatinib and bosutinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and saracatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and imatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and saracatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and imatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of imatinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib, bosutinib and saracatinib.

According to some embodiments of the present invention, the autoantigen is further used in combination with a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor.

According to particular embodiments of the present invention, such a compound is a cyclooxygenase inhibitor, such as a non-steroidal anti-inflammatory drug (NSAID). According to more specific embodiments of the present invention, the NSAID is selected from the group consisting of ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, salsalate (disalcid), piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib and nimesulide.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a propionic acid derivative such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin or loxoprofen.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an acetic acid derivative such as indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac or nabumetone.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a salicylate, such as aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, or salsalate.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an enolic acid derivative (oxicam), such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam or isoxicam.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an anthranilic acid derivative such as mefenamic acid, meclofenamic acid, flufenamic acid or tolfenamic acid.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a selective COX-2 inhibitor, such as celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, or etoricoxib.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is ibuprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is dexibuprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is naproxen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is fenoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is ketoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is dexketoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is flurbiprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is oxaprozin.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is loxoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is indomethacin.

According to other specific embodiments of the present invention, such a compound is a CTLA-4 compound, such as cytotoxic T lymphocyte-associated immunoglobulin 4 antigen.

According to more specific embodiments of the present invention, the CTLA-4 compound is abatacept.

According to other specific embodiments of the present invention, such a compound is a TNF alpha inhibitor such as adalimumab, sertolizumab, etanercept, golimumab or infliximab. According to more specific embodiments of the present invention, the TNF alpha inhibitor is adalimumab. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is sertolizumab. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is etanercept. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is golimumab. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is infliximab.

According to other specific embodiments of the present invention, the autoantigen is for the prevention and/or treatment of latent autoimmune diabetes, such as latent autoimmune diabetes in a GAD antibody positive individual.

According to specific embodiments of the present invention, the autoimmune disease is type 1 diabetes.

According to other specific embodiments of the present invention, the autoimmune disease is autoimmune diabetes.

According to other specific embodiments of the present invention, the autoimmune disease is latent autoimmune diabetes.

According to other specific embodiments of the present invention, the autoimmune disease is a relapse of autoimmune diabetes, such as a relapse of autoimmune diabetes in an individual (e.g., a human) with autoimmune diabetes who has undergone islet cell transplantation or other types of cell therapy, including stem cell therapy.

Thus, the present invention relates to a combination (e.g., a therapeutic combination) comprising at least one autoantigen and at least one compound that causes IL-10 induction.

More particularly, the present invention relates to a combination (e.g., a therapeutic combination) that comprises i) at least one autoantigen selected from the group consisting of insulin, insulin B chain, proinsulin and beta cell autoantigens, and ii) at least one IL-10 inducing compound selected from the group consisting of vitamin D, vitamin D analogues and tyrosine kinase inhibitors.

According to some embodiments of the present invention, at least one autoantigen is a beta cell autoantigen, such as glutamic acid decarboxylase (GAD), insulinoma antigen-2, ZnT8, islet-specific glucose-6-phosphate catalytic subunit protein (IGRP), or chromogranin A.

According to specific embodiments of the present invention, the beta cell autoantigen is GAD, such as GAD-65, including fragments thereof, derivatives thereof, or the nucleotide encoding it.

According to more specific embodiments of the present invention, the beta cell autoantigen is GAD-65, a fragment thereof, a derivative thereof, or a nucleotide encoding it. According to more specific embodiments of the present invention, the beta cell autoantigen is GAD-65. According to other specific embodiments of the present invention, the beta cell autoantigen is a fragment derived from GAD-65 (i.e., a GAD65 fragment).

According to other specific embodiments of the present invention, the beta cell autoantigen is insulinoma antigen-2.

According to other specific embodiments of the present invention, the beta cell autoantigen is ZnT8.

According to other specific embodiments of the present invention, the beta cell autoantigen is islet-specific glucose-6-phosphate catalytic subunit protein (IGRP).

According to other specific embodiments of the present invention, the beta cell autoantigen is chromogranin A.

According to some other embodiments of the present invention, the at least one autoantigen is insulin.

According to some other embodiments of the present invention, at least one autoantigen is the insulin B chain.

According to some other embodiments of the present invention, at least one autoantigen is proinsulin.

The combination of the present invention may include more than one autoantigen. Thus, according to some embodiments of the present invention, the combination includes at least two autoantigens. According to some other embodiments of the present invention, the combination includes at least three autoantigens. According to some other embodiments of the present invention, the combination includes at least four autoantigens.

Thus, according to specific embodiments of the present invention, said combination comprises at least GAD, such as GAD-65, and insulin. According to other specific embodiments of the present invention, said combination comprises at least insulinoma antigen-2 and insulin. According to other specific embodiments of the present invention, said combination comprises at least ZnT8 and insulin. According to other specific embodiments of the present invention, said combination comprises at least IGRP and insulin. According to other specific embodiments of the present invention, said combination comprises at least chromogranin A and insulin.

According to other specific embodiments of the present invention, said combination comprises at least a GAD, such as GAD-65, and an insulin B chain. According to other specific embodiments of the present invention, said combination comprises at least insulinoma antigen-2 and an insulin B chain. According to other specific embodiments of the present invention, said combination comprises at least ZnT8 and an insulin B chain. According to other specific embodiments of the present invention, said combination comprises at least IGRP and an insulin B chain. According to other specific embodiments of the present invention, said combination comprises at least chromogranin A and an insulin B chain.

According to other specific embodiments of the present invention, said combination comprises at least a GAD, such as GAD-65, and proinsulin. According to other specific embodiments of the present invention, said combination comprises at least insulinoma antigen-2 and proinsulin. According to other specific embodiments of the present invention, said combination comprises at least ZnT8 and proinsulin. According to other specific embodiments of the present invention, said combination comprises at least IGRP and proinsulin. According to other specific embodiments of the present invention, said combination comprises at least chromogranin A and proinsulin.

According to other specific embodiments of the present invention, the combination comprises at least insulin and the B chain of insulin. According to other specific embodiments of the present invention, the combination comprises at least insulin and proinsulin. According to other specific embodiments of the present invention, the combination comprises at least the B chain of insulin and proinsulin.

According to specific embodiments of the present invention, the combination comprises at least a GAD, such as GAD-65, insulin and the B-chain of insulin. According to another specific embodiment of the present invention, said combination comprises at least a GAD, such as GAD-65, insulin and proinsulin. According to other specific embodiments of the present invention, said combination comprises at least a GAD, such as GAD-65, the B-chain of insulin and proinsulin. According to other specific embodiments of the present invention, said combination comprises at least insulin, the B-chain of insulin and proinsulin.

According to other specific embodiments of the present invention, the combination comprises GAD, such as GAD-65, insulin, insulin B chain, and insulin B chain.

According to some embodiments of the present invention, at least one autoantigen, such as at least two autoantigens or at least three autoantigens, is formulated together with an excipient.

According to specific embodiments of the present invention, the excipient is aluminum hydroxide.

According to some embodiments of the present invention, at least one IL-10 inducing compound is vitamin D or a vitamin D analog. According to specific embodiments of the present invention, said at least one IL-10 inducing compound is vitamin D.

According to more specific embodiments of the present invention, at least one IL-10 inducing compound is 1,25-dihydroxyvitamin D.

According to some other embodiments of the present invention, at least one IL-10 inducing compound is a vitamin D analog, such as TX527.

According to some other embodiments of the present invention, at least one IL-10 inducing compound is a tyrosine kinase inhibitor, such as dasatinib, bosutinib, saracatinib, imatinib, sunitinib, or combinations thereof.

According to specific embodiments of the present invention, the tyrosine kinase inhibitor is dasatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is bosutinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is saracatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is imatinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is sunitinib.

According to other specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of at least two compounds selected from dasatinib, bosutinib, saracatinib, imatinib and sunitinib. For example, the tyrosine kinase inhibitor can be a combination of dasatinib and bosutinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and saracatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and imatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and saracatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and imatinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of bosutinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of imatinib and sunitinib. According to other more specific embodiments of the present invention, the tyrosine kinase inhibitor is a combination of dasatinib, bosutinib and saracatinib.

The combination of the present invention may comprise more than one IL-10 inducing compound. Thus, according to some embodiments of the present invention, said combination comprises at least two IL-10 inducing compounds. According to some other embodiments of the present invention, said combination comprises at least three IL-10 inducing compounds. According to some other embodiments of the present invention, said combination comprises at least four IL-10 inducing compounds.

Thus, according to specific embodiments of the present invention, said combination comprises at least vitamin D and a tyrosine kinase inhibitor. According to other specific embodiments of the present invention, said combination comprises at least a vitamin D analogue and a tyrosine kinase inhibitor.

According to more specific embodiments of the present invention, said combination comprises at least vitamin D and dasatinib. According to other more specific embodiments of the present invention, said combination comprises at least vitamin D and bosutinib. According to other more specific embodiments of the present invention, said combination comprises at least vitamin D and saracatinib. According to other more specific embodiments of the present invention, said combination comprises at least vitamin D and imatinib. According to other more specific embodiments of the present invention, said combination comprises at least vitamin D and sunitinib.

According to more specific embodiments of the present invention, said combination comprises at least a vitamin D analog and dasatinib. According to other more specific embodiments of the present invention, said combination comprises at least a vitamin D analog and bosutinib. According to other more specific embodiments of the present invention, said combination comprises at least a vitamin D analog and saracatinib. According to other more specific embodiments of the present invention, said combination comprises at least a vitamin D analog and imatinib.

According to other more specific embodiments of the present invention, said combination comprises at least a vitamin D analogue and sunitinib.

According to some embodiments of the present invention, said combination further comprises iii) a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a cyclooxygenase inhibitor, a CTLA-4 compound, or a TNF alpha inhibitor.

According to particular embodiments of the present invention, such a compound is a cyclooxygenase inhibitor, such as a non-steroidal anti-inflammatory compound (NSAID). According to more specific embodiments of the present invention, the NSAID is selected from the group consisting of ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, salsalate (disalcid), piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, aarecoxib, lumiracoxib, etoricoxib and nimesulide.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a propionic acid derivative such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin or loxoprofen.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an acetic acid derivative such as indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac or nabumetone.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a salicylate, such as aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid, or salsalate.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an enolic acid derivative (oxicam), such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam or isoxicam.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is an anthranilic acid derivative such as mefenamic acid, meclofenamic acid, flufenamic acid or tolfenamic acid.

According to some embodiments of the present invention, the cyclooxygenase inhibitor is a selective COX-2 inhibitor, such as celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, or etoricoxib.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is ibuprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is dexibuprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is naproxen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is fenoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is ketoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is dexketoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is flurbiprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is oxaprozin.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is loxoprofen.

According to more specific embodiments of the present invention, the cyclooxygenase inhibitor is indomethacin.

According to other specific embodiments of the present invention, such a compound is a CTLA-4 compound, such as cytotoxic T lymphocyte-associated immunoglobulin 4 antigen.

According to more specific embodiments of the present invention, the CTLA-4 compound is abatacept.

According to other specific embodiments of the present invention, such a compound is a TNF alpha inhibitor such as adalimumab, sertolizumab, etanercept, golimumab or infliximab. According to more specific embodiments of the present invention, the TNF alpha inhibitor is adalimumab. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is sertolizumab. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is etanercept. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is golimumab. According to other more specific embodiments of the present invention, the TNF alpha inhibitor is infliximab.

The combination of the present invention can be used in therapy, in particular for the purpose of preventing and/or treating an autoimmune disease. Thus, the combination of the present invention can be a therapeutic combination.

According to some embodiments of the present invention, the combination is intended for use as a medicine, for example for use in the prevention and/or treatment of an autoimmune disease.

According to specific embodiments of the present invention, the combination is intended for use in the prevention and/or treatment of type 1 diabetes mellitus.

According to other specific embodiments of the present invention, the combination is intended for use in the prevention and/or treatment of autoimmune diabetes.

According to other specific embodiments of the present invention, the combination is intended for use in the prevention and/or treatment of latent autoimmune diabetes.

Finally, according to other specific embodiments of the present invention, the combination is intended for use in the prevention and/or treatment of relapse of autoimmune diabetes, such as relapse of autoimmune diabetes in an individual (eg, a human) with autoimmune diabetes who has undergone islet cell transplantation or other types of cell therapy, including stem cell therapy.

According to another aspect, the present invention relates to the use of a composition or combination that comprises i) at least one autoantigen, such as at least one autoantigen detailed above, ii) at least one IL-10 inducing compound, such as at least one IL-10 inducing compound described above, and optionally iii) a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells detailed above, in the manufacture of a medicament.

According to another aspect, the present invention relates to a method(s) for the prevention and/or treatment of an autoimmune disease in a patient, said method(s) comprising administering to said patient at least one autoantigen, such as at least one autoantigen described above; and administering to said patient at least one IL-10 inducing compound, such as at least one IL-10 as described above.

According to some embodiments of the present invention, said methods further comprise administering to said patient a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, as described in detail above.

According to some embodiments of the present invention, the patient to be treated in accordance with the present invention is a mammal.

According to certain embodiments of the present invention, the patient to be treated in accordance with the present invention is a human.

According to some embodiments of the present invention, the patient to be treated in accordance with the present invention is an adult.

According to specific embodiments of the present invention, the patient to be treated in accordance with the present invention is an adult.

The medicinal products, pharmaceutical compositions or therapeutic combinations of the present invention may be in any form suitable for use in humans and/or animals, preferably in humans, including infants, children and adults, and may be prepared using standard techniques known to those skilled in the art. The medicinal product, (pharmaceutical) composition or therapeutic combination may be prepared using standard techniques known to those skilled in the art, for example from the contents of the monographs “Pharmaceutics: The Science of Dosage Forms”, Second Edition, Aulton, M.E. (ED. Churchill Livingstone, Edinburgh (2002); “Encyclopedia of Pharmaceutical Technology”, Second Edition, Swarbrick, J. and Boylan J.C. (Eds.), Marcel Dekker, Inc. New York (2002); “Modern Pharmaceutics”, Fourth Edition, Banker G.S. and Rhodes C.T. (Eds.) Marcel Dekker, Inc. New York 2002 y “The Theory and Practice of Industrial Pharmacy”, Lachman L., Lieberman H. And Kanig J. (Eds.), Lea & Febiger, Philadelphia (1986). The corresponding descriptions are incorporated herein by reference and form an integral part of the present invention. The terms “drug”, “pharmaceutical composition” and “pharmaceutical preparations” may be used interchangeably.

The pharmaceutical composition of the present invention can, for example, be administered parenterally, including by intramuscular, intraperitoneal or intravenous injection, by transmucosal or sublingual administration; or administered orally, including administered in the form of tablets, pills, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays, or administered in the form of a dry powder reconstituted in a liquid medium.

Similarly, at least one autoantigen used in accordance with the present invention can be, for example, administered parenterally, including by intramuscular, intraperitoneal or intravenous injection, by transmucosal or sublingual administration; or administered in oral form, including being administered in the form of tablets, pills, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays, or administered in the form of a dry powder reconstituted in a liquid medium.

Similarly, at least one IL-10 inducing compound used in accordance with the present invention can be, for example, administered parenterally, including by intramuscular, intraperitoneal or intravenous injection, by transmucosal or sublingual administration; or administered orally, including being administered in the form of tablets, pills, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays, or administered in the form of a dry powder reconstituted in a liquid medium.

Similarly, the therapeutic combination of the present invention may, for example, be administered parenterally, including by intramuscular, intraperitoneal or intravenous injection, by transmucosal or sublingual administration; or administered orally, including administered in the form of tablets, pills, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays, or administered in the form of a dry powder reconstituted in a liquid medium.

According to some embodiments of the present invention, at least one autoantigen is administered intrainguinally.

According to some embodiments of the present invention, at least one autoantigen is administered intradermally or subcutaneously, for example, in the stomach, close to the draining lymph nodes of the pancreas. According to specific embodiments of the present invention, at least one autoantigen is administered intradermally. According to other specific embodiments of the present invention, at least one autoantigen is administered subcutaneously.

The medicinal product, pharmaceutical composition or therapeutic combination according to the present invention may additionally contain one or more pharmaceutically acceptable excipients.

Carriers, diluents and excipients that are suitable for preparing the medicament, pharmaceutical composition or therapeutic combination of the present invention are well known to those skilled in the art, for example, from the Handbook of Pharmaceutical Excipients, Sixth Edition, Raymond C. Rowe, Paul J. Sheskey and Marian E Quinn (Eds.), American Pharmaceutical Association (July 2009), which is incorporated herein by reference and forms a part of this specification.

At least one autoantigen and at least one IL-10 inducing compound may be administered simultaneously or separately. According to some embodiments of the present invention, at least one autoantigen and at least one IL-10 inducing compound are administered simultaneously. According to some embodiments of the present invention, at least one autoantigen and at least one IL-10 inducing compound are administered separately.

According to some embodiments of the present invention, the at least one IL-10 inducing compound is administered prior to the administration of the at least one autoantigen. According to particular embodiments of the present invention, the at least one IL-10 inducing compound is administered at least one day, such as at least two days, at least three days, at least four days, at least five days, at least six days, or at least one week prior to the start of the first administration of the at least one autoantigen. Thus, according to such embodiments of the present invention, the course of the compound(s) that induce IL-10 is/are started at least one day, such as at least two days, at least three days, at least four days, at least five days, at least six days, or at least one week prior to the first administration of the at least one autoantigen.

According to more specific embodiments of the present invention, the at least one compound that causes IL-10 induction is administered at least one week, such as at least two weeks, before the start of the first administration of the at least one autoantigen. Thus, according to such embodiments of the present invention, the course of the compound(s) that causes IL-10 induction is started at least one week, such as at least two weeks, before the start of the first administration of the at least one autoantigen.

A compound that reduces the ability of dendritic cells to activate naive CD4+ T cells can be administered simultaneously or separately with at least one self-antigen and/or at least one IL-10 inducing compound. According to some embodiments of the present invention, a CTLA-4 compound is administered simultaneously with at least one self-antigen. According to some other embodiments of the present invention, a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells is administered separately from at least one self-antigen.

According to some embodiments of the present invention, at least one compound that induces IL-10 and a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells are administered simultaneously. According to some embodiments of the present invention, at least one compound that induces IL-10 and a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells are administered separately.

According to specific embodiments of the present invention, a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells, such as, for example, 10 mg/kg abatacept, is administered within +/- 7 days of the time of the first administration of at least one autoantigen.

According to some embodiments of the present invention, administration of at least one self-antigen and administration of a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells are repeated on Day 14, 28, and 45 +/- one week, as appropriate, to ensure blockade of CD28 on T cells.

According to some embodiments of the present invention, at least one dose of 2-20 mcg of GAD, such as GAD-65, optionally in the form of an aluminum hydroxide formulation, is administered at one time during treatment.

According to some embodiments of the present invention, at least one dose of 1000-2000 international units of vitamin D, such as vitamin D3, is administered at one time during treatment.

According to some embodiments of the present invention, APCs are made more tolerogenic by administering 7,000-70,000 international units of vitamin D, such as vitamin D3, per week for 1-10 weeks prior to the first administration of at least one autoantigen and for 4 weeks after the last administration of at least one autoantigen.

According to some embodiments of the present invention, administration of GAD, such as GAD-65, optionally in the form of an aluminum hydroxide formulation, is performed approximately one week after each administration of a compound that reduces the ability of dendritic cells to activate naive CD4+ T cells.

According to some embodiments of the present invention, at least one 400 mg dose of an NSAID, such as ibuprofen, is administered at one time during treatment.

The present invention involves an understanding of conventional molecular biology techniques, which include techniques for manipulating polynucleotides that are well known to those skilled in the art of molecular biology. Examples of such well-known techniques can be found in Molecular Cloning: A Laboratory Manual 2nd Edition, Sambrook et al., Cold Spring Harbor, N.Y. (1989). Examples of conventional molecular biology techniques include, but are not limited to, in vitro ligation, restriction endonuclease digestion, PCR, cell transformation, hybridization, electrophoresis, DNA sequencing, and the like.

The invention also involves an understanding of traditional immunobiological methods that are well known to those of ordinary skill in the field of immunology. The basic information and methods can be found in Current Protocols in Immunology, editors Bierer et al., 4 volumes, John Wiley & Sons, Inc., which includes discussions on the following concepts: Care and Handling of Laboratory Animals, Induction of the Immune Response, In Vitro Assays of Lymphocyte Function, In Vivo Assays of Lymphocyte Function, Immunofluorescence and Cell Sorting, Cytokines and Their Cellular Receptors, Human Immunological Studies, Protein Isolation and Analysis, Peptides, Molecular Biology, Biochemistry of Cell Activation, Complement, Innate Immunity, Animal Models of Autoimmune and Inflammatory Diseases (which includes chapters on the NOD model, the SLE mouse model (for lupus), and the induction of autoimmune diseases by depletion of regulatory T cells), Antigen Processing and Presentation, Engineering of Immune Molecules and Receptors, Ligand-Receptor Interactions in the Immune System, Microscopy and Abbreviations and terminology for common genes and proteins of the immune system, including the CD system for leukocyte surface molecules.

Examples

The following examples describe studies that can be conducted to establish the safety and efficacy of various aspects of the present invention. The beta cell autoantigen used in the examples is glutamic acid decarboxylase (GAD), and can be replaced by any other beta cell autoantigen as described herein.

Example 1

Clinical trial design in patients with recent-onset type 1 diabetes:

The study was a randomized, double-blind, placebo-controlled, multicenter, 4-arm clinical trial. Every morning, patients in Group A received 400 mg ibuprofen orally per day for 90 days. Beginning on Day 1, patients also received 2,000 international units of vitamin D orally per day for 15 months (e.g., 25 drops per day), as well as 2 subcutaneous abdominal injections of 20 mcg Diamyd® (GAD-based diabetes therapy) in a prime-boost regimen on Days 15 and 45.

Group B received 2000 international units of vitamin D orally per day for 15 months (eg, 25 drops per day) and 2 subcutaneous injections of 20 mcg diamide in a prime-boost regimen on Days 15 and 45.

Group C received 2000 IU of vitamin D orally per day for 15 months (e.g. 25 drops per day) and 2 subcutaneous injections of 20 mcg diamide at two different sites (giving a total of 40 mcg diamide per administration) in a prime-boost regimen on Days 15 and 45.

Group D received placebo.

Patients were followed for a total of 30 months with a blinding period of 6 months.

Description of treatment groups:

Patients are assessed for eligibility at a screening visit (Visit 1) 2–4 weeks before treatment initiation. At Visit 2 (Day 1, baseline), eligible patients are randomly assigned to one of 4 treatment groups:

Fifteen patients are given ibuprofen suspension orally every morning; 400 mg from Day 1 to Day 90 and from Day 1 to Day 450, and vitamin D drops orally at a dose of 2000 international units per day (e.g., 25 drops per day). In addition, one subcutaneous injection of 20 mcg diamide and one of placebo are administered at two different sites in the abdomen, each on Days 15 and 45, e.g., in a prime-boost dosing regimen (total dose of 40 mcg diamide).

Fifteen patients are given placebo orally as a suspension each morning from Day 1 to Day 90 and from Day 1 to Day 450, and vitamin D drops orally at a dose of 2000 international units per day (e.g., 25 drops per day). In addition, one subcutaneous injection of 20 mcg diamide and one of placebo are administered at two different abdominal sites, each on Days 15 and 45, e.g., in a prime-boost dosing regimen (total dose of 40 mcg diamide).

Fifteen patients are given placebo orally as a suspension every morning from Day 1 to Day 90 and from Day 1 to Day 450, and vitamin D drops orally at a dose of 2000 international units per day (e.g., 25 drops per day). In addition, two subcutaneous injections of 20 mcg diamide are administered at two different sites in the abdomen, each on Days 15 and 45, e.g., in a prime-boost dosing regimen (total dose of 80 mcg diamide).

Fifteen patients were assigned to receive placebo orally as a suspension each morning from Day 1 to Day 90, placebo orally as drops from Day 1 to Day 450, and 2 subcutaneous injections of placebo (administered at two different sites in the abdomen), each on Days 15 and 45.

Efficiency variables:

Primary and secondary efficacy endpoints include:

Change in C-peptide (90-min value and AUC _mean 0-120 min) during MMTT.

Proportion of patients with stimulated maximum C-peptide level greater than 0.2 nmol/L

Biomarkers, mechanistic data.

Fasting C-peptide, change between baseline and 6 months.

Security variables:

Safety assessment includes monitoring of injection site reactions, GAD65Ab titers, monitoring of adverse events (AEs), laboratory measurements, monitoring of vital signs, neurological assessments, and limited medical examination.

Results from 20 patients who completed a 6-month study show that improvement in beta cell function was observed for the group of patients who received treatment with diamide compared to the placebo treatment group.

GADA+ C-Peptide Visit 2 Visit 6 Max (6 months) -Max (baseline) Patient identification Visit 3 Visit 5 60 min 90 min 120 min 60 min 90 min 120 min Identified as placebo group 1st injection 3 months 3 678 228.5 0.23 0.28 0.27 0.13 0.11 0.11 -0.15 4 2053 2117 0.71 0.75 0.68 0.77 0.69 0.64 0.02 31 465.8 345.3 0.77 0.79 0.72 0.63 0.73 0.69 -0.06 63 1378 1187 0.86 1.00 1.01 0.60 -0.41 49 118.6 114 0.90 0.91 0.90 0.66 0.82 1.07 0.16 48 555 1187 1.29 1.40 1.88 1.40 1.55 1.58 -0.30

9 61.4 224 0.35 0.38 0.34 0.15 0.17 0.15 -0.21 Average(max-max) -0.14 Identified as an active group 1st injection 3 months 1 964 5460 0.91 0.96 1.03 0.95 1.44 1.47 0.44 6 992 2622 0.48 0.44 0.51 0.41 0.41 0.33 -0.10 17 266.7 2408 0.53 0.48 0.51 0.53 0.55 0.59 0.06 25 109.3 1823 0.61 0.70 0.70 0.77 0.81 0.70 0.11 28 393.7 1931 0.67 0.82 0.76 0.84 0.87 0.88 0.06 33 340.4 1095 0.58 0.64 0.64 0.61 0.62 0.66 0.02 35 125.8 4300 0.96 1.14 1.16 0.75 0.72 0.79 -0.37 40 604 3506 0.37 0.44 0.53 0.15 0.12 0.12 -0.38 41 2266 4230 0.52 0.42 0.40 0.20 0.17 0.13 -0.32 46 132.0 3485 0.65 0.82 0.88 0.32 0.49 0.51 -0.37 47 135.1 3574 1.49 1.71 1.58 1.08 1.30 1.66 -0.05 53 967 4104 0.90 0.84 0.85 0.97 0.94 0.78 0.07 54 162.4 2035 0.77 0.69 0.68 0.81 0.87 1.01 0.24 Average(max-max) 0.04538

Example 2

A pilot clinical trial to preserve residual insulin secretion in adults with recent-onset type 1 diabetes, with treatment by injecting GAD antigen (Diamyd®) into the lymph nodes. (DIAGNODE)

1.1. History of the issue and justification

The incidence of type 1 diabetes mellitus (T1D) in Sweden is the highest in the world after Finland and is increasing rapidly. T1D is by far the most common chronic, severe, life-threatening disease in children and adolescents, and the incidence of type 1 diabetes is also high in young adults. The disease is showing potential to become an extremely serious global problem. The disease is characterized by a lack of insulin. Even if a few patients have quite impressive residual beta-cell function at diagnosis (1), the deficiency soon becomes quite pronounced and eventually beta-cell function disappears completely (2,3). Residual insulin secretion is crucial. Rarely, beta-cell function improves so quickly after diagnosis that glucose metabolism is normalized and insulin is not required for some time, i.e. the patient goes into a state of so-called complete remission (4). As long as the patient is in complete remission, there is no need for active treatment, except perhaps some advice on healthy lifestyle, exercise, and diet. There are no symptoms, no acute complications, and if someone remains in complete remission, it is unlikely that they will ever develop complications. Minor abnormalities in glucose or lipid metabolism may increase the risk of developing macrovascular complications in the same way as in individuals with latent diabetes mellitus or impaired glucose tolerance. Complete remission is rare, but partial remission is not (4). During this period, the patient usually has near-normal blood glucose values, no mild hypoglycemia, and no cases of ketoacidosis. Quality of life is very good because the patient feels well, the children grow normally and few if any dietary restrictions are necessary, patients can engage in a variety of physical activities without developing hypoglycemia and have good home blood glucose tests. Only a small residual insulin secretion is sufficient to reduce the risk of ketoacidosis (5). In addition, the DCCT study showed that even with a very modest residual insulin secretion, the response to beta-cell stimulation with serum C-peptide > 0.20 pmol/mL plays an important role in preventing complications (6). This effect may be related to the fact that residual insulin secretion is likely to facilitate the achievement of good blood glucose balance, but it is also possible that C-peptide itself has a physiological function. In fact, C-peptide has been reported to affect vascular permeability, reduce leakage from retinal blood vessels, and not least have a positive effect on nerve function (7), although the effect of C-peptide itself is still debated.

1.1.1 Factors influencing the natural development of the disease

At diagnosis of T1D, it has been estimated that 80-90% of the beta cells in the pancreas are destroyed. However, the evidence is limited and the main problem may well be deterioration of function. There is also a large variability between patients, with some patients having fairly good residual insulin secretion and others not. Soon after diagnosis, particularly when active insulin treatment is given, there is an increase in C-peptide production and at the same time insulin sensitivity improves. Good metabolic control probably improves the environment and metabolism for the beta cells and beta cell function is preserved, which in turn improves metabolic control and vice versa. The intensity of the autoimmune process plays a role and children probably have a more aggressive immune process than adults with type 1 diabetes, but it remains difficult to predict the course of the disease. Some studies suggest that high concentrations of autoantibodies are accompanied by a more rapid loss of insulin secretion, while other studies do not note such a connection, rather the opposite. No specific signs of cell-mediated immunity that allow predicting the loss of beta cells have been found so far, but our own studies have shown that the pathological process is associated with deviations of the T-helper-1 (Th-1) immune system with an increase in the number of certain cytokines, such as IFNg, and a decrease in the amount of IL-10, IL-13.

Effect of insulin treatment on beta cell function.

Active insulin treatment during the early stage of the disease has long been used to prolong partial remission, a finding that can be supported and justified by the increased residual insulin secretion (2). Intensive treatment appears to improve residual beta-cell function, at least for some time (8), but it may also have long-term beneficial effects (9). Active treatment has not only been shown to prevent or delay diabetes in experimental animals, but studies have shown that such treatment can prevent the development of diabetes in high-risk individuals (10). However, when attempts were made to carry out diabetes prevention studies on a larger scale, parenteral insulin treatment was found to fail to prevent the development of diabetes (11). Treatment with oral insulin may have an effect (12), and further studies are needed.

1.1.2 Interventions

In the 1970s, it became clear that T1D was an autoimmune disease and immune interventions were tried. We conducted the first immune intervention studies in the world in children with diabetes, after we had already used plasmapheresis to treat newly diagnosed children and adolescents with some positive effects 30 years earlier (13). As a side effect of this treatment, a new 64kD protein was found in the plasma (14), which was later found to be glutamic acid decarboxylase (GAD). The breakthrough that was used as a proof of concept for immune intervention was cyclosporine, which clearly slows down the autoimmune destructive process and improves residual insulin secretion, while other immunosuppression studies had minimal effect, especially in children (15, 16, 17), or showed too serious side effects or risks (18, 19). In an attempt to regulate the immune system, the present inventors used photopheresis. Although clear effects on the immune system were demonstrated in a double-blind, placebo-controlled study (20), the clinical effect was minimal and almost no improvement in residual beta-cell function was seen (21). Thus, in the absence of successful immune intervention, the present inventors' interest was directed to protective agents such as nicotinamide and diazoxide, which had no effect or only temporary effects (22, 23, 24).

As knowledge of the immune process that leads to beta cell destruction has increased, it has become possible to target immune interventions more precisely to target important T cells. In an attempt to block the destructive immune process, promising studies have been conducted using anti-CD3 antibodies. Results from both the North American and French studies with anti-CD3 antibodies have shown that it is possible to block the destructive autoimmune process and thereby at least delay the decline in beta cell function (25, 26). The decline in residual insulin secretion was significantly slowed, but unfortunately it appears that the decline was simply delayed for a year, and then the decline in C-peptide paralleled the declining curve in the placebo group. In addition, most patients experienced some degree of cytokine release syndrome (CRS), which can be significant, and most patients also experienced a range of side effects. The present authors participated in one of the last two phase III clinical trials (the Protegé trial) that failed to meet the primary endpoint, although the most intensively treated group did show some preservation of residual insulin secretion and a reduction in insulin requirements to achieve good HbA1c levels (27; Sherry, Hagopian, Ludvigsson et al Lancet 2011). More research is needed, but it is difficult to believe that this treatment alone will become an accepted solution for general clinical use. It is even less likely that such a treatment will be accepted as a preventive treatment for healthy individuals, many of whom will never develop diabetes.

1.1.3 Immune therapy using autoantigens

In the treatment of allergic diseases, immunotherapy using small amounts of disease-specific antigen has been used effectively for many years. The mechanism of this treatment remains unclear, although immune regulation of the immune response and induction of regulatory cells have been proposed. In autoimmune diseases, none of these treatments have been successful, but attempts should continue (28). Experiments in diabetes-prone animals have shown that treatment with heat shock protein can delay or reverse the onset of diabetes. The use of the peptide DiaPep277 in a study of adults showed significant preservation of insulin secretion with virtually no side effects (29). However, more recent studies in children and adolescents with T1D (30) have shown no effect. Studies using DiaPep277 in the treatment of so-called LADA (latent autoimmune diabetes in adults) are ongoing, and preliminary results (presented at IDF Dubai in December 2011 and at ADA in June 2012) suggest that treatment with DiaPep277 may preserve beta cell function in adults with mild type 1 diabetes. However, the results are somewhat unclear, as only a weak preservation of C-peptide was observed after glucagon stimulation, but no effect was observed after a meal tolerance test, and no differences in immune markers were observed between the active treatment and placebo groups. Not only has active insulin treatment been shown to prevent or delay diabetes in experimental animals, but preliminary open-label studies have shown that such treatment can prevent the development of diabetes in a high-risk individual (10). Insulin, the undisputed specific beta-cell autoantigen, has been unsuccessfully administered parenterally (DPT) to prevent diabetes in high-risk individuals, although oral administration of insulin for the same purpose may have little effect (12).

1.1.4 Previous clinical studies with GAD-alum

1.1.4.1 GAD vaccination

GAD (glutamic acid decarboxylase) can be considered as an autoantigen, since it is produced with increased release in islet cells in response to beta cell stimulation. This protein has been shown to have a significant effect on the autoimmune immune process (31, 32, 33, 34). Several studies have shown that GAD is indeed able to prevent the development of diabetes mellitus in experimental animals (35-42). The similarity of GAD to viral proteins may be important for the therapeutic effect. The observed effect, even after the onset of the immune process, suggests that the same effect could be expected in humans after the onset of the immune process. In a phase II study in LADA patients, the administration of a single low dose of 20 μg of the diamide resulted in an improvement in beta cell function for up to 2 years, compared with the placebo treatment group, without any side effects. Other doses were also tested: 4 μg showed no effect, 100 μg showed the same effect as 20 μg, while 500 μg showed no effect. None of the doses showed any side effects even after several years of observation (43). An association with the change in the CD4+CD25+/CD25-CD4- cell ratio was found, indicating the mechanism of the effect. With this promising background, we initiated a phase II study in patients with type 1 diabetes mellitus aged 10-18 years who had recently developed diabetes. Based on the idea that treatment had previously been effective in patients with slowly progressive LADA, we included patients with up to 18 months of T1D diabetes for the intervention. Patients were randomly assigned to receive 20 μg GAD-alum (diamide) subcutaneously on Days 1 and 30 or placebo. After 30 months, the effect was still significant and clearly statistically and clinically significant (44), with about half the amount of C-peptide reduced in the GAD-treated group compared to the placebo group. In patients with diabetes duration <3 months, a surprisingly good response was observed, with no or minimal decline in beta-cell function during the follow-up at the end of the first 15 months. Almost complete response was observed in patients with vaccination duration <6 months. Moreover, unlike other interventions, this effect was obtained without any side effects at all, making the treatment very encouraging! Even after 48 months, patients treated for <6 months showed significant preservation of C-peptide and no side effects (45). So far, treatment with GAD seems very promising. Two phase III trials were conducted, one European with Johnny Ludvigsson (JL) as Principal Investigator and one in the US with Jerry Palmer as Principal Investigator and JL as a co-investigator. In the European study 334 patients were recruited into three groups, one group received GAD-alum (diamide) 20 mcg on Days 1, 30, 90 and 270, another group received GAD-alum 20 mcg on Days 1 and 30 and placebo on Days 90 and 270, and the third group received placebo on Days 1, 30, 90 and 270. The primary endpoint, serum C-peptide AUC after a meal tolerance test (MTT) at 15 months was not met! (AUC peptide, p=0.1; fasting C-peptide, p=0.07) (46). This forced the company (Diamyd Medical+Johnson & Johnson) to close the phase III trial early. However, the phase III study did reveal some positive effects. For example, statistically significant efficacy was seen in several pre-specified subgroups. In addition, 45 Swedish patients were seen by their physician at month 30, when the study was stopped, and the 15 patients who received two doses of 20 mcg GAD-alum (diamyd) showed significant preservation of C-peptide at month 30, compared to placebo! What is particularly noteworthy is that the Swedish patients were the ones who did not show efficacy after month 15, while efficacy was seen after month 15 in non-Scandinavian patients.

1.1.4.2 Possible reasons for different results between phase II and phase III

In phase III randomization, patients receiving active drug were more common in the 10-11 year age group than in the 16-18 year age group, while placebo was more common than active drug in the older age group. This may have influenced the outcome. Patients in phase II were treated in March-April, and those in phase III who were treated in March-April also showed a significant effect of treatment with GAD. No vaccinations were allowed in phase II, while influenza vaccinations were allowed in phase III. Unfortunately, the H1N1 flu epidemic meant that almost all patients were vaccinated, many of them together with GAD vaccination.

In Sweden and Finland, the vaccine contains squalene, which is suspected of influencing the immune system towards autoimmunity, and in these two countries, no efficacy was observed with GAD treatment, while efficacy was significant in other European countries. Patients in Sweden who had not received an influenza vaccine at the time of GAD treatment had a higher efficacy with GAD treatment (46).

1.1.4.3 Ongoing DIABGAD-1 study

Since January 2013, the phase II study DIABGAD-1 is ongoing in Sweden. The trial is being conducted in 60 patients aged 10-18 years with recent onset of type 1 diabetes and who are being treated in a double-blind, randomized, placebo-controlled study with 20 μg and 40 μg GAD-alum, respectively, given twice at 30-day intervals in combination with 2000 units/day vitamin D for 450 days, and with ibuprofen 400 mg/day for 90 days. Recruitment has been completed and is currently closed. 60 patients have been randomized and 4 patients are being screened, pending screening results. There have been no serious adverse events considered related to the study drug and only minor adverse events unrelated to the study drug, with the exception of mild transient reactions at the injection site of GAD-alum. An interim analysis is planned for all patients after 6 months of observation, and extended analyses will be performed after 15 and 30 months.

1.1.5. Immunotherapy in the lymph node

Antigen therapy aims to present antigen to T cells in the lymph nodes in order to obtain a new balance of the immune system and tolerance to the antigen. In the treatment of autoimmune diseases, antigen has so far been administered either orally, intranasally or subcutaneously to present the antigen to antigen-presenting/dendritic cells, which in turn are expected to present the antigen to T cells of the immune system. However, animal studies have shown that intralymphatic injections induce a strong and T cell-related response (47,48), and allergy studies in the clinical setting have shown that antigen/allergen presentation directly to the lymph nodes is probably more effective than traditional administration (49). Lower doses of allergen can be used, the number of treatments can be significantly reduced, and no treatment-related adverse events have been reported. The inguinal lymph nodes are easily accessible in patients and the pain associated with the injection is estimated to be less than that associated with venous puncture (50). Against this background, the aim of the present inventors is to study whether the same approach can be used in patients with autoimmune type 1 diabetes mellitus. For ethical reasons, we will conduct the first pilot clinical study in adults.

1.2 Hypotheses

The encouraging results of the phase II study using GAD and the partially positive results of the phase III European study support the concept that the administration of GAD-alum (diamide) can reduce the autoimmune process and help preserve residual insulin secretion. As previous studies indicate, the dose should be between 20 and 100 mcg of diamide, smaller doses of 3 mcg administered three times should be sufficient if administered directly into the lymph nodes. Injection of GAD-alum (diamide) directly into the lymph nodes does not produce any serious side effects, produces immunological effects and (as will be shown in future studies) increases efficacy.

2. Risk-benefit analyses

The incidence of type 1 diabetes mellitus after Finland is higher in Sweden than in any other country in the world. The incidence has been steadily increasing for decades. The disease cannot be cured and cannot be prevented. Despite very difficult, intensive, expensive treatment, many patients experience life-threatening serious acute and late complications, and mortality has increased significantly. At diagnosis, many patients have a small residual secretion of insulin. As long as this condition persists, it is much easier to maintain stable blood glucose levels, and the occurrence of hypoglycemia is reduced, as is the risk of developing ketoacidosis. The patient's quality of life, as well as the quality of life of parents of children with diabetes, is better as long as some residual secretion of insulin is preserved.

Type 1 diabetes in adults differs from the disease in children and adolescents because the disease process is often milder, the decline in residual insulin secretion is slower, and blood glucose levels are easier to control. However, there are many similarities, similar treatments and complications, and preserving beta cell function is also very important in adults.

It is obvious that there is a great advantage in preserving residual insulin secretion, and therefore therapies aimed at preserving this function justify treatment that is quite difficult, even dangerous and expensive. Thus, it was considered justified to treat type 1 diabetes at the beginning of the disease using drugs such as monoclonal antibodies against the CD3 receptor, which causes adverse effects in almost all patients, and in some - even quite serious side effects and risks. Even purely cytostatic drugs were used.

In the present study, the present inventors are using GAD-alum (diamide) 4 mcg x 3, a treatment used in much higher doses in children and adults with virtually no side effects, which have been observed in thousands of patients per year. In this study, the present inventors plan to use a very low dose, which means that the overall risk is expected to be even lower, but the injection will be made directly into the lymph node, which may cause local reactions. The effect on the immune system may be more pronounced, but should not lead to any side effects. Previous allergy studies (where one of the participants in the collaborative study, Helene Zachrisson, was injected into the lymph node with allergen formulations together with aluminum hydroxide) did not show any negative effects, either systemically or locally). For safety reasons, since this is the first ever pilot clinical trial for this type of autoantigen therapy in lymph nodes, the present inventors are testing primarily on adults who can give their free informed consent. Even though type 1 diabetes in adults is somewhat milder than in younger patients, it is important to preserve beta cell function, and therefore the proposed treatment may have greater therapeutic value in adult patients as well.

When all the pros and cons are taken together, there is a clear possibility of significant therapeutic benefit for both the patients in the study and for patients in future studies, and the risk is very small. If these studies lead to the development of a good treatment, this will be of great importance to a large number of patients.

3. The purpose of this study

The purpose of this pilot clinical study is to obtain information on whether GAD-alum can be administered into the lymph nodes without causing serious treatment-related side effects, to allow future phase II studies using the same approach to improve the efficacy in preserving residual insulin secretion in type 1 diabetes. Thus, we want to see if this treatment produces any side effects and how the dosing regimens affect the immune system, induce the desired Th2 shift, increase T regulatory cells, and hopefully show signs of preserving residual beta cell function. Based on the short-term results of this pilot clinical study (6 months of follow-up), we may wish to conduct an expanded phase II study to include younger patients and provide more robust information. The main long-term goal is to find a treatment method at the onset of type 1 diabetes in young patients that is acceptable to patients, safe, and that can preserve residual insulin secretion and improve the quality of life of patients with less acute complications and, in the long term, reduce the risk of late complications.

4. Objectives of the present invention

Goals:

To assess the safety of delivering diamide directly to the lymph nodes and to evaluate the immune response (51-54) and the effect on maintaining endogenous insulin secretion measured at baseline and after 6, 15 and 30 months.

5. Contingent

Adult patients with recent-onset type 1 diabetes at Linköping University Hospital were provided with information about the study and asked to participate in the study.

5.1 Inclusion criteria

1. Informed consent given by patients and guardians/parents.

2. Type 1 diabetes according to the ADA classification with diabetes duration <6 months.

3. Age 18.00-29.99 years at the time of diagnosis of type 1 diabetes.

4. Fasting C-Peptide ≥0.12 nmol/ml.

5. Positive GADA but <50,000 arbitrary units.

6. Women must agree to avoid pregnancy and have a negative urine pregnancy test.

7. Patients must agree to use adequate contraception for up to 1 year after the last administration of GAD-alum/placebo.

5.2 Exclusion criteria:

1. Previous or current treatment with immunosuppressant therapy (although topical or inhaled steroids are acceptable).

2. Continuous treatment with any anti-inflammatory drug (sporadic treatment due to headache or high fever for several days is allowed).

3. Treatment with any other anti-diabetic drugs other than insulin, administered orally or by injection.

4. History of anemia or significantly abnormal hematology results on screening.

5. History of epilepsy, traumatic brain injury or acute cerebrovascular accident, or clinical evidence of continuous activity of neuromotor units in proximal muscles.

6. Clinically significant history of acute reaction to vaccines or other drugs in the past.

7. Treatment with any vaccine, including influenza vaccine, within 4 months prior to the planned first dose of study drug or planned treatment with any vaccine up to 4 months after the last injection of study drug.

8. Participation in other clinical trials with new chemical substances within the previous 3 months.

9. Inability or unwillingness to comply with the provisions of this protocol.

10. History of alcohol or drug abuse.

11. Significant illness other than diabetes within 2 weeks prior to first dose.

12. Confirmed presence of human immunodeficiency virus (HIV) or hepatitis virus.

13. Women during lactation or pregnancy (the possibility of pregnancy should be excluded by on-site urine βHCG analysis within 24 hours prior to starting treatment with GAD-alum).

14. Men or women who do not wish to use adequate contraception for up to 1 year after the last treatment with GAD-alum.

15. The presence of a concomitant serious illness or condition, including active skin infections, precluding subcutaneous injection, which, in the opinion of the investigator, makes the patient unsuitable for the study.

16. Inability, in the opinion of the investigator, to follow instructions and/or comply with the study protocol.

5.3 Volunteer recruitment and screening

Patients who are eligible to participate in the study are given an explanation of the study and are given written patient information. Patients are then given time to review the nature of the study and are given the opportunity to ask questions of the research team. If patients then agree to participate in the study, they personally sign and date the written informed consent. Patients are then given a copy of the signed and dated patient information/informed consent form.

5.4 Exclusion of patients from the study

In accordance with the Declaration of Helsinki, the investigator must explain to the patient that he/she has the right to withdraw from the study at any time and that this will not in any way prejudice his/her future treatment. However, unless safety concerns arise, the present inventors plan to follow up the patients throughout the study period, for the purpose of analyzing efficacy and safety variables and for those patients who withdraw from the study. The reasons for any withdrawal from the study must be recorded in the appropriate clinical trial patient report form (CRF).

There are different categories of patients who terminated their participation in the study early: complete withdrawal (i.e. cessation of administration of the investigational product, as well as assessment of efficacy and safety).

Standard reasons for withdrawal from further study participation and discontinuation of follow-up visits (<e.g. for blood tests>) may include the following:

Patient's decision (withdrawal of consent to participate in the study)

The patient was lost to follow-up.

Common reasons for discontinuing further study product intake but continuing follow-up visits and safety assessments may include:

Undesirable side effects

Patient's request

Researcher's decision

Patient lost to follow-up/failure to attend

Concomitant disease

Patient's pregnancy

Thus, intra-lymph node administration of GAD-alum should not be administered to a patient if the patient has experienced the following after inclusion in the study:

- brain damage, epilepsy, traumatic brain injury, neurological disease

- any active, severe hormonal disease other than type 1 diabetes

- other severe autoimmune disease (except celiac disease, which is acceptable for inclusion)

- immunosuppressive treatment

- oncological disease, treatment of oncological disease

- any other antidiabetic drugs except insulin

- any vaccination

- drug/alcohol abuse

or if the patient

- becomes pregnant or no longer wishes to use reliable contraception during the study.

However, whenever a patient is withdrawn from the study, or for any reason fails to attend the observation visit, a final study assessment should be made for that patient (visit <>) - indicating the reason(s) why the patient was withdrawn from the study. All documentation concerning the patient should be as complete as possible. Withdrawals due to failure to attend should be investigated by the investigator to determine the reason for failure to attend. Withdrawals due to comorbidities or adverse events should be fully documented as an entry in the clinical trial patient case report, with additional information if available and/or necessary.

6. Medical procedures

6.1 Clinical trial plan and treatment

The study is a single-center, open-label, pilot clinical trial of GADA-positive T1D patients of both sexes aged 18.00 to 29.99 years diagnosed with T1D within 6 months at screening (Visit 1) and fasting C-peptide levels equal to or greater than 0.12 nmol/L. A total of approximately 5 patients will be recruited at one site in Linköping, Sweden. Patients will be assessed for eligibility at a screening visit (Visit 1) 10-21 days before treatment initiation. Eligible patients will be assigned a sequential number and the screening number will be used as patient identification throughout the study.

Patients who are accepted into the study will then be enrolled in the study to receive the study drug at subsequent visits as outlined in Table 1 below. Patients will be followed for the entire 30-month study period, which includes 8 visits to the study center.

Table 2 below provides an overview of the study visits.

Table 1. Patient visit schedule, visit intervals and study drug administrations

Study Screening Intervention Follow-up observation Days -10 to -21 Screening Day 1 Initial Level Day 30±3 Day 6±3 Day 180±14 Month 6 Day 450±14 Month 15 Day 900±14 Month 30 Visit 1 Visit 2 Visit 3 Visit 4 Visit 6 Visit 7 Visit 8 GAD-alum Placebo GAD-alum treatment GAD-alum treatment GAD-alum treatment

6.2 Examinations and procedures

1. Standard insulin treatment, education and psychological support for newly diagnosed patients with type 1 diabetes.

2. Normalization of fluid balance, electrolytes and acid-base balance.

3. Follow-up information about the study.

4. After patients have given their informed consent, no later than 120 days after diagnosis, screening with fasting venous sampling is performed in patients who meet the criteria other than C-peptide and GADA concentrations (Visit 1).

5. At baseline (Visit 2), 6, 15 and 30 months, residual endogenous insulin secretion is assessed using the MMTT method. At each visit, HbA1c, safety (hematology and chemistry), autoantibody titers (GAD65, IA-2), immunology are monitored with subsequent blood sampling. Exogenous insulin dose/24 h, adverse events and concomitant treatments are recorded during the examination at each study visit.

6. Self-reported hypoglycemia (defined as need for assistance from others and/or seizures and/or unconsciousness) recorded at each visit.

7. Any symptoms or signs of other medical problems should be considered at the discretion of the clinician. Investigations should be carried out in accordance with section 7 in Table 2 below and in the order set out in the Case Report Form (CRF).

6.2.1 All visits, visits 1 to 7

It should be noted that the patient must attend all study visits in the morning after an overnight fast (> 10 hours, water is allowed). For patients with signs of infection (including fever), the entire visit should be postponed for 5 days or until the patient has recovered.

6.2.2 GAD-alum administration, visits 2, 3 and 4

Following administration, the patient must remain in close proximity to the study site for the next hour, and the injection site will be examined by the investigator/nurse 1 hour after the injection.

6.2.3 Food Tolerance Test (FTT), Visits 2, 5, 6 and 7

MMTT should be performed according to the instructions set out in the CRF. The patient should:

Arrive at the research site after an overnight fast (> 10 hours), i.e. the patient cannot eat, but is allowed to drink water.

Do not take short-acting/direct-acting insulin within 6 hours before MMTT. The patient may take basal insulin the day/night before, but not in the morning before MMTT.

Patients with CSII (insulin pump) should continue to take their basal insulin dose but not add a bolus dose during the last 6 hours before MMTT.

Have a fasting glucose level in the range of 4-12 mmol/L as measured by a home blood glucose meter on the morning of the test. If the patient does not meet all of the above criteria, the MMTT should be rescheduled and the patient should return to the test site within 5 days if possible.

If patients need to eat or take insulin for safety reasons, the visit should also be rescheduled.

6.3 Laboratory tests and studies:

1. Immunological tests:

a. Autoantibodies (anti-GAD65, anti-insulin, anti-IA-2, ZnT8)

b. The corresponding cytokines and chemokines are determined (see below)

c. T cells are classified and studied (see below)

2. Genetics:

a. HLA and genes associated with diabetes development are determined

b. Exploring the data set to determine the importance of genes associated with diabetes

3. Virus tests:

a. Genetic, immunological and microbiological tests may be performed.

4. Diabetes status:

a. HbA1c

b. Fasting glucose and C-peptide levels

c. Meal-stimulated glucose and C-peptide levels

5. Taking blood samples for safety purposes:

a. Hematology

b. Chemical analysis

6.4 Patient card

A complete review of the patient's previous medical history is performed by the investigator and documented in the CRF medical record.

All pre-existing conditions/diseases will be reflected on the CRF History page during the Screening Visit (Visit 1).

The date of diagnosis of type 1 diabetes in the patient and family history of type 1 diabetes are also documented.

6.5 Physical examination, including neurological examination

At the screening visit (Visit 1), the patient will undergo a general physical examination and neurological examination, and any findings will be documented as pre-existing conditions on the CRF history page.

During subsequent study visits, the patient will be assessed for any new medical conditions or worsening of existing conditions. Any change in pre-existing conditions or new conditions should be entered on the AE page in the CRF, and any medications entered on the relevant medication pages.

In addition to a limited physical examination by a physician, patients undergo a standardized clinical neurological examination at screening, 0, 6, 15, and 30 months. Neurological tests are performed to detect possible subtle signs of neuromuscular disease, such as decreased muscle strength, balance, and coordination.

Neurological examination includes:

Limb reflexes

Romberg (balance and coordination)

Straight line movement, 2 meters (balance and coordination)

Standing on 1 leg, left and right, 15 sec on each leg (balance and coordination)

Finger-nose test (coordination)

Facial expressions (cranial nerves)

Babinski reflex (central function)

Muscle strength (hand grip), biceps, triceps, distal extensors and flexors

At the investigator's discretion, these examinations may also be repeated between scheduled visits. Screening for neurological disease using electroencephalogram (EEG) is not included due to low sensitivity and specificity. However, if any evidence of neurological impairment is detected, the patient should be referred to a neurologist for further evaluation.

6.7 Concomitant drug therapy

Any concomitant medication used during the study, whether considered by the investigator to be study-relevant or not, must be recorded in the concomitant medication log in the CRF. Please see also section 8.5 below.

7. Schedule of procedures

Table 3 DIAGNODE-1 Pilot Study, Schedule of Study Events, T1D Patients

Research period Screening Intervention Follow-up observation Visit number 1 2 3 4 5 6 7 Time (Day/Week/Month) Day From -10 to -21 Day 1 Initial Level Day 30±3 Day 60±3 M6 (D180±14) M15 (D450±14) M30 (D900±14) Informed consent × Treatment with CAD-alum (diamide) × × × Anamnesis × Physical examination × × × × × × × Neurological examination × × × × × × × Concomitant medications × × × × × × × Height × Weight, Body Mass Index × × × × × × × Urine Pregnancy Test (Women) × × × Inspection of the injection site ^a × × × Insulin dose × × × × × × × Self-referral for hypoglycemia ^b × × × × × × Side effects × × × × × ×

Blood samples for safety purposes: *Hematology × × × × × × × *Chemical analysis × × × × × × × Autoantibodies: *GADA × × × × × × × *Diabetes-associated antibodies × × × × × × × Blood tests to determine diabetes status: *HbA1c × × × × × × × Fasting Glucose/C-Peptide × × × × × × × Glucose/C-peptide MMTT × × × × Blood samples for genetics, immunology × × × × × × Vitamin D in serum × Blood samples for biobank × × × × × × ×

^a Inspection of the injection site before and 60 minutes after the injection, performed by an investigator or nurse

^b Hypoglycemia is defined as a need for assistance from others and/or seizures and/or unconsciousness

7.1 Visits

The first visit, the screening visit (Visit 1), should be scheduled 10 to 21 days before the scheduled Visit 2 (baseline). Visits 3 and 4 should then be scheduled at a visit interval of ±3 days (second and third GAD-alum administration), ±14 days for Visits 5, 6, and ±30 days for Visit 7. Please note that all visits should be calculated from the baseline visit (Visit 2) according to the visit schedule. Please note that visits must be scheduled at a visit interval to comply with the study protocol.

Please see Tables 1 and 2 above for patient visit schedules, visit intervals, and study medication.

8. Study drug

8.1 Study drug

The study uses medicinal substances from the following sources:

Study drug: GAD-alum (diamide), 4 mcg x 3 (prescribed three times with an interval of one month)

Supplier of the study drug: Diamyd Medical AB, Stockholm, Sweden.

8.2 Delivery

GAD-alum (Diamide) is a product supplied by Diamyd Medical. It is supplied as a pre-packaged drug by Diamyd Medical to a local pharmacy network. All dosing will be performed in the hospital and will only be performed by trained and authorized personnel participating in the study. The study drug will be labeled with information in accordance with local regulations. GAD-alum will be stored in a refrigerator at 2-8°C in a secure area (e.g., a locked cabinet or drug room) where it will be protected from unintentional use. All study drugs will be labeled with information in accordance with local regulations.

8.3 Dosage and administration of the drug

GAD-alum: 4 mcg is injected into the lymph nodes in the groin area (using ultrasound technology) three times at one-month intervals.

8.4 Duration of treatment

See 8.3

8.5 Concomitant treatment

No systemic immunomodulatory drug or other antidiabetic drugs other than insulin, whether currently on the market or not, are approved.

9. Response variables and outcomes

9.1 Exploratory evaluation of effectiveness

9.1.1. Efficiency variables

Because this is a pilot study, the primary efficacy endpoint is not defined; however, the present inventors will monitor:

Change in fasting C-peptide level and C-peptide level (90 min value and AUC _mean 0-120 min) during MMTT from baseline to 6, 15 months and to 30 months, respectively.

Th2-biased cellular immune response, seen for example as increased ratio of IL-5, 10, 13 versus IFN-gamma, TNF-alpha, IL-1beta, IL-17, and increased T-regulatory cells. Change between baseline and follow-up visits.

Inflammatory markers, eg, TNF-alpha, IL-1 beta, IL-2, IL-17. Change between baseline and follow-up visits.

Hemoglobin A1c (HbA1c). Change between baseline and follow-up visits.

Exogenous insulin dose per kg body weight and 24 h; change between baseline and follow-up visits.

10. Statistical methodology and data processing

10.1 Clinical trial plan

The DIAGNODE-1 study is a pilot, open-label, phase I interventional study.

Study participants: patients with newly diagnosed classic type 1 diabetes mellitus: N=5. Age 18-29.99 years. Recruited from an endocrinology clinic in Sweden

10.2 Sample size estimation

Power Analysis: A formal power analysis was not conducted for this experimental study.

10.3 Statistical Analysis Plan

In brief, the following analyses are planned:

For all continuous variables, the following descriptive statistics are displayed: number of observations (n), mean, standard deviation, minimum, median, and maximum. All categorical variables will be displayed with frequencies and percentages. Tabulation of descriptive statistics will be separated by visits. Where appropriate, basic (screening) descriptive statistics will also be included.

Group data and other baseline characteristics

Group data and baseline characteristics will be presented using descriptive statistics (summary tables).

Safety variables and efficacy data

AE/SAE data will be presented using standardized tabulation of frequency and incidence rates for all observed AEs/SAEs. Frequencies and incidence rates are calculated on a per-patient basis. Adverse events will be summarized by body system, causality, and severity. Other safety data will be presented using descriptive statistics.

Efficacy data regarding C-peptide and the immune system, as well as adverse events and other safety data will be summarized narratively.

After 6 months, safety data will be analyzed. (The results will be used to develop the study plan for the Phase II DIAGNODE trial.)

10.4 Study sample

Analysis of all randomized patients.

Patients will be included in the set of all randomized patients who received study drug at least once for the efficacy analysis if they received at least 1 dose of all study drugs in their group and were assessed at a later visit.

Individuals who complied with the protocol and completed the study

To qualify as per-protocol completers, patients must have followed the study protocol without any major violations. Any missed examinations may be rescheduled for a later date, but no more than 1 visit may be missed.

General population

Any patient who withdraws from the study after receiving at least one treatment (Visit 2) will be included in the safety analysis (adverse events and safety parameters). Data for all patients will be presented, and a list of withdrawn patients will be provided, with all reasons that led to discontinuation from the clinical trial.

10.5 Data collection/case study patient records

Case Report Forms (CRFs) will be provided to record the data obtained for each patient. Because it is important to ensure that relevant data are collected in a timely manner, the investigator or authorized representative should complete the CRFs as soon as possible. When the clinical investigator requests additional data or clarifications on a CRF, the request should be satisfactorily responded to within the allotted time.

It is the responsibility of the investigator to ensure that the case report forms are completed correctly. The investigator signs the approval sheet to confirm that the case report form is accurate and complete.

To ensure their legibility, CRFs should be completed in block letters using a black or blue ballpoint pen (not a pencil, felt-tip pen, or fountain pen). Any corrections to the CRFs should be made by the investigator or a person designated by the investigator. One line should be completed using the original entry. The correction should be dated and initialed. Incorrect data should not be corrected with correction fluid or destroyed or made illegible in any way. Even if there are no changes from the previous survey, in the interest of completeness of data collection, questions that are repeated in each section of the CRFs should be answered in full. A reasonable explanation should be provided by the investigator for any missing data.

10.6 Data processing

The data will be coded and entered into a computer database. Data processing, including data quality control, will comply with regulatory requirements (e.g. International Conference on Harmonization [ICH] and Good Clinical Practice [GCP]).

11. Regulatory and administrative procedures

Any regulatory requirements must be met before the trial can begin. The sponsor of the clinical trial applies for official approval from the relevant authorities. Study sites, clinical trial resources, laboratories and all data (including raw data) and documents must be available for inspection by authorities.

11.2 Information for patients/Informed consent

The investigator is responsible for providing patients with complete and adequate oral and written information about the features, purpose, possible risks, and benefits of the study. Patients must also be informed that they are free to withdraw from the study at any time. Patients must be given reasonable time to read and understand the information before signing. The investigator is responsible for obtaining signed informed consent from all patients before including a patient in any study-related procedures. A copy of the patient information and the Informed Consent Form will be given to patients.

11.4 Patient Treatment Plan

All patients will continue to receive standard treatment for type 1 diabetes during the study. After an individual completes the study, the patient will return to the standard treatment they were receiving before participating in the study.

Example 3

The example therapeutic regimens have an "orthogonal" effect and reduce T1D autoimmunity in the long term. The immune system is suppressed by etanercept, which in turn suppresses inflammation around beta cells, while the beta cell autoantigen (GAD) is presented by dendritic cells, whose ability to induce tolerance is improved by vitamin D treatment.

Study: An open-label study to evaluate the tolerability of combination therapy consisting of GAD-alum (Diamyd®), etanercept and vitamin D in children and adolescents with newly diagnosed type 1 diabetes.

Active ingredients: recombinant human glutamic acid decarboxylase (rhGAD65), calciferol (vitamin D), etanercept.

Study stage: Phase IIa

Goals:

Evaluation of tolerability of combination therapy using rhGAD65, vitamin D and etanercept.

Evaluation of how the above treatments affect the immune system and endogenous insulin secretion.

Clinical study plan:

The study is a multicenter, open-label, pilot clinical trial. All patients receive 2000 international units of vitamin D orally daily for 15 months from Day 1, and from Days 1-90, etanercept (Enbrel) subcutaneously at a dose of 0.8 mg/kg body weight (maximum 50 mg) once a week, and two subcutaneous injections of 20 mcg diamide in a "prime-boost" regimen on Days 30 and 60. Patients are assessed for drug tolerability for 6 months (main study period, 6 visits) and then for an additional 24 months (extension study period, 3 visits). The total treatment period is 30 months.

Subject Selection: Patients must be between 8.00 and 17.99 years of age and have been diagnosed with type 1 diabetes mellitus (T1D) within 100 days prior to screening. Patients are eligible for enrollment if fasting C-peptide is ≥0.12 nmol/L (0.36 ng/mL) and elevated GAD65 antibody levels are present.

Planned number of patients: Approximately 20 patients will be enrolled.

Description of treatment groups:

There is one treatment arm. Patients are assessed for eligibility at a screening visit (Visit 1) 2–4 weeks before treatment. At Visit 2 (Day 1), eligible patients begin treatment as described above.

End points

Primary endpoints:

To assess the tolerability of combination therapy using diamide, vitamin D and etanercept at 6 months (main study period), 9, 15 and 30 months (extended study period)

Variables for assessing tolerability:

Injection site reactions

Infections

Occurrence of adverse events (AEs)

Occurrence of serious adverse events

Physiological and neurological assessments

Laboratory measurements (biochemistry and hematology), including determination of calcium and vitamin D in the blood serum

Titer GAD65AB (GADA)

Secondary endpoints:

To assess how the above treatments affect the immune system and endogenous insulin secretion after 6 months (main study period), 9, 15 and 30 months (extended study period).

Variables for assessing the impact on the immune system:

Inflammatory markers, in particular TNF-alpha, IL-1 beta, IL-2, IL-17

Th2-biased cell-mediated immune response, observed, for example, as an increase in the ratio of IL-5,10,13, compared to IFN-gamma, TNF-alpha, IL-1 beta, and IL-17.

Increase in T-regulatory cells

Variables for assessing the influence of endogenous insulin secretion:

C-peptide (90 min value and AUC _mean 0-120 min) during MMTT

Proportion of patients with stimulated maximum C-peptide level greater than 0.2 nmol/l

Fasting C-peptide

Hemoglobin A1c (HbA1c)

Dose of exogenous insulin per kg of body weight and 24 hours

Sample size:

There is no actual sample size calculation in this open-label pilot study, but rather an assessment of whether the treatment is tolerable and does not cause adverse effects on beta cell function and/or the immune system.

All variables will be summarized descriptively.

Example 4

Study Title: Effect of GABA or GABA/GAD combination on the progression of type 1 diabetes mellitus in children

Research Stage: Human Pilot Study

Goals:

To evaluate the safety and impact of GABA treatment on the preservation of residual insulin secretion in new-onset type 1 diabetes.

To evaluate the safety and effect of treatment with two doses of GAD-alum (Diamyd®) plus GABA on the preservation of residual insulin secretion in new-onset type 1 diabetes.

Clinical Study Design: The study is a randomized, double-blind, placebo-controlled, three-arm clinical trial. Patients receive one of:

i) oral GABA at a dose calculated per kilogram, twice daily for 12 months plus 2 subcutaneous injections of 20 mcg diamide in a prime-boost regimen for 30 days

ii) orally GABA, in a dose calculated per kilogram, twice daily for 12 months

iii) placebo.

Patients are followed for a total of 12 months.

Subject selection: Patients must be between 4 and 17 years of age and have been diagnosed with type 1 diabetes mellitus (T1D) within the previous 4 weeks prior to randomization. Patients will be eligible to participate in the study if elevated levels of the GAD65 antibody are present.

Planned number of patients: Approximately 75 patients will be enrolled.

Description of treatment groups:

Patients will be assessed for eligibility prior to randomization. At Visit 1 (Day 1, baseline), eligible patients will be randomly assigned to one of 3 treatment groups:

25 patients are given oral GABA twice daily at a dose per kilogram, from Day 1 through Month 12. In addition, 2 subcutaneous injections of 20 mcg diamide (GAD-alum) are given on Day 1 and Month 1, 1 prime dose, and 1 booster dose (total dose is 40 mcg diamide).

Twenty-five patients are assigned to receive oral GABA twice daily at a dose per kilogram from Day 1 through Month 12. In addition, two subcutaneous injections of diamide placebo are given on Day 1 and Month 1.

25 patients are assigned to receive oral placebo GABA twice daily at a dose per kilogram, from Day 1 through Month 12. In addition, 2 subcutaneous injections of placebo diamide are assigned on Day 1 and Month 1.

Primary endpoint:

To evaluate the effects of GABA and the combination of GABA+GAD-alum on pancreatic beta-cell function, as measured by meal-stimulated C-peptide secretion levels, compared with an age-matched placebo control group, before and after treatment for one year.

Secondary endpoints:

To evaluate the effect of GABA and GABA+GAD-alum combination on autoimmune diabetes autoantibodies: GAD-65, ICA512 and zinc transporter 8 (ZnT8A) and the effect on HbA1c, fasting and stimulated glucose and glucagon levels, fasting C-peptide and the amount of insulin used daily by participants, compared with baseline at subsequent visits

Safety assessment of GABA and the combination GABA/GAD-alum.

Safety

Safety assessment includes monitoring of injection site reactions, occurrence of adverse events (AEs), laboratory measurements, neurological assessment, and exercise restriction.

Sample size:

The sample size for the proposed study is 75 children; 50 in the treatment groups and 25 in the placebo group. For the primary comparison of change from baseline in C-peptide values at 12 months between these groups, assuming an α of 0.05 and a mean (SD) C-peptide AUC of 1.0 (0.4), this sample size provides ~40% power to detect a 25% difference and ~97% power to detect a 50% difference. Adverse events and other data will be summarized narratively.

Example 5

Active Ingredient Name: Recombinant human glutamic acid decarboxylase (rhGAD65)

Study Title: Double-blind, randomized, investigator-initiated study to determine the safety and effect of Diamyd® on the progression of type 1 diabetes in children with multiple islet cell autoantibodies.

Goals:

The primary objective is to demonstrate that Diamyd® is safe for children at risk of developing type 1 diabetes. Patients will be followed for 5 years.

A secondary objective is to evaluate whether Diamyd® can delay or halt the autoimmune process leading to clinical type 1 diabetes in children with ongoing persistent beta cell autoimmunity as indicated by multiple positive islet cell autoantibodies.

Clinical study plan:

The study is a randomized, double-blind, placebo-controlled, single-center, two-arm clinical trial. Study participants will receive 2 subcutaneous injections of either Diamyd® 20 mcg or placebo in a prime-boost regimen for 30 days. Study participants will be followed for 5 years.

Post-diagnostic investigation protocol (PDIP)

Given the shelf life of the study product, children diagnosed with clinical type 1 diabetes during the study period may be offered to continue the study as part of the post-diagnostic infusion protocol (PDIP) and receive an additional 2 injections of Diamyd® regardless of which treatment group they were randomly assigned to in the prevention portion of the study.

Patients' choice:

Patients must be >4.00 years of age, be positive for GADA, and have at least one additional autoantibody associated with type 1 diabetes (IA-2 Ab > 5, ZnT8R/W/Q/A Ab > 72, or IAA > 0.8).

Planned number of patients: 50 patients will be enrolled.

Description of treatment groups:

Patients will be assessed for study eligibility at a screening visit (Visit 0) approximately 30 days prior to their first injection. At Visit 1 (Day 1), eligible patients will be randomly assigned to one of 2 treatment groups:

25 patients are given 2 subcutaneous injections of 20 mcg Diamyd® on Days 1 and 30, i.e. 1 primary and 1 booster dose.

25 patients are given 2 subcutaneous injections of placebo, one each on Day 1 and Day 30.

Post-diagnostic investigation protocol (PDIP)

For 4 months from the date of diabetes diagnosis, participants will receive one injection of Diamyd® 20 mcg on Day 1 and post-diagnosis continuation, followed by a second injection of Diamyd® on Day 30 in a prime-boost regimen (the total prescribed dose will be 40 mcg Diamyd® for participants who were pre-diagnosed as receiving placebo, and the total prescribed dose will be 80 mcg Diamyd® for participants who were pre-diagnosed as receiving Diamyd®).

Sample size:

Up to 50 children will be invited to participate in DIAPREV-IT 2. It is expected that 50% of children with more than one positive islet cell autoantibody will develop type 1 diabetes within 5 years.

Tests:

Analysis of the obtained data will be continued to address both the safety and effectiveness of treatment.

Example 6

Study Title: Double-blind, randomized, investigator-initiated study to determine the safety and effect of Diamyd® in combination with vitamin D on the progression of type 1 diabetes in children with multiple islet cell autoantibodies.

Active Ingredient Name: Recombinant Human Glutamic Acid Decarboxylase (rhGAD65), Calciferol (Vitamin D3)

Goals:

The primary objective is to evaluate whether Diamyd® in children receiving relatively high doses of vitamin D can delay or halt the autoimmune process leading to clinical type 1 diabetes in children with ongoing persistent beta cell autoimmunity as indicated by multiple positive islet cell autoantibodies.

The secondary objective is to demonstrate that Diamyd® is safe in children at risk of developing type 1 diabetes.

Clinical study plan:

The study is a randomized, double-blind, placebo-controlled, single-center, two-arm clinical trial. Study participants will receive 2 subcutaneous injections of either Diamyd® 20 mcg or placebo in a prime-boost fashion for 30 days. All study participants will receive additional vitamin D at a daily dose of 2,000 international units throughout the study period (regardless of which treatment group they are randomly assigned to). Study participants will be followed for 5 years.

Post-diagnostic investigation protocol (PDIP)

Given the shelf life of the study product, children diagnosed with clinical type 1 diabetes during the study period may be offered to continue the study as part of the post-diagnostic investigation protocol (PDIP) and receive an additional 2 Diamyd® injections, regardless of which treatment group they were randomly assigned to in the prevention portion of the study. All children who are enrolled in the PDIP will be discontinued from the initial prevention study and will undergo a thorough safety and efficacy assessment as per the PDIP or 15 months after the first Diamyd® injection in the PDIP.

Patients' choice:

Patients must be >4.00 years of age, be positive for GADA, and have at least one additional autoantibody associated with type 1 diabetes (IA-2A, ZnT8R/W/QA, or IAA).

Planned number of patients: Approximately 80 patients will be enrolled.

Description of treatment groups:

Patients will be assessed for study eligibility at a screening visit (Visit 0) approximately 30 days prior to their first injection. At Visit 1 (Day 1), eligible patients will be randomly assigned to one of 2 treatment groups:

Approximately 40 patients will receive 2 subcutaneous injections of 20 mcg Diamyd® on Days 1 and 30, i.e. 1 primary and 1 booster dose.

Approximately 40 patients are given 2 subcutaneous injections of placebo, one each on Day 1 and Day 30.

Patients in both treatment groups were additionally prescribed vitamin D3 at a daily dose of 2000 international units for the entire study period of 5 years.

Post-diagnostic investigation protocol (PDIP)

For 4 months from the date of diagnosis of clinical type 1 diabetes, participants receive one injection of Diamyd® 20 mcg on Day 1 in the PDIP, followed by a second injection of Diamyd® on Day 30 in a prime-boost regimen, regardless of which treatment group they were randomly assigned to in the prevention portion of the study (before diagnosis).

End points:

Primary endpoint:

Proportion of patients diagnosed with clinical type 1 diabetes mellitus treated in the Diamyd® group compared with the placebo group five years after the first injection.

Secondary endpoints:

To assess safety and change in metabolic status from normal to impaired glucose metabolism in a group of children with normal glucose metabolism at baseline, and progression of metabolic status in children with impaired glucose metabolism at baseline screening, in non-diabetic children with multiple islet cell autoantibodies treated with Diamyd ^® compared with placebo.

Variables for safety assessment:

Injection site reactions

Occurrence of adverse events (AEs)

Laboratory tests (biochemistry and hematology, including complete blood count (CBC)), including serum calcium and vitamin D3

Urine analysis

Physical examination, including neurological assessments

Epitope-specific GADA titer, isotypes and subtypes, and anti-idiotypic autoantibodies to GADA

Metabolic status:

A transition from normal to abnormal glucose metabolism, defined as any of the following:

a) F-glucose ≥ 6.1 mmol/l

b) Maximum p-glucose after 30, 60, 90 minutes ≥ 11.1 mmol/l in OGTT

c) 120 min p-glucose ≥ 7.8 mmol/l in OGTT

d) HbA1c ≥ 39 mmol/mol

Impaired glucose metabolism should be confirmed at the second visit. This endpoint will be used in the group of children with normal glucose metabolism at initial screening.

Progression of impaired glucose metabolism from one or more of the above variables to additional evidence of impaired glucose metabolism confirmed at visit 2. This endpoint will be used in the group of children with impaired glucose metabolism at baseline.

Exploratory endpoints:

Proportion of patients diagnosed with clinical type 1 diabetes mellitus after 1, 2, 3 and 4 years of follow-up.

Time from baseline visit to diagnosis of clinical type 1 diabetes.

Change from baseline in the following key metabolic variables at different time points: HbA1c, first phase insulin response and K-value from IvGTT, AUC of p-glucose and C-peptide after OGTT, 120 min glucose and C-peptide after OGTT, C-peptide insulin and fasting glucose.

Change in other metabolic variables from baseline: AUC of C-peptide, glucose and insulin from IvGTT, AUC of insulin from OGTT, change in maximum p-glucose from OGTT.

Sample size:

Up to 80 children will be recruited for DIAPREV-IT 2. It is expected that 50% of untreated children with multiple autoantibodies will develop type 1 diabetes within 5 years. This rate has previously been reported as being observed in relatives of people with diabetes in the general population. If 20% of treated children develop type 1 diabetes in the same time period, this would give a power of 82% with alpha = 5% in a group of 40 + 40 = 80 children. A P value of < 0.05 will be used as the significance level.

Tests:

Analyses of the study data will be conducted to determine both the safety and effectiveness of the treatment. A statistical analysis plan (SAP) will be developed prior to statistical analysis.

Analyses will be performed using parametric statistics. If normality criteria for variables are not met (e.g., Kolmogorov-Smirnov test), nonparametric statistics such as Wilcoxon will be used. Four-field tables will be analyzed using Fisher's exact test. Time to diabetes will be analyzed using biological parameter analyses such as Kaplan-Meier distribution function estimation and Cox proportional hazards model. Efficacy analyses will be performed for both the per-protocol (PP) and the intention-to-treat (ITT) set. Last observation carried forward will be used for ITT.

Adverse events and other safety data will be summarized descriptively.

Example 7

Study Title: Phase II, randomized, double-blind, placebo-controlled, multicenter, two-arm study of multiple escalating doses of ^Diamyd® in combination with vitamin D to evaluate safety, immune response, and diabetes status in young adults with newly diagnosed type 1 diabetes.

Name of active ingredients: recombinant human glutamic acid decarboxylase (rhGAD65) and calciferol (vitamin D)

Goals:

The main goal

Safety assessment of multiple escalating doses of ^Diamyd® in combination with vitamin D.

Secondary goals

Evaluation of the effect on the immune system of administration of Diamyd ^® in multiple increasing doses in combination with vitamin D.

Comparison of diabetes status parameters for Diamyd and placebo before and after administration of Diamyd ^® with several increasing doses in combination with vitamin D.

Clinical study plan:

The study is a randomized, double-blind, placebo-controlled, multicenter, two-arm clinical trial. Eligible patients receive vitamin D orally once daily for 1 month prior to the first Diamyd ^® injection. Beginning at Month 1 (baseline), all patients receive a maintenance dose of vitamin D for an additional 5 months.

Weekly subcutaneous injections of Diamyd ^® (or placebo) in escalating doses will be administered beginning at Month 1 (baseline) for 13 weeks (dose escalation), followed by the maximum dose at 2, 4, and 8 week intervals (beginning at Week 15 through Week 27) (maintenance dose). The maintenance dose will then be administered every 8 to 12 weeks for 1 year. Patients will be assessed for safety and immunologic parameters at Month 6, followed by diabetes status variables at Month 15 and Month 30. The total study duration is 30 months.

Subject selection: Patients must be 18 to 30 years of age and have a diagnosis of type 1 diabetes mellitus (T1D) within the previous 6 months at the time of screening. Patients will be eligible for enrollment if they have a fasting C-peptide level ≥0.12 nanomol/L (0.36 ng/mL) and have elevated GAD65 antibody levels.

Planned number of patients: Approximately 40 patients will be enrolled.

Description of treatment groups:

Patients will be assessed for study eligibility at a screening visit (Days -14-28) 2 to 4 weeks prior to randomization. All eligible patients will be given 7,000 IU of vitamin D orally once daily for 1 month from Day 1 to maintain vitamin D levels >70 nM/L until the first Diamyd ^® /placebo injection. Beginning at Month 1 (baseline), all patients will receive an additional 2,000 IU of vitamin D daily for an additional 5 months.

On Day 1, eligible patients are randomly assigned to one of 2 treatment groups:

Approximately 20 patients will be assigned to receive subcutaneous injections of Diamyd ^® at escalating doses starting at baseline (Month 1) as follows: 0.4; 0.8; 2; 3.2; 4; 6.4; 8; 12; 16; 20; 24, 32, 40 mcg Diamyd ^® weekly, where 40 mcg Diamyd ^® will then be administered from Week 15 to Week 27 at 2, 4, and 8 week intervals. 40 mcg Diamyd ^® will then be administered every 8 to 12 weeks for 1 year. Vitamin D will be administered as described above (starting at Day 1).

Approximately 20 patients will be assigned to receive subcutaneous placebo injections with the same timing schedule as above. Vitamin D will be administered as described above (without placebo for vitamin D).

Patients will be followed for three subsequent visits at Month 6, Month 15, and Month 30.

End points:

Primary endpoint:

Evaluation of the safety of multiple escalating doses of ^Diamyd® in combination with vitamin D treatment after 6, 15 and 30 months.

Variables for safety assessment:

Injection site reactions

Occurrence of adverse events (AEs)

Laboratory measurements (biochemistry and hematology)

Urine analysis (microalbuminuria, creatinine)

Physical examination, including neurological assessment

Titer GAD65AB (GADA)

Secondary endpoints:

To evaluate the effects of immune system and diabetes variables between Diamyd and placebo before and after administration of multiple increasing doses of Diamyd® in combination with vitamin D treatment at Months 15 and 30.

Variables for assessing the impact on the immune system:

Inflammatory marker (TNF-alpha, IL-1 beta, IL-2, IL-17)

Th2-biased immune cellular response (observed as an increase in the ratio of IL-5,10,13, compared to IFN-gamma, TNF-alpha, IL-1 beta and IL-17)

Increase in T-regulatory cells

Variables for assessing the influence of endogenous insulin secretion:

Hemoglobin A1c (HbA1c), change between baseline and follow-up visits.

Exogenous insulin dose per kg body weight and 24 h, change between baseline and follow-up visits.

Number of patients who self-reported hypoglycemia.

Fasting C-peptide, change between baseline and follow-up visits.

AUC _mean C-peptide at 0-120 min during MMTT, change between baseline and follow-up visits.

Measurement of C-peptide at 30, 60, 90 and 120 min during MMTT

Maximum C-peptide level during MMTT, change between baseline and follow-up visits.

Proportion of patients with stimulated maximum C-peptide level exceeding 0.2 nmol/L.

Sample size:

No real sample size calculations will be made, as this study is only needed to see if the various treatments are safe and do not adversely affect beta cell function and/or the immune system. Adverse events and other safety data will be summarized narratively. Immunologic parameters and diabetes status variables will be summarized narratively.

Claims (12)

1. A method for the prevention and/or treatment of autoimmune diabetes, comprising administering to a patient a composition, wherein said composition contains at least one beta-cell autoantigen, which is insulinoma antigen-2, by injection directly into a lymph node. 2. The method according to claim 1, wherein the beta cell autoantigen is administered in an amount of 1-15 μg, more preferably in the range of 2-10 μg, and most preferably in the range of 2-5 μg per injection. 3. The method according to claim 1 or 2, comprising administering a composition comprising a beta cell autoantigen at least 2 times, more preferably at least 3 times, and most preferably at least 4 times, wherein each administration is carried out at least 14 days apart, more preferably at least 30 days apart. 4. The method according to any one of claims 1-3, further comprising administering to the patient a cyclooxygenase inhibitor. 5. The method according to claim 4, wherein the cyclooxygenase inhibitor is selected from the group consisting of ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, acetylsalicylic acid, diflunisal (dolobid), salicylic acid, salsalate (disalcid), piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib and nimesulide. 6. The method of any one of claims 1-5, further comprising administering to the patient a CTLA4 compound, such as abatacept. 7. The method according to any one of claims 1-6, further comprising administering to the patient a TNF-alpha inhibitor. 8. The method according to claim 7, wherein the TNF-alpha inhibitor is selected from the group consisting of adalimumab, sertolizumab, etanercept, golimumab and infliximab. 9. The method according to any one of claims 1-8, further comprising administering to the patient vitamin D, vitamin D analogs, tyrosine kinase inhibitors, gamma-aminobutyric acid or a gamma-aminobutyric acid analog and/or exposing the patient to UVB radiation. 10. The method according to claim 9, wherein the administration of vitamin D and/or vitamin D analogues and/or exposure to UVB radiation is carried out for 7 to 90 days prior to the administration to the patient of a composition containing at least one beta cell autoantigen. 11. The method according to claim 9 or 10, wherein the method comprises administering vitamin D and/or vitamin D analogues in an amount of 7000-70000 international units per week for 3-48 months. 12. The method according to any one of claims 1-11, wherein the autoimmune diabetes is type 1 diabetes.