EA043223B1 - GENOTYPE STRATIFICATION IN THE TREATMENT AND PREVENTION OF DIABETES - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to the field of medical treatment, and in particular, to methods of immunotherapeutic treatment of patients based on the genetic profile of these patients, as well as compounds and compositions for use in such methods.

State of the art

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by immune-mediated destruction of insulin-secreting cells, beta cells, of the pancreas. T1DM often has an early onset, already in childhood.

Production of autoantibodies to beta cells causes destruction of beta cells. The process of destruction takes place over many years and eventually leads to metabolic disorders. These disorders first manifest as impaired glucose tolerance and progression to symptomatic hyperglycemia. Antibodies that have been identified in association with the development of T1DM are antibodies to insulin (IAA), GAD65 (GADA, including truncated GADA or tGADA), IA2 (IA-2A), and ZnT8 (ZnT8A).

It has been suggested that administration of glutamic acid decarboxylase formulated with alum may preserve beta cell function in patients with recent onset T1DM (Ludvigsson et al. N Engl J Med. 2012 Feb 2;366(5):433- 42).

Insulin has also been used as an antigenic component in immunotherapy for T1DM (Ali et al, Sci Transl Med. 2017 Aug 9;9(402)).

The 6-year incidence of diabetes-associated autoantibodies in children at genetic risk is described in Krischer et al. (Diabetologia. 2015 May;58(5):980-7).

It is known in the art, inter alia from EP 1755631 and EP 3151853, immunomodulation with a therapeutic drug for the treatment of diabetes and the prevention of autoimmune diabetes.

The essence of the invention

The number and type of antibodies are predictive of the progression of diabetes.

Patients with different genotypes have different sets and production of antibodies leading to the destruction of beta cells. This, in turn, will lead to a difference in the progression of the disease, on the basis of which the stimulation of antibodies in the patient occurs.

By identifying the genotype and measuring and quantifying anti-GAD65 and insulin antibodies at the same time, a personalized vaccine can be obtained to prevent or treat T1DM.

Thus, in one aspect, the invention relates to a method for treating or preventing an autoimmune disease in a human, comprising determining a human HLA haplotype; and exposing a person to a treatment regimen based on said haplotype.

In one embodiment, the method includes determining a human HLA haplotype, determining the specificity of initially occurring autoantibodies associated with an autoimmune disease in a human; and exposing the individual to a treatment regimen based on said haplotype and specificity of said initially emerging autoantibodies.

In one embodiment, the disease is autoimmune diabetes, such as type 1 diabetes.

In one embodiment, a human having the HLA DR3-DQ2 haplotype and who optionally develops GADA first is treated with at least the GAD autoantigen.

In one embodiment, a human having an HLA DR3/4-DQ2/8 genotype is treated with both the GAD autoantigen and the insulin autoantigen in single or separate formulations.

In one embodiment, a human having the DR4-DQ8 haplotype but not the DR3-DQ2 haplotype, and who optionally develops insulin autoantibodies first, is treated with at least the insulin autoantigen.

In one embodiment, a human having haplotype DR4-DQ8 but not DR3-DQ2 is treated with alum alone.

In one embodiment, a human having an HLA haplogenotype of DR8/4-DQ4/8 and who optionally develops insulin autoantibodies first is treated with at least the insulin autoantigen.

In one embodiment, a person having the DQ2 genotype and who first develops anti-GAD antibodies is treated with a GAD antigen.

In one embodiment, a human having the HLA-DR4/4-DQ8/8 genotype is treated with the antigen insulin.

In one embodiment, a human having the HLA-DR8/4-DQ4/8 genotype is treated with the antigen insulin.

In one embodiment, a human having the HLA-DR3/3-DQ2/2 genotype is treated with a GAD antigen.

In one embodiment, a human having the HLA-DR3/4-DQ2/8 genotype is subjected to

- 1 043223 antigen GAD.

In one embodiment, administration of the GAD antigen is via one of the subcutaneous, intradermal, or intralymphatic routes.

In one embodiment, the GAD antigen is formulated with alum.

In one embodiment, administration of the insulin autoantigen is by oral, sublingual, intramuscular, intradermal, or intralymphatic routes.

In one embodiment, the insulin antigen is prepared as a formulation with alum or as saline.

In one embodiment, administration of alum alone is by subcutaneous, intradermal, or intralymphatic route.

In one aspect, the invention relates to a GAD for use as an autoantigen in the method as described above.

In one aspect, the invention relates to insulin for use as a self-antigen in the method as described above.

In one aspect, the invention relates to the use of GAD in the preparation of a pharmaceutical composition for use in the method as defined above.

In one aspect, the invention relates to the use of insulin in the preparation of a pharmaceutical composition for use in the method as defined above.

Definitions

All terms within the scope of the present invention are intended to have the meaning given to them by one of ordinary skill in the art, in the context of the disclosure, to avoid confusion, several terms are specifically defined below.

The term alum refers to aluminum hydroxide, which is widely used as an adjuvant in pharmaceutical compositions for use in immunotherapy. Alum only refers to a composition containing alum but not an antigen.

The term GAD refers to the glutamic acid decarboxylase protein and includes GAD isoforms such as GAD38, GAD65 and GAD67 as well as fragments thereof.

The term insulin when used for insulin as a self-antigen should be construed to include preproinsulin, proinsulin, as well as fragments thereof.

The term T1D includes all types of diabetes where autoantibodies to beta-cell autoantigens are present, such as type 1 diabetes mellitus (T1DM), type 1.5 diabetes, LADA, LADY, SPIDDM, PIDM, and others.

Terms in the singular form are to be construed as including both the singular and the plural.

All references in this document are expressly and in their entirety incorporated by reference.

Detailed description

The etiology of T1DM has not yet been elucidated. However, autoantibodies against insulin (IAA), GAD65 (GADA), IA-2 (IA-2A), or ZnT8 (ZnT8A) have been noted in pathogenesis.

Recent studies have found that there are two large groups of children who develop autoantibodies to islet cells; one in which IAA is present as the first autoantibody, and this group often includes young children with HLA genotypes containing the DR4-DQ8 haplotype, and the other group is often several older children who first develop GADA or tGADA, often DR3- DQ2 haplotype 2 prompted the present inventors to test the efficacy of GAD-alum versus placebo in these HLA groups (see Example 2).

The HLA (human leukocyte antigen) family of genes provides a group of proteins known as the HLA complex. The HLA complex helps the immune system distinguish self from non-self. Within the HLA complex, there are three main groups, MHC (major histocompatibility complex) class I, MHC class II, and MHC class III. Class II MHC genes include HLA-D genes.

An additional TEDDY (Environmental Determinants of Diabetes in Young Adults) study was conducted to identify genetic and environmental factors that explain the initiation of the first autoantibody against islet cells.

The study examined the development of autoimmunity against islet cells and T1DM. The study included children - from newborns to children aged 15 years with a genetic predisposition to T1DM.

Participants made clinical visits every 3 months. At each visit, blood was analyzed for GADA, IAA, IA-2A, ZnT8A, DNA, mRNA, infectious agents, HbA1c, PBMC, erythrocytes, plasma/serum at storage. The analysis was also performed on urine samples, nasal swabs, tap water, nail clippings, and salivary cortisol. Fecal samples were collected once a month for the first 48 months and then quarterly.

In addition to the analyzes mentioned above, surveys were conducted regarding maternal diet during pregnancy (FFQ of individual foods), smoking; negative life events, parental anxiety, depression, reported infections, drugs, immunizations, family

- 2 043223 anamnesis, DNA of first level relatives (FDR), assessment of physical activity. We also conducted a re-recruitment of retired people.

The aim was to investigate prenatal factors that may influence the risk of HLA-dependent autoantibodies. Non-diabetic mothers of singletons (n=6.947) completed the questionnaire 3-4.5 months after pregnancy. Lower birth weight was defined as being below 25% of the birth weight of TEDDY children.

Maternal factors such as smoking, BMI, alcohol use during the 3rd trimester (>2 times/month), and maternal infections (lower respiratory tract infection, skin infection or rash, genital infection). After adjusting for country, FDR T1DM, HLA genotypes, and sex, none of these maternal factors were associated with the first anti-islet autoantibody.

Among FDR children, IAA alone as the first autoantibody was more common than GADA alone at an early age, but not at an older age.

Compared with children with the HLA-DR3/4 genotype, children with the HLA-DR3/3 genotype showed a reduced risk of developing IAA alone as the first anti-islet autoantibody.

Among children with HLA-DR4/4 and HLA-DR4/8 genotypes, there was a reduced risk of GADA alone as the first anti-islet autoantibody.

Among girls, there was a reduced risk of only IAA as the first anti-islet autoantibody, but not only GADA as the first anti-islet autoantibody.

Among children born with lower birth weight, there was a reduced risk of GADA alone as the first anti-islet autoantibody.

The occurrence of IAA alone was more common in boys and is associated with HLA-DR4/4-DQ8/8 and HLA-DR8/4-DQ4/8.

Occurrence of GADA alone was not associated with sex, but is associated with HLA-DR3/3-DQ2/2 and HLA-DR3/4DQ2/8, and is less common in low birth weight infants.

It can be concluded that only GADA as the first anti-islet cell autoantibody occurs primarily in people with the HLA-DQ2 haplotype. Maternal factors affecting birth weight may influence the emergence of GADA as the first autoantibody.

Humans with the HLA-DR4/4-DQ8/8 and HLA-DR8/4-DQ4/8 genotypes are usually found to have IAA as the first anti-islet autoantibody first.

Humans with the HLA-DR3/3-DQ2/2 and HLA-DR3/4-DQ2/8 genotypes are usually found to have GADA as the first anti-islet autoantibody first.

Surprisingly, the present inventors were able to show that administration of the GAD T1DM autoantigen to patients with an HLA haplotype associated with GADA as the first autoantibody can be successfully used in T1DM treatment/prevention regimens. Thus, the decision on the antigen to be used can be made based on the patient's genotype.

In addition, it can be shown that alum alone can delay the decline of stimulated Speptide in patients with the DR4-DQ8 haplotype but not the DR3-DQ2 haplotype.

According to the present invention, the autoantigen can be administered by intralymphatic injection, injection directly into the lymph node, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, intranasal, transmucosal or sublingual application; or orally, including administration as tablets, pellets, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays, or liquid reconstituted dry powder forms.

In one embodiment of this invention, administration of the self-antigen is carried out directly into the lymph nodes or into the lymphatic system, allowing the resident APCs to present the antigenic peptides to the immune system. If administration of self-antigens is administered directly to a lymph node or to the lymphatic system, the dose should preferably be between 1 and 15 μg per self-antigen, more preferably between 2 and 10 μg per self-antigen, or 2-5 μg per self-antigen. Preferably the finished form is in the form of alum.

In some embodiments, at least one self-antigen is administered to the intragroin, lymph node region, or intralymphatic route. In some embodiments, the volume for intra-inguinal injection of antigens is between 0.05 and 0.2 ml, more preferably between 0.05 and 0.15 ml.

In some embodiments, where at least one antigen is administered to the region of the lymph node via or intralymphatic injection, the preferred dosage is between 1-15 micrograms, more preferably between 2-10, and most preferably between 2-5 micrograms per injection and autoantigen, and such introduction occurs 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, more preferably with an interval of at least 30 days.

In some embodiments, at least one antigen and at least one IL-10 inducing compound are administered simultaneously. According to some options

At least one antigen and at least one IL-10 inducing compound are administered separately.

The method may further include pre-treatment of the individual to control serum vitamin D levels, and such pre-treatment may include administering to said patient vitamin D and/or vitamin D analogs and/or exposure to ultraviolet B light, preferably for 7 -90 days prior to administration of a composition containing at least one beta cell autoantigen as described in EP 3151853.

Examples

The examples below are aimed at further elucidating the invention. The examples are not to be construed as limiting the scope of the invention, which is defined in the appended claims.

Example 1. II Phase TrialNet.

Wherrett DK et al. (Lancet, 2011 Jul 23;378(9788):319-27) reported in three groups (A: 3 scx20 µg GAD alum (n=48); B: 2x20 µg GAD alum and 1 alum (n=49) and C: 3x alum (n=48), a randomized trial in patients with diagnosed T1D within 100 days and aged 3-45 years, which at 1 year 2-h C-peptide AUC adjusted for age, sex, and C value -peptide baseline was 0.412 nmol/l (0.349-0.478) in the GAD-alum group, B 0.382 nmol/l (0.322-0.446) in the GAD-alum plus alum group, and C 0.413 nmol/l (0.351-0.477) in the alum, corresponding to a one-year mean C-peptide loss of 44%, 42%, and 41% of the mean baseline for GAD-alum x3, GAD-alum x2, and alum x3, respectively. α-peptide baseline and one year later reflected the control group results of three other TrialNet studies in patients treated within 3 months of diagnosis, which also demonstrate that alum alone does not affect the loss of insulin secretion at 12 months.

So the 12-month adjusted decline is 3.7%, 3.5% and 3.42% per month. Unadjusted values for up to 24 months are shown in the table below. In it, groups A, B and C showed a decrease of 58.3%, 62.3%, 55.1% over two years or by 2.42%, 2.6%, 2.3% per month. The alums were marginally better.

Table 1

Average C-peptide AUC over 24 months

Time A1*3 GAD-A1*2 GAD-A1*3 Average time* (months) Average C-peptide Average time* (months) Average C-peptide Average time* (months) Average C-peptide original level -0.83 0.697 -0.736 0.660 -0.75 0.736 3 months 2.87 0.637 2.88 0.561 2.90 0.683 6 months 6.12 0.537 6.03 0.530 5.98 0.545 9 months 9.07 0.477 9.07 0.431 9.00 0.521 12 months 12.0 0.418 12.0 0.350 12.0 0.448 18 months 18.0 0.365 18.1 0.284 18.0 0.334 24 months 24.1 0.313 24.0 0.249 23.8 0.307

However, hypothesizing that GAD as an immunomodulatory antigen is effective in HLA groups in which anti-GAD antibodies are usually found first, and that the insulin antigen family is effective in haplotypes in which anti-insulin antibodies often appear first, the present inventors now turn again to the data. study, and unexpectedly found that GAD-alum was nearly twice as effective as alum alone (change/month 2.35% vs. 4.45%) in the DR3-DQ2 haplotype group, which typically had anti-GAD antibodies first. . In addition, the efficacy of GAD-alum in groups that include DR4-DQ8, which usually has IAA first (although DR3/DR4-DQ2/DQ8 may have either GADA or IAA first) only had a 40% advantage. And of particular interest, only the alum adjuvant (placebo) was more effective than the active drug in the DR4-DQ8 haplotype group, which is reported to include patients who often have IAA first.

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table 2

Change in stimulated C-peptide over 1 year depending on haplotype Quantity Group AL Change Change Steam. participation & DQ 7 with DQ2 3 injections 20 mcg GADA Steam. C-pept. AUC for 1 year (%) -22.03 C-pept. month (%) -1.84 AUC in 7 with DQ2 GAD alum 2 injections 20 mcg GADA -34.33 -2.86 14s DQ2 GAD alum plus alum alone Average GADA -28.18 -2.35 4 with DQ2 active groups 3 injections only GADA -53.37 -4.45 19cDQ8 alum 3 injections 20 mcg ΙΑΑ -42.84 -3.57 13 with DQ8 GAD alum 2 injections 20 mcg ΙΑΑ -37.21 -3.10 32 with DQ8 GAD alum plus alum alone Average ΙΑΑ -40.55 -3.38 16cDQ8 active groups 3 injections only ΙΑΑ -22.48 -1.87 alum Example 2: DiaPrevIt. In a randomized double blind placebo-controlled study in 50 healthy children with

multiple islet autoantibodies but no insulin-dependent diabetes mellitus (DiaPREV-IT, ClinicalTrials.gov Identifier: NCT01122446), found that prophylactic treatment with GADalum had no effect on the progression and onset of clinical T1D.

The study was performed in children aged 4-17.9 years (median age 5.2) with GADA and at least one additional anti-islet autoantibody. Enrollment in the study was completed in 2012, and the follow-up period was 5 years. Eligible children from the Diabetes Prediction in Skane (DiPiS), The Environmental Determinants of Diabetes in the Young (TEDDY) studies received two subcutaneous injections of 20 mcg GAD alum (n=25) or placebo (n=25) 30 days apart . Among the exclusion criteria was a positive reaction to HLA DQB 1*06:02.

Anti-islet cell autoantibodies predict the clinical onset of T1D, and a child with more than one anti-islet cell autoantibody has a 70% risk of developing T1D within 10 years. The sample size of 50 children randomized 1:1 to treatment with either GAD-alum (active) or alum alone (placebo) was based on the assumption that 50% of untreated children with more than one autoantibody would have clinical T1D during period of 5 years. however, it was surprisingly found that although half of the children had impaired baseline glucose metabolism, indicating a very high risk of clinical disease progression, only 18 of 50 (36% ), not 50% as expected. Against the background of the reported overall non-significant results, a study was conducted to assess which, if any, subgroups could explain the lower than expected incidence of severe cases of T1D.

It was found that 12 of the 18/50 children in whom T1D progressed during the 5-year follow-up period (170-1830 days after the first injection of study drug) were girls with a higher rate of progression than boys (p = 0.012). The progression of T1D was not affected by the status of first-degree relatives (p = 0.925), while children with impaired glucose metabolism at baseline, in whom plasma glucose was determined 120 minutes after OGTT > 7.8 and < 11.1 mmol / l, plasma glucose >11.1 mmol/L at 30, 60 and/or 90 minutes after OGTT and/or FPIR <30 in IvGTT had a higher rate of progression than children with normal glucose tolerance (p=0.013).

- 5 043223

Time to clinical diagnosis was independent of treatment in the full group (p=0.573) or in the stratum groups with 2 or 3-6 autoantibodies (p=0.957 and 0.628, respectively). In addition, time to diabetes was not significantly affected by treatment in the group with normal and impaired glucose metabolism (p=0.359 and p=0.376, respectively) or gender (p=0.079 boys, p=0.400 girls, respectively).

Recent studies have shown that there are two large groups of children who develop autoantibodies to islet cells; one in which IAA is present as the first autoantibody, and this group often includes young children with HLA genotypes, including the DR4-DQ8 haplotype, and another group of often slightly older children who first develop GADA or tGADA, often DR3- DQ2 haplotype 2, prompting the present inventors to test the efficacy of GAD-alum in these HLA groups compared to placebo. In this study, including only people at high risk for clinical T1D HLA, six different HLA genotypes were presented. Of these, three groups are DQ 2/2; DQ 2/X; and DQ X/X included too few patients (n=2, 2, 1) to be included in any analysis. The three remaining HLA groups included DQ 2/8 (n=25), of which 10 patients received GAD-alum and 15 received placebo; DQ 8/X (n= 11) where six patients received GAD alum and five received placebo; and DQ 8/8 (n=9), where five people received GAD alum and 4 received placebo.

Surprisingly, it was found that in 25 patients in the DQ2/8 group, two of 10 patients in the GAD-alum group (20%) developed T1D over a period of five years compared to eight of 15 (53%) people who received placebo (Table 3), indicating that GAD alum is effective in delaying T1D in high-risk individuals with the DQ2/8 haplotype. Similar results are not seen in either the DQ 8/X group or the DQ 8/8 group, where placebo performed better than patients treated with GAD-alum.

Table 3

25 people with positive DQ2/DQ8 # of people who developed insulin-dependent diabetes mellitus in the group GAD-alum (n=10) # of people who developed insulin-dependent sugar diabetes in the placebo group (n=15) time period (year) 1 1 0 2 0 4 3 0 3 4 0 1 5 1 0 1-5 2 (20%) 8 (53%)

Experiment 3. DiagNode-1.

As described in Tavira et al. Journal of Diabetes Research, Volume 2018, Article ID 9391845), an open study was conducted in 12 patients with T1D, where 4mcg of GAD formulated with alum was injected directly into the inguinal lymph node three times with an interval of 30 days between injections. According to the subject matter of this invention, it was found that treatment of patients having the DR3-DQ2 haplotype resulted in better HbA1c- and stimulated C-peptide area under the curve data than patients without the DR3-DQ2 haplotype.

Table 4

Patient ID-# Ale baseline (B) А1s% In 15 months. group risk Haplogenot sp Stimulated C-peptide baseline (AUC-B) AUC% AUC B after 15 months 1 52 78.8 Neutral DR3- DQ2/DR11- DQ7 0.364 107.56 2 58 93.1 taller DR4/4- 0.853 79.77

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other DQ8/8 3 66 78.8 Higher different DR4/10- DQ8/5.1 0.426 82.11 4 68 60.3 Reduced DR11/13- DQ7v/6.3 0.744 108.40 5 103 43.8 Higher different DR3/4- DQ2/7 0.415 85.84 6 78 56.4 Higher different DR3/10- DQ2/5.1 0.375 71.67 7 41 90.2 high DR3/4- DQ2/8 8 37 124.3 Neutral DR4/10- DQ7/5.1 0.518 104.83 9 66 60.6 high DR3/4- DQ2/8 0.518 76.57 10 54 94.4 Higher different DR4/4- DQ8/8 0.356 62.11 eleven 37 105.4 Higher different DR3/13- DQ2/6.4 0.853 61.73 12 50 98 Higher different DR4/10- DQ8/5.1 0.69 54.38

Table 5

DR Group Pat ID Alc% B after 15 months AUC% AUCB at 15 months DR3s-pat #11 1+5+6+7+9 65.964 85.41 DR3s 1+5+6+7+9+11 72.53 80.67 DR4s 2+3+8+10+12 97.73 76.63

CLAIM

Claims (6)

1. A method for the treatment or prevention of autoimmune diabetes in humans, including determining the human HLA haplotype; and exposing a human having the HLA DR3-DQ2 haplotype and presenting GAD A before treatment to treatment with the GAD autoantigen formulated with alum. 2. The method of claim 1 wherein the autoimmune diabetes is selected from type 1 diabetes mellitus, type 1.5 diabetes, LADA, LADY, SPIDDM and PIDM. 3. The method of claim 1 or 2, wherein the human having the HLA DR3/4-DQ2/8 genotype is treated with both the GAD autoantigen and the insulin autoantigen in the same or separate formulations. 4. The method according to claim 1 or 2, wherein the human having the HLA-DR3/3-DQ2/2 genotype is treated with the GAD autoantigen. 5. The method according to any one of paragraphs. 1-4, in which the administration of the GAD antigen is carried out by one of the subcutaneous, intradermal or intralymphatic methods. 6. The method of any one of claims 1-5, wherein the GAD autoantigen is selected from the group consisting of GAD38, GAD65 and GAD67.