**4. Antigen specific therapies**

#### **4.1. Insulin**

Targeting of B-cells is achieved by an antibody against the CD20 molecule, a cell surface phosphoprotein that is expressed during the mid-stages of B-cell development but which does not occur on hematopoietic stem cells or normal plasma cells [11]. Anti CD20 antibody is approved for the treatment of B-cell lymphomas and was used in a study of patients with recent onset of T1D (median time interval between diagnosis of T1D and first infusion 81 days). Four consecutive infusions were given over an interval of 22 days. The results of this trial assessed 12 month after study begin resemble those obtained by anti CD3 treatment- slower decrease of C-peptide levels, lower levels of glycated hemoglobin and lower requirement for exogenous insulin in the treated vs. the placebo group. Although not statistically significant, subgroup analysis again tended to suggest a better response in children and adolescents. Side effects included fever, rash and pruritus as consequence of the 'cytokine storm' (or cytokine release syndrome) triggered by the first injection of the antibody. Again these effects were transient and did not reappear when subsequent doses of the anti CD20 antibody were

It is not known whether the effects of T- or B-cell targeting with antibodies can be prolonged or increased if the treatment is given repeatedly or if T-and B-cell targeting are combined. The guess here is that an increased risk of adverse side effects might counterbalance positive effects gained by repeated or combined administration of T or B-cell depleting antibodies and/or that repeated administration become less efficient because the immune system activates counter‐

**3.1. Autologous nonmyeloablative stem cell transplantation and treatment with cyclosporin**

Before reviewing antigen specific immunotherapies two studies with very broad targeting of the immune system shall be mentioned. In one study peripheral hematopoietic stem cells of patients with recent onset of T1D were mobilized, harvested and frozen before immune ablation was achieved by administration of high dose cyclophosphamide and anti thymocyte globulin. The previously harvested hematopoietic stem cells were then infused. During the time needed for the immune system to regenerate extensive supportive care including antibacterial, antiviral and antifungal prophylaxis as well as patient isolation in rooms equipped with air filters was required. This approach resulted in reversal of T1D in the majority of the patients. 12 of the 23 patients participating in this trial became independent from exogenous insulin and this state lasted for 14 to 53 months while 8 patients relapsed and resumed insulin use at low doses [13]. In the insulin-independent group C-peptide levels at 24 and 36 months post transplantation of stem cells had increased while values of glycated hemoglobin had decreased significantly compared to pre transplantation values. The rationale for this study was the possible reconstitution of immune tolerance after an 'immunologic reset' by high dose immunosuppression followed by autologous hematopoietic stem cell transplan‐ tation [14]. However, it is known from the NOD mouse that the self-reactive tendency of the immune system cannot be eliminated permanently by this approach. Once the immune system

administered [12].

520 Type 1 Diabetes

acting mechanisms.

**3. General immunosuppression**

Although it appears from the approaches presented above that the more severe the therapeutic intervention the better its success antigen specific therapies remain attractive conceptually because they allow for an intervention that is more precisely targeted. Rather than targeting all T-cells (or the immune system in its entirety) the idea is to apply an approach that controls only those T-cells that are self-reactive. Here an important question concerns the specificity of the self-reactive T-cells that 'merit' control. It is obvious that insulin is considered a major selfantigen as it is the defining protein of the beta cells that are impaired and destroyed during the pathogenesis of T1D. There are many experimental findings that confirm this view such as the presence of anti-insulin autoantibodies as a proven prediction tool for the assessment of diabetes risk in the pre-clinical state. Furthermore, among the group of beta cell proteins that have been studied to date as potential self-antigens insulin is one of the few that fulfills a formal requirement for a beta cell protein to be considered a self antigen: insulin specific Tcell clones and lines derived from NOD mice can reliably transfer diabetes to NOD-SCID recipients. Another question concerns how these self-reactive T-cells can be targeted and here the mechanisms that mediate oral or nasal tolerance offer a possible approach. Oral or nasal tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen via the oral or nasal route. The mechanisms of oral tolerance are thought to have evolved in order to generate peripheral tolerance to external agents that gain access to the body via a natural route (the digestive or respiratory tract). As consequence these external agents are 'seen' by the immune system as internal components that become part of self. Two different but not mutually exclusive mechanisms have been defined that can mediate oral tolerance, depending on the amount of antigen administered orally: Induction/activation of regulatory T-cells has been reported to occur when low doses are given whereas induction of anergy or deletion of T-cells appears to be the main mechanism involved when higher doses are administered [17]. According to this schematic, if insulin specific self-reactive or autoaggressive T-cells were to be targeted, feeding of insulin would result - upon presentation of insulin by specialized gut-associated antigen presenting cells - in the activation of insulin-specific regulatory T-cells in the gut. These T-cells then migrate to the pancreatic lymph nodes where they encounter epitopes derived from endogenous insulin and become reactivated. This leads to the secretion of IL-10 and TGFβcytokines, which can attenuate the ongoing inflammatory process. Because it is mediated by cytokines this mechanism would not only target insulin reactive T-cells but would suppress T-cells with other specificities as well. A degree of specificity would be generated because both types of T-cells -autoagggressive and regulatory- would become activated in the same location (pancreatic lymph nodes or islet infiltrates) but not in other sites. This is one reason why antigen specific therapies thought to rely on T-regulatory cells might be better applied before the onset of T1D. Once islets have been destroyed the pancreatic lymph nodes can no longer activate T regulatory cells because beta cell antigens are no longer presented.

Oral administration of insulin has been tested as intervention in patients with recent onset of T1D [18] and oral as well as nasal insulin have been given to persons at risk of developing T1D in order to assess the potential of this approach to prevent or delay the onset of the disease. The 'Diabetes Prevention Trial-Type 1' (DPT1) screened first and second-degree relatives of patients with T1D for the presence of islet cell antibodies. Relatives who had anti islet cell and anti insulin antibodies but a normal glucose tolerance and first phase response to intravenous insulin were projected to have a 5-year risk of 26-50%. 372 of these individuals were random‐ ized in the oral insulin study. (DPT1 also included a group of individuals classified as having a risk of greater than 50% who received intravenous instead of oral insulin [19]). The followup in the oral insulin study was 4.3 years. During this time there appeared no differences between placebo and control groups. The average proportion of subjects who progressed to diabetes was 6.4% per year in the oral insulin group and 8.2% per year in the placebo group. However, upon subgroup analysis there appeared to be a beneficial effect in those individuals who had a higher anti insulin autoantibody titer (≥80nU/ml, n=263). In this group the propor‐ tion who developed diabetes was 6.2% per year in the oral insulin group and 10.4% in the placebo group [20]. This effect became even more pronounced if analysis was confined to those with an anti insulin autoantibody titer of >300nU/ml (n=132] with a projected delay of the disease of almost 10 years [21]. These findings were encouraging but since the subgroup analyses had not been prespecified they could not be considered a positive outcome. Another important result this trial yielded was the confirmation that the parameters used to predict development of T1D in relatives of individuals with the disease were sufficient and accurate. Risk was projected to be 26-50% whereas the actual observed value was 35% over 5 years. Accurate risk prediction is essential for the design of further prevention trials, one of which is currently ongoing and builds on the hypotheses generated by the evaluation of the oral insulin DPT1 trial (better efficiency of the treatment in individuals with higher anti insulin autoanti‐

A second prevention trial used nasal instead of oral insulin and a screening and staging approach different from the DPT1. In this case cord blood samples of infants were tissue typed for the presence of the T1D susceptibility allele HLA-DQB1. Carriers of this allele and an additional cohort consisting of their siblings were repeatedly tested for the presence of T1Dassociated autoantibodies. Individuals of the two cohorts who were positive for two or more autoantibodies but free of clinical diabetes were invited to participate in the prevention trial. Individuals enrolled in this trial were younger than those in the DPT1 (1.6-5.2 years vs. 7-14

cohort were randomized to receive intranasal insulin or placebo with a median duration of the intervention of 1.8 years. This trial failed to demonstrate a positive effect of intranasal insulin in all analyzed groups. The annual rate of progression to diabetes in the HLA-DQB1+

was 16.8% for the group receiving intranasal insulin vs. 15.3% for the placebo group. In the sibling cohort these values were 10.8% vs. 6.0% respectively. In contrast to DPT1 a subgroup analysis of individuals with high anti insulin autoantibody titers did not show any benefit of intranasal administration of insulin. Although this trial failed to demonstrate positive effects of intranasal insulin it showed that by screening for HLA risk alleles a cohort with a disease risk similar to that of first-degree relatives could be identified from the general population [22].

cohort and 40 individuals of the sibling

Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/54717 523

cohort

years in the DPT1). 224 individuals of the HLA-DQB1+

body titers).

Anergy or deletion of insulin reactive T-cells might also be achieved by oral administration of insulin with the latter mechanism potentially leading - through the presence of debris from apoptotic insulin specific T-cells - to the generation of T regulatory cells according to the process discovered to be activated by i.v. administration of anti CD3 antibodies. It should be mentioned in advance, that in the studies administering oral or nasal insulin presented below, parameters that would indicate which, if any, of the proposed mechanism (tolerance/anergy/ activation of T-regulatory cells) had been triggered were not acquired.

Oral administration of insulin has been tested as intervention in patients with recent onset of T1D [18] and oral as well as nasal insulin have been given to persons at risk of developing T1D in order to assess the potential of this approach to prevent or delay the onset of the disease. The 'Diabetes Prevention Trial-Type 1' (DPT1) screened first and second-degree relatives of patients with T1D for the presence of islet cell antibodies. Relatives who had anti islet cell and anti insulin antibodies but a normal glucose tolerance and first phase response to intravenous insulin were projected to have a 5-year risk of 26-50%. 372 of these individuals were random‐ ized in the oral insulin study. (DPT1 also included a group of individuals classified as having a risk of greater than 50% who received intravenous instead of oral insulin [19]). The followup in the oral insulin study was 4.3 years. During this time there appeared no differences between placebo and control groups. The average proportion of subjects who progressed to diabetes was 6.4% per year in the oral insulin group and 8.2% per year in the placebo group. However, upon subgroup analysis there appeared to be a beneficial effect in those individuals who had a higher anti insulin autoantibody titer (≥80nU/ml, n=263). In this group the propor‐ tion who developed diabetes was 6.2% per year in the oral insulin group and 10.4% in the placebo group [20]. This effect became even more pronounced if analysis was confined to those with an anti insulin autoantibody titer of >300nU/ml (n=132] with a projected delay of the disease of almost 10 years [21]. These findings were encouraging but since the subgroup analyses had not been prespecified they could not be considered a positive outcome. Another important result this trial yielded was the confirmation that the parameters used to predict development of T1D in relatives of individuals with the disease were sufficient and accurate. Risk was projected to be 26-50% whereas the actual observed value was 35% over 5 years. Accurate risk prediction is essential for the design of further prevention trials, one of which is currently ongoing and builds on the hypotheses generated by the evaluation of the oral insulin DPT1 trial (better efficiency of the treatment in individuals with higher anti insulin autoanti‐ body titers).

all T-cells (or the immune system in its entirety) the idea is to apply an approach that controls only those T-cells that are self-reactive. Here an important question concerns the specificity of the self-reactive T-cells that 'merit' control. It is obvious that insulin is considered a major selfantigen as it is the defining protein of the beta cells that are impaired and destroyed during the pathogenesis of T1D. There are many experimental findings that confirm this view such as the presence of anti-insulin autoantibodies as a proven prediction tool for the assessment of diabetes risk in the pre-clinical state. Furthermore, among the group of beta cell proteins that have been studied to date as potential self-antigens insulin is one of the few that fulfills a formal requirement for a beta cell protein to be considered a self antigen: insulin specific Tcell clones and lines derived from NOD mice can reliably transfer diabetes to NOD-SCID recipients. Another question concerns how these self-reactive T-cells can be targeted and here the mechanisms that mediate oral or nasal tolerance offer a possible approach. Oral or nasal tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen via the oral or nasal route. The mechanisms of oral tolerance are thought to have evolved in order to generate peripheral tolerance to external agents that gain access to the body via a natural route (the digestive or respiratory tract). As consequence these external agents are 'seen' by the immune system as internal components that become part of self. Two different but not mutually exclusive mechanisms have been defined that can mediate oral tolerance, depending on the amount of antigen administered orally: Induction/activation of regulatory T-cells has been reported to occur when low doses are given whereas induction of anergy or deletion of T-cells appears to be the main mechanism involved when higher doses are administered [17]. According to this schematic, if insulin specific self-reactive or autoaggressive T-cells were to be targeted, feeding of insulin would result - upon presentation of insulin by specialized gut-associated antigen presenting cells - in the activation of insulin-specific regulatory T-cells in the gut. These T-cells then migrate to the pancreatic lymph nodes where they encounter epitopes derived from endogenous insulin and become reactivated. This leads to the secretion of IL-10 and TGFβcytokines, which can attenuate the ongoing inflammatory process. Because it is mediated by cytokines this mechanism would not only target insulin reactive T-cells but would suppress T-cells with other specificities as well. A degree of specificity would be generated because both types of T-cells -autoagggressive and regulatory- would become activated in the same location (pancreatic lymph nodes or islet infiltrates) but not in other sites. This is one reason why antigen specific therapies thought to rely on T-regulatory cells might be better applied before the onset of T1D. Once islets have been destroyed the pancreatic lymph nodes can no longer

522 Type 1 Diabetes

activate T regulatory cells because beta cell antigens are no longer presented.

activation of T-regulatory cells) had been triggered were not acquired.

Anergy or deletion of insulin reactive T-cells might also be achieved by oral administration of insulin with the latter mechanism potentially leading - through the presence of debris from apoptotic insulin specific T-cells - to the generation of T regulatory cells according to the process discovered to be activated by i.v. administration of anti CD3 antibodies. It should be mentioned in advance, that in the studies administering oral or nasal insulin presented below, parameters that would indicate which, if any, of the proposed mechanism (tolerance/anergy/

A second prevention trial used nasal instead of oral insulin and a screening and staging approach different from the DPT1. In this case cord blood samples of infants were tissue typed for the presence of the T1D susceptibility allele HLA-DQB1. Carriers of this allele and an additional cohort consisting of their siblings were repeatedly tested for the presence of T1Dassociated autoantibodies. Individuals of the two cohorts who were positive for two or more autoantibodies but free of clinical diabetes were invited to participate in the prevention trial. Individuals enrolled in this trial were younger than those in the DPT1 (1.6-5.2 years vs. 7-14 years in the DPT1). 224 individuals of the HLA-DQB1+ cohort and 40 individuals of the sibling cohort were randomized to receive intranasal insulin or placebo with a median duration of the intervention of 1.8 years. This trial failed to demonstrate a positive effect of intranasal insulin in all analyzed groups. The annual rate of progression to diabetes in the HLA-DQB1+ cohort was 16.8% for the group receiving intranasal insulin vs. 15.3% for the placebo group. In the sibling cohort these values were 10.8% vs. 6.0% respectively. In contrast to DPT1 a subgroup analysis of individuals with high anti insulin autoantibody titers did not show any benefit of intranasal administration of insulin. Although this trial failed to demonstrate positive effects of intranasal insulin it showed that by screening for HLA risk alleles a cohort with a disease risk similar to that of first-degree relatives could be identified from the general population [22]. Thus even if these two trials largely failed in their primary aim they nevertheless clearly demonstrated the ability to accurately predict disease risk, which is essential to optimize the timing of preventative therapies.

**4.3. 60kDa heat shock protein (DiaPep277)**

A third antigen tested for its therapeutic value in human T1D, is the 60kDa heat shock protein (hsp60). While GAD and especially insulin are specifically expressed in pancreatic islets this is not the case for hsp60, which is expressed throughout the body. Although anti hsp60 autoantibodies can be detected in patients at the onset of T1D they are not useful as predictive markers for disease beyond what can be achieved by measuring anti insulin or anti GAD titers. If hsp60 is an autoantigen in T1D and is widely expressed throughout the body one would expect to find inflammation driven by hsp60-reactive T-cells in other organs as well. However this is not the case and raises the question as to whether there are beta cell/islet intrinsic factors

Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/54717 525

In therapeutic applications hsp60 is not given as a whole protein but as a peptide derived from the native sequence of human heat shock protein 60. The sequence of this peptide was first identified in the NOD mouse with the help of diabetogenic T-cell clones responding to the *M. tuberculosis* hsp60. Heat shock proteins are highly conserved proteins and it was discovered that these T- cell clones cross-reacted with the human - and presumably with the mouse form - of hsp60 and specifically recognized an epitope in the C-terminal part of hsp60, which was termed peptide277. Vaccination of NOD mice with peptide277 in mineral oil delayed T1D [26]. Since the sequence of this peptide contained two cysteine residues a more stable form was subsequently generated in which the cysteine residues were replaced by valine. The more stable form of peptide277 was also effective in delaying T1D in NOD mice and was termed DiaPep277 [27]. These studies suggested that the mechanisms mediating the effects of vaccination with DiaPep277 might be similar to the ones proposed for vaccination with GAD (e.g. induction of T regulatory cells). It has become evident however that DiaPep277 (and hsp60) can also exert direct effects on the immune system. Hsp60 can activate B-cells via the Toll like receptor 4 (TLR4), which respond by producing IL-10 [28]. Furthermore, TLR4 activation by hsp60 also occurs in macrophages and dendritic cells promoting pro-inflamma‐ tory effectors. At the same time hsp60 can also induce anti-inflammatory effects promoted through TLR2. It is reported DiaPep277 does not engage TLR 4 but only TLR2, which leads to

that set this site apart immunologically from other parts of the body.

the generation of a T-cell mediated anti-inflammatory environment [29].

Several phase II trials have been conducted with DiaPep277 in patients with T1D. In one of these trials that focused on the changes in immunological parameters after treatment, Dia‐ Pep277 was administered subcutaneously in a 10% lipid preparation with the placebo group receiving mannitol in 10% lipid preparation. Three different doses of DiaPep277 were tested (0.2mg, 1mg and 2.5mg). Four injections of the drug or the placebo were given over a timeframe of 12 months and a total of 48 patients were enrolled with onset of T1D between 200 and 800 days before start of treatment. Glucagon-stimulated C-peptide production significantly decreased over 12 months in all groups except the group receiving Diapep277 at 2.5mg. The decrease in C-peptide production over 12 months was significantly less in the 2.5mg than in the placebo group. Absolute daily insulin dosage did not decrease over time in any of the groups [30]. These results are in accordance with an earlier trial that found a significantly higher stimulated C-peptide concentration in the treatment vs. placebo group at 6 and 10 months after start of treatment. This earlier trial also found a significantly reduced insulin

#### **4.2. Glutamic acid decarboxylase**

Besides oral or nasal administration of an autoantigen there is in T1D models another approach to induce tolerance. In this case a candidate autoantigen is injected subcutaneously together with the adjuvant alum. Although using an islet autoantigen as a vaccine to prevent or ameliorate disease might appear strange it has nevertheless been shown in the mouse model of T1D that this approach can be effective. The idea is that such a vaccination either activates regulatory T-cells or that it converts autoaggressive T-cells to a non-destructive phenotype. This approach has been tested in humans with GAD65, which is the 65 kd isoform of the autoantigen glutamic acid decarboxylase. In contrast to the prevention studies with oral and nasal insulin the trials with GAD65 have been conducted in individuals with recent onset of T1D. A phase II trial tested the safety and efficacy of vaccination with human GAD65 in alum (two subcutaneous vaccinations with 20μg GAD one month apart) in patients with recent onset of T1D (n=70). Results of this study were reported 30 months and again 4 years after treatment. Of the subgroups prespecified in the protocol (HLA classification, age, sex, baseline GAD autoantibody levels) only duration of T1D had a significant influence on the efficacy of the vaccination. In the patients vaccinated less than 6 month after diagnosis of T1D both fasting and stimulated C-peptide secretion decreased significantly less in the GAD-alum group than in the placebo group by month 30 and this positive effect was retained at 4 years after treatment. There was no significant difference between the GAD-alum and the placebo group for patients treated 6 month or more after diagnosis. As expected, vaccination with GAD-alum lead to strong increase of the GAD autoantibody titers, which was sustained to month 30 and a neurological assessment was performed because of concerns that this might lead to stiff-man syndrome. However, there were no notable neurological differences between treatment and placebo group. In accordance with the B-cell responses anti GAD cytokine responses assessed in PBMCs of treatment and control groups at 15 months showed a significantly increased release of most of the tested cytokines (IL-5, 10, 13, 17, IFN-γ and TNFα) in the GAD-alum group. Furthermore increased GAD-induced levels of FOXP3, a transcription factor associated with T regulatory cells, was found in the GAD-alum group [23] [24]. Given the findings of this trial a second study (phase III) was conducted, which enrolled patients within 3 month of the diagnosis of T1D. Patients were randomly assigned to receive one of three study treatments: either two (n=108) or four vaccinations with GAD-alum (n=111) or a vaccination with the adjuvant alum alone (placebo group, n=115). This trial with a follow up time of 15 months failed to show improvements in stimulated C-peptide levels after either the two or the fourdose vaccination when compared to the placebo group. Pooling of data from both groups with GAD vaccinations failed to show a significant effect on stimulated C-peptide levels compared to the control group [25].

#### **4.3. 60kDa heat shock protein (DiaPep277)**

Thus even if these two trials largely failed in their primary aim they nevertheless clearly demonstrated the ability to accurately predict disease risk, which is essential to optimize the

Besides oral or nasal administration of an autoantigen there is in T1D models another approach to induce tolerance. In this case a candidate autoantigen is injected subcutaneously together with the adjuvant alum. Although using an islet autoantigen as a vaccine to prevent or ameliorate disease might appear strange it has nevertheless been shown in the mouse model of T1D that this approach can be effective. The idea is that such a vaccination either activates regulatory T-cells or that it converts autoaggressive T-cells to a non-destructive phenotype. This approach has been tested in humans with GAD65, which is the 65 kd isoform of the autoantigen glutamic acid decarboxylase. In contrast to the prevention studies with oral and nasal insulin the trials with GAD65 have been conducted in individuals with recent onset of T1D. A phase II trial tested the safety and efficacy of vaccination with human GAD65 in alum (two subcutaneous vaccinations with 20μg GAD one month apart) in patients with recent onset of T1D (n=70). Results of this study were reported 30 months and again 4 years after treatment. Of the subgroups prespecified in the protocol (HLA classification, age, sex, baseline GAD autoantibody levels) only duration of T1D had a significant influence on the efficacy of the vaccination. In the patients vaccinated less than 6 month after diagnosis of T1D both fasting and stimulated C-peptide secretion decreased significantly less in the GAD-alum group than in the placebo group by month 30 and this positive effect was retained at 4 years after treatment. There was no significant difference between the GAD-alum and the placebo group for patients treated 6 month or more after diagnosis. As expected, vaccination with GAD-alum lead to strong increase of the GAD autoantibody titers, which was sustained to month 30 and a neurological assessment was performed because of concerns that this might lead to stiff-man syndrome. However, there were no notable neurological differences between treatment and placebo group. In accordance with the B-cell responses anti GAD cytokine responses assessed in PBMCs of treatment and control groups at 15 months showed a significantly increased release of most of the tested cytokines (IL-5, 10, 13, 17, IFN-γ and TNFα) in the GAD-alum group. Furthermore increased GAD-induced levels of FOXP3, a transcription factor associated with T regulatory cells, was found in the GAD-alum group [23] [24]. Given the findings of this trial a second study (phase III) was conducted, which enrolled patients within 3 month of the diagnosis of T1D. Patients were randomly assigned to receive one of three study treatments: either two (n=108) or four vaccinations with GAD-alum (n=111) or a vaccination with the adjuvant alum alone (placebo group, n=115). This trial with a follow up time of 15 months failed to show improvements in stimulated C-peptide levels after either the two or the fourdose vaccination when compared to the placebo group. Pooling of data from both groups with GAD vaccinations failed to show a significant effect on stimulated C-peptide levels compared

timing of preventative therapies.

524 Type 1 Diabetes

**4.2. Glutamic acid decarboxylase**

to the control group [25].

A third antigen tested for its therapeutic value in human T1D, is the 60kDa heat shock protein (hsp60). While GAD and especially insulin are specifically expressed in pancreatic islets this is not the case for hsp60, which is expressed throughout the body. Although anti hsp60 autoantibodies can be detected in patients at the onset of T1D they are not useful as predictive markers for disease beyond what can be achieved by measuring anti insulin or anti GAD titers. If hsp60 is an autoantigen in T1D and is widely expressed throughout the body one would expect to find inflammation driven by hsp60-reactive T-cells in other organs as well. However this is not the case and raises the question as to whether there are beta cell/islet intrinsic factors that set this site apart immunologically from other parts of the body.

In therapeutic applications hsp60 is not given as a whole protein but as a peptide derived from the native sequence of human heat shock protein 60. The sequence of this peptide was first identified in the NOD mouse with the help of diabetogenic T-cell clones responding to the *M. tuberculosis* hsp60. Heat shock proteins are highly conserved proteins and it was discovered that these T- cell clones cross-reacted with the human - and presumably with the mouse form - of hsp60 and specifically recognized an epitope in the C-terminal part of hsp60, which was termed peptide277. Vaccination of NOD mice with peptide277 in mineral oil delayed T1D [26]. Since the sequence of this peptide contained two cysteine residues a more stable form was subsequently generated in which the cysteine residues were replaced by valine. The more stable form of peptide277 was also effective in delaying T1D in NOD mice and was termed DiaPep277 [27]. These studies suggested that the mechanisms mediating the effects of vaccination with DiaPep277 might be similar to the ones proposed for vaccination with GAD (e.g. induction of T regulatory cells). It has become evident however that DiaPep277 (and hsp60) can also exert direct effects on the immune system. Hsp60 can activate B-cells via the Toll like receptor 4 (TLR4), which respond by producing IL-10 [28]. Furthermore, TLR4 activation by hsp60 also occurs in macrophages and dendritic cells promoting pro-inflamma‐ tory effectors. At the same time hsp60 can also induce anti-inflammatory effects promoted through TLR2. It is reported DiaPep277 does not engage TLR 4 but only TLR2, which leads to the generation of a T-cell mediated anti-inflammatory environment [29].

Several phase II trials have been conducted with DiaPep277 in patients with T1D. In one of these trials that focused on the changes in immunological parameters after treatment, Dia‐ Pep277 was administered subcutaneously in a 10% lipid preparation with the placebo group receiving mannitol in 10% lipid preparation. Three different doses of DiaPep277 were tested (0.2mg, 1mg and 2.5mg). Four injections of the drug or the placebo were given over a timeframe of 12 months and a total of 48 patients were enrolled with onset of T1D between 200 and 800 days before start of treatment. Glucagon-stimulated C-peptide production significantly decreased over 12 months in all groups except the group receiving Diapep277 at 2.5mg. The decrease in C-peptide production over 12 months was significantly less in the 2.5mg than in the placebo group. Absolute daily insulin dosage did not decrease over time in any of the groups [30]. These results are in accordance with an earlier trial that found a significantly higher stimulated C-peptide concentration in the treatment vs. placebo group at 6 and 10 months after start of treatment. This earlier trial also found a significantly reduced insulin requirement at 10 months and observed that individuals with higher C-peptide concentration at the time of initiation of treatment showed better preservation of C-peptide concentrations 10 months later [31]. Therefore the rule that the earlier treatment is started the more efficient it tends to be also applies to this approach. The former study was accompanied by an extensive evaluation of immunological parameters before, during and after treatment. As expected, it was observed that immunological responses were quantitatively and qualitatively highly diverse among the subjects. Nevertheless, after development of new methods to evaluate the results obtained from proliferation and cytokine release experiments, some interesting information could be derived. An IL-10 response but not a proliferative response to DiaPep277 before initiation of treatment, and a decrease or loss of proliferative response subsequent to treatment, appeared to provide a correlate for clinical efficiency. These biomarkers might reflect some kind of tolerance to DiaPep277 (hsp60) and appear to be associated with improved clinical outcome. These findings imply that the status of the immune response prior to therapy may be predictive for treatment outcome. Proliferative responses after treatment with DiaPep277 were frequently specific for hsp60 in that responses to GAD or tetanus toxoid were not or only weakly altered [32]. Treatment with DiaPep277 therefore appeared immunologi‐ cally effective and specific. One phase III trial with DiaPep277 was recently concluded and awaits publication of the results and another phase III trial is currently underway.

beneficial to some systems (e.g. T-cells) might be detrimental to other affected cells (e.g. beta cells, endothelia). The clinical outcome might thus depend on the sum of all these effects and

Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/54717 527

The analysis of the trials presented here suggests that treatment efficacy can differ from subgroup to subgroup. This indicates that there might not be a single therapeutic approach that fits all. Rather the observations suggest that it may be necessary to establish an individual profile that goes beyond the standard parameters such as sex, age, family history, time of diagnosis of T1D, HLA type, and autoantibody profile for each person intending to undergo an immune therapeutic intervention. These parameters might include the spectrum of T-cell responses to beta cell autoantigens (in terms of proliferation as well as of cytokine release), characterization of the gut flora [36; 37], imaging of islet inflammation [38] type and time of prior vaccinations and infections, season [25], and might even include psychological parame‐ ters such as familial stress levels [39]. As new approaches are translated from the pre clinical stage to individuals at risk of developing T1D or to patients already suffering from the disease the palette of possible interventions will grow more diverse. Obtaining highly differentiated profiles may refine the process of matching the time point and the type of immune intervention

might not be predictable.

to an individual and thus optimize outcome.

Address all correspondence to: Werner.gurr@yale.edu

Yale University, Dept. of Internal Medicine/Endocrinology, New Haven, USA

CD3 monoclonal antibody. *J Immunol* 144:16-22, 1990

overtly diabetic NOD mice. *J Immunol* 158:2947-2954, 1997

[1] Jenkins MK, Chen CA, Jung G, Mueller DL, Schwartz RH: Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-

[2] Chatenoud L, Thervet E, Primo J, Bach JF: Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. *Proc Natl Acad Sci U S A*

[3] Chatenoud L, Primo J, Bach JF: CD3 antibody-induced dominant self tolerance in

**5. Conclusion**

**Author details**

Werner Gurr\*

**References**

91:123-127, 1994

What could be reasons for the limited success of the antigen specific therapies presented above? From a conceptual point of view there is a concern that in these therapies there is always a risk that administration of the candidate autoantigen does not lead to attenuation of the autoim‐ mune reaction but rather leads to its exacerbation. This is especially the case when autoantigens are administered with an adjuvant such as was done in the GAD-alum trials. We have observed while studying the Reg proteins as potential autoantigens in T1D that vaccination of NOD mice with an N-terminal fragment of RegII in alum leads to acceleration of T1D instead of prevention [33]. A similar observation was made in BB rats, which like the NOD mice spon‐ taneously develop T1D. Here insulin given orally with an *E.coli*-derived endotoxin-free bacterial adjuvant containing acidic glycolipoproteins lead to an acceleration of the disease compared to the group receiving oral insulin alone [34]. Although the GAD-alum studies did not show any acceleration of T1D in the treated groups, it is noteworthy that in the T1D prevention trial with nasal insulin the subgroup of children who presented with three or four types of autoantibodies before the start of the treatment had an unadjusted hazard ratio of insulin vs. placebo of 1.50. This hazard ratio implied a possible risk of an accelerated effect on the onset of T1D in this cohort. It should also be noted that mechanisms involving the activation of regulatory T-cells such as suggested by the findings of the GAD-alum study and considered to be an important factor in oral tolerance generation may not necessarily have only beneficial effects on T1D. Regulatory T-cells are thought to exert their effects via cytokines (e.g. IL-10 or TGF-β), which might on the one hand attenuate self-reactive effector T-cells. But on the other hand these cytokines might also negatively impact beta cell biology and accelerate beta cell destruction by enhancing insulitis through modulation of the release of other cytokines and the islet microvasculature [35]. Cytokines are molecules with a broad range of effects that may differ depending on the target cells. Therefore a therapy that relies on the alteration of cytokine profiles as important effector mechanism carries the risk that these alterations although beneficial to some systems (e.g. T-cells) might be detrimental to other affected cells (e.g. beta cells, endothelia). The clinical outcome might thus depend on the sum of all these effects and might not be predictable.
