**2. T or B-cell targeting**

#### **2.1. Therapy with anti-CD3 antibodies**

That treatment with antibodies directed against CD3, a component of T-cell receptor complex, might have potential in the treatment of autoimmune disease was evident since the discovery *in vitro* that immobilized anti CD3 antibody could render T-cells non-responsive or anergic to subsequent stimulation by antigen [1]. Studies in NOD mice revealed that treatment with a hamster antibody against CD3 could reverse diabetes even when it was given at a stage when the mice were already hyperglycemic. This was an important feature in light of the constraints of immunotherapy applied in human patients as outlined above. It was also in contrast to many other promising therapy approaches tested in the NOD mouse that needed to be administered preventively before the onset of overt clinical signs of T1D in order to be effective. Adding to the attractiveness of this approach was the observation that a single course of treatment lasting for 5 days was sufficient to reverse hyperglycemia for several months and that treated NOD mice were resistant to the induction of disease by spleen cells from diabetic donors [2; 3]. This indicated that rather than a passive mechanism such as a blockade of the CD3 receptor some kind of active mechanism of regulation must have been involved in the generation of the clinical effect. Over the years this mechanism has been studied in great detail in mouse models and it has become clear that rather than induction of non-responsiveness in the T-cells a complex cascade of effector mechanisms is initiated by the anti CD3 treatment. It was discov‐ ered that binding of the antibody to the CD3 receptor transmits a strong activation signal to the T-cell, which triggers a 'cytokine storm' and then death of T-cells. The ensuing cellular debris is 'cleaned up' by macrophages, which release IL-6. The increased levels of IL-6 together with TGFβ create an environment that favors the development of a subset of T-cells charac‐ terized by the secretion of the cytokine IL-17. As these T-cells circulate through the body they pass the small intestine. Epithelial cells at this site respond to the presence of IL-17 with the increased expression of the chemokine CCL20. Since the IL-17 releasing T-cells have a receptor for this chemokine they accumulate in the small intestine. It appears that once these IL-17 releasing T-cells have reached the small intestine they assume a phenotype that allows them to suppress the proliferation of other T-cells and consequently to also attenuate self-reactive T-cells [4]. Thus the impact of a short treatment with anti CD3 antibodies can induce immune suppression that lasts much longer than the presence of the antibody in the system.

Statistically significant reduction in daily insulin needs vs. the corresponding placebo group were reported for patients whose residual beta cell function at baseline was above the median of all patients as well as for patients whose age was below the median age. These patients also had a slower decrease in C-peptide levels than the placebo group i.e. their residual beta cell function was maintained for longer than that of the placebo group. Levels of HbA1c (glycated hemoglobin) were also positively affected by the treatment but this occurred again only in younger patients [5; 6]. The dependency of treatment efficacy on age and on the residual beta cell mass (correlated to the time interval between diagnosis of T1D and treatment start) was also observed in a recent published study that compared different dose regimens of an anti CD3 antibody [7]. The fact that anti CD3 treatment was more effective in younger patients was explained by the age-dependency of the insulitis process. Islet inflammation was detected in children but rarely in adolescents and adults. Furthermore, late onset T1D patients are presumed to suffer from a less severe form of the disease. Therefore a more pronounced loss of residual beta cell function was observed in the younger placebo subgroup compared to the older placebo subgroup and the effect of the anti CD3 treatment consequently became more

Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/54717 519

Side effects induced by the treatment with anti CD3 antibody occurring in most patients are transient and are in accordance with the mechanism of action of this approach. Fever, which might be explained by the 'cytokine storm' triggered by the antibody treatment, a syndrome similar to acute mononucleosis correlating with an increase in EBV copies which may be a result of the activation-induced T-cell death upon anti CD3 administration are some of the adverse events reported for this treatment. Lymphomas as consequence of the anti CD3

While it has been established in the mouse that T-cells are necessary and sufficient to cause the disease the role of B-cells in the pathogenesis of T1D is more indirect. Islet antigen selfreactive T-cells both of the helper and cytotoxic type from diabetic NOD mice can transfer the disease to NOD-SCID recipients whereas B-cells are unable to do so. The contribution of Bcells to the pathogenesis of human T1D is likely also to be more indirect. This is suggested by a report of a child with X-linked agammaglobulinemia who developed T1D [8]. Nevertheless, B-cells must participate in the pathogenesis of T1D because it is possible to prevent the disease by B-cell depletion and it has been shown that B-ells are necessary for the initiation of insulitis in the NOD mouse [9; 10]. B-cells are very efficient antigen presenting cells particularly after they have been activated, which could occur in the accumulation of inflammatory cells in the islets during the pathogenesis of T1D. The rationale for the use of B-ell depletion in humans would therefore be a reduction of antigen presentation, which would result in less T-cell activation and an attenuated inflammatory process. It could also include the elimination of cytokines produced by B-cells that might be damaging to the islets and reduce further recruitment of immune cells to the islets. It is also possible that depletion of B-cells with an antibody initiates complex mechanisms similar to those thought to underlie the effects of

clearly visible in younger patients[6].

treatment have not been observed.

treatment with anti CD3 antibody.

**2.2. B-lymphocyte depletion in patients with recent onset of T1D**

Among immune therapies with broader specificity applied to patients with T1D the treatment with anti CD3 antibodies has been tested most extensively despite side effects such as 'cytokine storm' and a selection of recently evaluated trials is summarized below.

All anti CD3 treatments have been given to patients with recent onset of T1D and in accordance with the constraints outlined above, statistically significant increases in the reversal of disease i.e. full independence from exogenous insulin within the study time frame are not observed. However, differences between treatment and placebo groups have been observed and most prominently where results of patient subpopulations are analyzed. This was shown in a trial that observed treated patients and the placebo group for up to four years after treatment with a humanized anti CD3 antibody which was given over a period of six consecutive days. Statistically significant reduction in daily insulin needs vs. the corresponding placebo group were reported for patients whose residual beta cell function at baseline was above the median of all patients as well as for patients whose age was below the median age. These patients also had a slower decrease in C-peptide levels than the placebo group i.e. their residual beta cell function was maintained for longer than that of the placebo group. Levels of HbA1c (glycated hemoglobin) were also positively affected by the treatment but this occurred again only in younger patients [5; 6]. The dependency of treatment efficacy on age and on the residual beta cell mass (correlated to the time interval between diagnosis of T1D and treatment start) was also observed in a recent published study that compared different dose regimens of an anti CD3 antibody [7]. The fact that anti CD3 treatment was more effective in younger patients was explained by the age-dependency of the insulitis process. Islet inflammation was detected in children but rarely in adolescents and adults. Furthermore, late onset T1D patients are presumed to suffer from a less severe form of the disease. Therefore a more pronounced loss of residual beta cell function was observed in the younger placebo subgroup compared to the older placebo subgroup and the effect of the anti CD3 treatment consequently became more clearly visible in younger patients[6].

Side effects induced by the treatment with anti CD3 antibody occurring in most patients are transient and are in accordance with the mechanism of action of this approach. Fever, which might be explained by the 'cytokine storm' triggered by the antibody treatment, a syndrome similar to acute mononucleosis correlating with an increase in EBV copies which may be a result of the activation-induced T-cell death upon anti CD3 administration are some of the adverse events reported for this treatment. Lymphomas as consequence of the anti CD3 treatment have not been observed.

#### **2.2. B-lymphocyte depletion in patients with recent onset of T1D**

**2. T or B-cell targeting**

518 Type 1 Diabetes

**2.1. Therapy with anti-CD3 antibodies**

That treatment with antibodies directed against CD3, a component of T-cell receptor complex, might have potential in the treatment of autoimmune disease was evident since the discovery *in vitro* that immobilized anti CD3 antibody could render T-cells non-responsive or anergic to subsequent stimulation by antigen [1]. Studies in NOD mice revealed that treatment with a hamster antibody against CD3 could reverse diabetes even when it was given at a stage when the mice were already hyperglycemic. This was an important feature in light of the constraints of immunotherapy applied in human patients as outlined above. It was also in contrast to many other promising therapy approaches tested in the NOD mouse that needed to be administered preventively before the onset of overt clinical signs of T1D in order to be effective. Adding to the attractiveness of this approach was the observation that a single course of treatment lasting for 5 days was sufficient to reverse hyperglycemia for several months and that treated NOD mice were resistant to the induction of disease by spleen cells from diabetic donors [2; 3]. This indicated that rather than a passive mechanism such as a blockade of the CD3 receptor some kind of active mechanism of regulation must have been involved in the generation of the clinical effect. Over the years this mechanism has been studied in great detail in mouse models and it has become clear that rather than induction of non-responsiveness in the T-cells a complex cascade of effector mechanisms is initiated by the anti CD3 treatment. It was discov‐ ered that binding of the antibody to the CD3 receptor transmits a strong activation signal to the T-cell, which triggers a 'cytokine storm' and then death of T-cells. The ensuing cellular debris is 'cleaned up' by macrophages, which release IL-6. The increased levels of IL-6 together with TGFβ create an environment that favors the development of a subset of T-cells charac‐ terized by the secretion of the cytokine IL-17. As these T-cells circulate through the body they pass the small intestine. Epithelial cells at this site respond to the presence of IL-17 with the increased expression of the chemokine CCL20. Since the IL-17 releasing T-cells have a receptor for this chemokine they accumulate in the small intestine. It appears that once these IL-17 releasing T-cells have reached the small intestine they assume a phenotype that allows them to suppress the proliferation of other T-cells and consequently to also attenuate self-reactive T-cells [4]. Thus the impact of a short treatment with anti CD3 antibodies can induce immune

suppression that lasts much longer than the presence of the antibody in the system.

storm' and a selection of recently evaluated trials is summarized below.

Among immune therapies with broader specificity applied to patients with T1D the treatment with anti CD3 antibodies has been tested most extensively despite side effects such as 'cytokine

All anti CD3 treatments have been given to patients with recent onset of T1D and in accordance with the constraints outlined above, statistically significant increases in the reversal of disease i.e. full independence from exogenous insulin within the study time frame are not observed. However, differences between treatment and placebo groups have been observed and most prominently where results of patient subpopulations are analyzed. This was shown in a trial that observed treated patients and the placebo group for up to four years after treatment with a humanized anti CD3 antibody which was given over a period of six consecutive days. While it has been established in the mouse that T-cells are necessary and sufficient to cause the disease the role of B-cells in the pathogenesis of T1D is more indirect. Islet antigen selfreactive T-cells both of the helper and cytotoxic type from diabetic NOD mice can transfer the disease to NOD-SCID recipients whereas B-cells are unable to do so. The contribution of Bcells to the pathogenesis of human T1D is likely also to be more indirect. This is suggested by a report of a child with X-linked agammaglobulinemia who developed T1D [8]. Nevertheless, B-cells must participate in the pathogenesis of T1D because it is possible to prevent the disease by B-cell depletion and it has been shown that B-ells are necessary for the initiation of insulitis in the NOD mouse [9; 10]. B-cells are very efficient antigen presenting cells particularly after they have been activated, which could occur in the accumulation of inflammatory cells in the islets during the pathogenesis of T1D. The rationale for the use of B-ell depletion in humans would therefore be a reduction of antigen presentation, which would result in less T-cell activation and an attenuated inflammatory process. It could also include the elimination of cytokines produced by B-cells that might be damaging to the islets and reduce further recruitment of immune cells to the islets. It is also possible that depletion of B-cells with an antibody initiates complex mechanisms similar to those thought to underlie the effects of treatment with anti CD3 antibody.

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 administered [12].

has regenerated autoimmune responses are eventually re-established and islet destruction resumes. This process is reflected by the prolonged but not permanent state of independence from exogenous insulin experienced by the majority of the treated patients in this trial. The results of this study raise the questions whether the increase in C-peptide levels and inde‐ pendence from exogenous insulin was due to a process of regeneration of islets or due to an attenuation of the inflammatory environment the islets were exposed to. Since the majority of the insulin-free patients discontinued insulin use between 3 days before stem cell transplan‐ tation (i.e. during the process of immune ablation) and 34 days after transplantation of stem cells it is reasonable to assume that the latter mechanism was dominant at least initially because this time span would appear to be too short to allow extensive regeneration of islets. There was however one patient who achieved insulin independence 610 days after stem cell trans‐ plantation and in this case regeneration of islets may have played a role and it is possible that this process also contributed during later stages to the increased C-peptide levels observed in the patients with long term independence from exogenous insulin. If attenuation of the inflammatory environment that islets are exposed to is an important early effector mechanism and if insulitis in humans is predominantly found in children but seldom in adolescents and adults then this approach would be expected to work best in children with recent onset of T1D. However none of the patients in this study was younger than 14 years and therefore the best

Immunotherapies for Type 1 Diabetes http://dx.doi.org/10.5772/54717 521

The above-mentioned trial could not have been designed as a controlled and blinded study and it is possible that that some of the remissions observed were not related to the treatment but represent spontaneous remissions. However, the close correlation of the observed remissions with the immune ablative treatment and their duration argued for a genuine effect of the treatment. There are results from a double blind and placebo- controlled study applying broad targeting of the immune system with cyclosporine. They show a statistically significant increase in complete as well as complete and partial remissions at the 9th month in the treated vs. placebo group with the effects being more pronounced in the subgroup with whole blood

Certainly immune ablation followed by autologous stem cell transplantation even if it has to be performed only every 3-4 years is not something that could be considered suitable for repeat administration. This also holds in regards to the continued administration of cyclosporine to patients with T1D especially since even a short lasting course of the drug (12.5 +/- 4 months) accelerated the rate of progression of the urinary albumin excretion rate and tended to induce

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

possible effect might not have been achieved.

cyclosporine levels of ≥300ng/ml [15].

a loss in kidney function [16].

**4.1. Insulin**

**4. Antigen specific therapies**

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‐ acting mechanisms.
