Objective
Durable prevention or remission of T1D have not been achieved so far. There is thus an urgent need to optimize therapies and their associated factors, with the objective of halting the pathological process before the complete destruction of pancreatic beta cells. Humanized monoclonal anti-CD3 antibodies (Abs) have been extensively studied in several clinical trials, and provided some of the best results in both recent-onset T1D patients and at-risk individuals. However, further investigations are required to improve the safety profile, extend the indications of this treatment to earlier disease stage, and achieve long-term efficacy. In this translational perspective, combination therapies with autoantigens and applications through less invasive mucosal routes are very attractive.
Thus, we propose to test the preclinical efficacy of anti-CD3 Ab administration combined to insulin (more exactly its preproinsulin PPI precursor form) through the intranasal route, which has already been exploited in T1D trial. To that aim, we have developed a fusion protein using PPI fused to an antibody Fc fragment (PPI-Fc) to increase delivery and transfer across nasal mucosa through binding to the neonatal Fc receptor. Our objectives are:
1) Define an optimal dose of intranasal PPI-Fc and a sub-therapeutic dose of intranasal anti-CD3, i.e. providing on its own a partial protection from diabetes development and limiting side-effects in the NOD mouse model of T1D.
2) Assess the capacity of the intranasal PPI-Fc/anti-CD3 combination to efficiently and durably prevent T1D onset through synergistic effects, and identify the cellular and molecular immune mechanisms induced by the combined therapy, in comparison to each mono-therapy.
Background Rationale
Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of pancreatic β cells by the immune system, in particular by autoreactive T lymphocytes, leading to dysregulated glucose homeostasis. T1D is triggered by complex genetic and environmental factors, progressing from asymptomatic to autoantibody-positive to overt dysglycemia. The number of children and adolescents with type 1 diabetes (T1D) is increasing at a rate of ~4%/year in most industrialized countries, and affects 1.2 million persons under 20 years worldwide in 2021, with 150,000 new cases each year (International Diabetes Federation, 10th report 2021). Moreover, the peak of T1D incidence continues to shift toward a younger age of onset, frequently under 5 years. The constraints of insulin therapy, the daily threat of hypoglycemia and the associated risk of late complications (retinopathy, nephropathy, neuropathy, cardiovascular diseases) put a significant burden on patient quality of life, families and health care systems. Therefore, rather than treating disease symptoms, with exogenous insulin injections developing effective immunotherapies targeting the underlying autoimmune mechanisms to prevent T1D in identified at-risk individuals is critical to impact these incidence trends.
In this context, prevention strategies with β-cell antigens Ags (GAD, insulin) are attractive due to their selectivity and safety, but have failed so far to restore tolerance in T1D trials. Indeed, numerous immunotherapies with the potential to halt the autoimmune aggression have been evaluated in the clinic, but overall provided limited effectiveness to efficiently and durably prevent or treat T1D. Thus, improvements may arise from combination therapies using therapeutics agents exhibiting complementary mode of action. Another path to optimization is to use non-invasive administration route, which would help wider applicability to at-risk but as yet clinically healthy, and mostly pediatric, populations.
In this perspective, we propose to combine the main autoantigen insulin with monoclonal anti-CD3 antibodies (Abs), and use the intranasal route of administration. To optimize this strategy, we have developed a fusion protein composed of the preproinsulin (PPI) precursor form of insulin fused to an antibody Fc fragment (PPI-Fc) to increase delivery and transfer across nasal mucosa through binding to the neonatal Fc receptor. In addition, anti-CD3 Abs showed promise for T1D treatment. as shown by significant preservation of residual β-cell functions in recent-onset diabetic patients, and an impressive 24-month delay in the median time to T1D diagnosis in at-risk autoantibody-positive individuals. In all these trials, anti-CD3 Abs were injected by the intravenous route and was associated with some moderate side-effects namely fever, head¬ache, gastrointestinal symptoms, skin rash, and lymphopenia. Therefore, the stake is to improve safety, efficacy and overall acceptability of anti-CD3 Ab therapy to accelerate and expand application to T1D prevention. In this context, intranasal delivery of anti-CD3 Abs may provide several therapeutic advantages over the classical intravenous route. It is less invasive, stimulates tolerogenic mechanisms associated with the nasal mucosa, and may be better tolerated, and thus more suited for prolonged or repeated treatment courses. All these features are key for T1D prevention trials mostly targeting pediatric populations. Accordingly, intranasal anti-CD3 therapy significantly prevent T1D onset in the NOD mouse model of T1D, without displaying side-effects. Of importance, although unsuccessful, intranasal administration of insulin has already proven safe in the clinic, both in recent-onset diabetic and aAb+ at-risk patients.
We thus propose to combine intranasal insulin with intranasal anti-CD3 Ab in NOD mice, with the objective of improving safety and efficacy through synergistic effects, and thus prevent diabetes development.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of pancreatic β cells by the immune system, in particular by autoreactive T lymphocytes. Despite significant advances in the understanding of T1D pathogenesis, there is still no cure for the disease, whose incidence is steadily increasing in industrialized countries, with a peak shifting toward a younger age of onset. Therefore, developing effective immunotherapies targeting the underlying autoimmune mechanisms to prevent T1D is critical to impact these incidence trends.
In this context, prevention strategies using β-cell antigens Ags (GAD, insulin) are attractive due to their selectivity and safety, but have failed so far to restore tolerance in T1D trials. These therapies may be optimized through combination with other therapeutics agents exhibiting complementary effects. Another path to improvement is to develop non-invasive administration route, which would help wider applicability to at-risk but as yet clinically healthy, and mostly pediatric, populations.
In this perspective, we propose to focus on the intranasal route of administration that has been already used in the context of clinical T1D (but without success so far) and to combine insulin with monoclonal anti-CD3 antibodies (Abs), which target autoreactive T cells. Anti-CD3 therapy holds promise for T1D treatment. Intravenous injection of anti-CD3 Abs showed significant preservation of residual β-cell functions in recent-onset diabetic patients, and an impressive 24-month delay in the median time to T1D diagnosis in at-risk autoantibody-positive individuals, accompanied however by mild side-effects. The promising clinical outcome of anti-CD3 therapy invites further improvements of its safety, efficacy and overall acceptability to accelerate and expand application to T1D prevention. We hypothesize that combining anti-CD3 Abs to insulin, both through the nasal route, will provide synergistic effects and better efficacy while reducing anti-CD3 doses, thus limiting unwanted side effects and facilitating multiple treatment courses. To further optimize this strategy, we will use the preproinsulin (PPI) precursor form of insulin fused to an antibody Fc fragment (PPI-Fc) that we have developed to increase transfer across nasal mucosa through binding to the neonatal Fc receptor. The present project is composed of two aims that will be investigated in the NOD mouse model of T1D.
First, we will first identify an optimal dose of intranasal PPI-Fc and a sub-therapeutic dose of intranasal anti-CD3, i.e. providing on its own a partial protection from diabetes development and limiting side-effect in prediabetic NOD mice. We will also follow delivery and transfer of anti-CD3 and PPI-Fc across nasal mucosa and their local and systemic bio-distribution.
Second, we will assess T1D prevention with intranasal PPI-Fc/anti-CD3 combination, using the doses defined above. Diabetes incidence will be monitored and the therapeutic mechanisms at play will be identified, and compared to treatment with anti-CD3 or PPI-Fc mono-therapy.
Thus, the proposed combination therapy, using biological tools and therapeutic protocols (nasal insulin, Fc-protein therapeutics and anti-CD3 Abs), that have already been translated individually to the clinic, may represent a novel strategy for T1D prevention.
Anticipated Outcome
The first steps toward therapeutic optimization is to define the most suitable doses of intranasal PPI-Fc and anti-CD3 antibody. We thus expect to identify an optimal dose of intranasal PPI-Fc and a sub-therapeutic dose of intranasal anti-CD3, i.e. providing on its own efficient delivery and transfer through the mucosal FcRn and a partial protection from diabetes development and limiting side-effect in prediabetic NOD mice. We will also determine the mechanisms used by anti-CD3 and PPI-Fc to cross nasal mucosa and be delivered locally and/or systemically.
We next expect our nasal PPI-Fc/anti-CD3 combination strategy to efficiently and durably inhibit the infiltration of the pancreas by autoreactive T cells, halt the destruction of β cells and prevent diabetes development. Based on our preliminary results and our expertise in immunotherapies using Fc-coupled antigens and anti-CD3 antibody, we are confident that the intranasal route will provide the required safety profile and synergy of immunomodulatory and antigen-specific effects (compared to treatment with anti-CD3 or PPI-Fc mono-therapy). Indeed, it is unlikely that the limited anti-CD3 systemic bio-availability possibly observed will induce major side effects at the selected doses, in line with previous publications in experimental multiple sclerosis, lupus and arthritis. While inducing some systemic modulation of immune responses, we anticipate that the major effect of concomitant intranasal anti-CD3 and PI-Fc administration may be to boost the local intranasal induction of PPI-specific regulatory mechanisms that will spread to the target pancreas organ.
Thus, the proposed combination therapy, using biological tools and therapeutic protocols (nasal insulin, Fc-protein therapeutics and anti-CD3 Abs), that have already been translated individually to the clinic, may represent a novel strategy for T1D prevention.
Relevance to T1D
There is an urgent need to develop immunotherapies targeting the autoimmune mechanisms responsible for the destruction of insulin-producing beta cells, before the appearance of hyperglycemia. However, treating at-risk patients require a high safety profile as mostly children and adolescents are concerned. In addition, repeated treatments may be require to achieve sustained efficacy. In this context, immunotherapies using β-cell antigens (GAD, insulin) are attractive due to their selectivity and safety, but have failed so far to restore tolerance in T1D trials.
This project addresses these medical needs by proposing an innovative combination strategy using anti-CD3 Abs and PPI-Fc fusion protein, that may synergistically act on autoimmune T cells and dampen the pathological process, and by using the non-invasive intranasal route of administration. Mucosal (nasal or oral) anti-CD3 combination therapies have not been tested to date, neither in the mouse nor in the human. Intranasal anti-CD3 delivery provides several therapeutic advantages over alternative administration routes. Compared to oral anti-CD3, it is not subjected to gastric degradation. Compared to intravenous anti-CD3 used so far in T1D clinical trials, it is less invasive, does not induce systemic T-cell depletion and inflammatory side-effects, is better suited for prolonged or repeated treatment courses, and may stimulate physiological mucosal tolerogenic mechanisms. All these features are key for T1D preventative purposes mostly concerning pediatric, clinically healthy or prediabetic populations.
Of importance, these biological tools and therapeutic protocols have already been individually translated in the clinic and well tolerated (nasal insulin, Fc-protein therapeutics and anti-CD3 Abs). This supports the feasibility of applying intranasal PPI-Fc/anti-CD3 therapy for T1D prevention.