First-in-human, double-blind, randomized phase 1b study of peptide immunotherapy IMCY-0098 in new-onset type 1 diabetes: an exploratory analysis of immune biomarkers.

BACKGROUND: IMCY-0098, a synthetic peptide developed to halt disease progression via elimination of key immune cells in the autoimmune cascade, has shown a promising safety profile for the treatment of type 1 diabetes (T1D) in a recent phase 1b trial. This exploratory analysis of data from that trial aimed to identify the patient biomarkers at baseline associated with a positive response to treatment and examined the associations between immune response parameters and clinical efficacy endpoints (as surrogates for mechanism of action endpoints) using an artificial intelligence-based approach of unsupervised explainable machine learning. METHODS: We conducted an exploratory analysis of data from a phase 1b, dose-escalation, randomized, placebo-controlled study of IMCY-0098 in patients with recent-onset T1D. Here, a panel of markers of T cell activation, memory T cells, and effector T cell response were analyzed via descriptive statistics. Artificial intelligence-based analyses of associations between all variables, including immune responses and clinical responses, were performed using the Knowledge Extraction and Management (KEM®) v 3.6.2 analytical platform. RESULTS: The relationship between all available patient data was investigated using unsupervised machine learning implemented in the KEM® environment. Of 15 associations found for the dose C group (450 µg subcutaneously followed by 3 × 225 µg subcutaneously), seven involved human leukocyte antigen (HLA) type, all of which identified improvement/absence of worsening of disease parameters in DR4+ patients and worsening/absence of improvement in DR4- patients. This association with DR4+ and non-DR3 was confirmed using the endpoints normalized area under the curve C-peptide from mixed meal tolerance tests where presence of DR4 HLA haplotype was associated with an improvement in both endpoints. Exploratory immune analysis showed that IMCY-0098 dose B (150 µg subcutaneously followed by 3 × 75 µg subcutaneously) and dose C led to an increase in presumed/potentially protective antigen-specific cytolytic CD4+ T cells and a decrease in pathogenic CD8+ T cells, consistent with the expected mechanism of action of IMCY-0098. The analysis identified significant associations between immune and clinical responses to IMCY-0098. CONCLUSIONS: Promising preliminary efficacy results support the design of a phase 2 study of IMCY-0098 in patients with recent-onset T1D. TRIAL REGISTRATION: ClinicalTrials.gov NCT03272269; EudraCT: 2016-003514-27.

First-in-human, double-blind, randomized phase 1b study of peptide immunotherapy IMCY-0098 in new-onset type 1 diabetes.

BACKGROUND: Type 1 diabetes (T1D) is a CD4+ T cell-driven autoimmune disease characterized by the destruction of insulin-producing pancreatic ß-cells by CD8+ T cells. Achieving glycemic targets in T1D remains challenging in clinical practice; new treatments aim to halt autoimmunity and prolong ß-cell survival. IMCY-0098 is a peptide derived from human proinsulin that contains a thiol-disulfide oxidoreductase motif at the N-terminus and was developed to halt disease progression by promoting the specific elimination of pathogenic T cells. METHODS: This first-in-human, 24-week, double-blind phase 1b study evaluated the safety of three dosages of IMCY-0098 in adults diagnosed with T1D < 6 months before study start. Forty-one participants were randomized to receive four bi-weekly injections of placebo or increasing doses of IMCY-0098 (dose groups A/B/C received 50/150/450 µg for priming followed by three further administrations of 25/75/225 µg, respectively). Multiple T1D-related clinical parameters were also assessed to monitor disease progression and inform future development. Long-term follow-up to 48 weeks was also conducted in a subset of patients. RESULTS: Treatment with IMCY-0098 was well tolerated with no systemic reactions; a total of 315 adverse events (AEs) were reported in 40 patients (97.6%) and were related to study treatment in 29 patients (68.3%). AEs were generally mild; no AE led to discontinuation of the study or death. No significant decline in C-peptide was noted from baseline to Week 24 for dose A, B, C, or placebo (mean change - 0.108, - 0.041, - 0.040, and - 0.012, respectively), suggesting no disease progression. CONCLUSIONS: Promising safety profile and preliminary clinical response data support the design of a phase 2 study of IMCY-0098 in patients with recent-onset T1D. TRIAL REGISTRATION: IMCY-T1D-001: ClinicalTrials.gov NCT03272269; EudraCT: 2016-003514-27; and IMCY-T1D-002: ClinicalTrials.gov NCT04190693; EudraCT: 2018-003728-35.

Retroviral vectors induce epigenetic chromatin modifications and IL-10 production in transduced B cells via activation of toll-like receptor 2.

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2(d)) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor [mouse cationic amino acid transporter 1 (mCAT1)] and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination.

In vivo induction of type 1-like regulatory T cells using genetically modified B cells confers long-term IL-10-dependent antigen-specific unresponsiveness.

Regulatory T cells (Tregs) hold much promise for the therapy of allergy and autoimmunity, but their use is hampered by lack of Ag specificity (natural Tregs) and difficulty to expand in vitro or in vivo (adaptive Tregs). We designed a method for in vivo induction of Ag-specific Tregs, in BALB/c H-2d, that share characteristics with type 1 Tregs (Tr1). A retroviral vector was constructed encoding a major T cell epitope of a common allergen, Der p 2, fused to an endosomal targeting sequence (gp75) for efficient MHC class II presentation. B cells transduced with such construct were adoptively transferred to BALB/c mice before or after peptide immunization. Long-lasting Ag-specific immune tolerance was achieved in both cases. Genetically modified B cells constitutively expressed the transgene for at least 3 mo. B cells from IL-10(-/-) mice were unable to induce tolerance. Upon transfer, B cells induced Foxp3(-)CD4(+) T cells showing phenotypic and functional characteristics comparable to Tr1-cells, including production of IL-10 but not of TGF-beta, and high expression of CTLA-4. Adoptive transfer of such T cells conferred unresponsiveness to allergen immunization and prevented the development of Der p 2-induced asthma. Functional Tr1-like cells can therefore be induced in vivo using retrovirally transduced B cells.

Increased synapse formation obtained by T cell epitopes containing a CxxC motif in flanking residues convert CD4+ T cells into cytolytic effectors.

The nature of MHC class II-binding epitopes not only determines the specificity of T cell responses, but may also alter effector cell functions. Cytolytic CD4+ T cells have been observed primarily in anti-viral responses, but very little is known about the conditions under which they can be elicited. Their potential as regulators of immune responses, however, deserves investigations. We describe here that inclusion of a thiol-disulfide oxidoreductase motif within flanking residues of class II-restricted epitopes results, both in vitro and in vivo, in elicitation of antigen-specific cytolytic CD4+ T cells through increased synapse formation. We show that both naïve and polarized CD4+ T cells, including Th17 cells, can be converted by cognate recognition of such modified epitopes. Cytolytic CD4+ T cells induce apoptosis on APCs by Fas-FasL interaction. These findings potentially open the way towards a novel form of antigen-specific immunosuppression.

Regulatory CD4+ T cells in allergic asthma.

Active suppression by regulatory T cells (T(regs)) appears to play a key role in the downregulation of T-cell responses to foreign antigens. Several subtypes of T(regs) have been described but their mechanisms of action remain unclear. Recent data demonstrate that the suppressive capacity of natural T(regs) could be associated with cytotoxicity due to the release of granzymes, which are capable of apoptosis induction in target effector T lymphocytes and in antigen-presenting cells, such as dendritic cells. The mechanism of such nonspecific T(regs) is discussed. Peptide immunotherapy is thought to induce regulatory cells capable of suppressing autoimmune and allergic diseases. We have recently optimized a vaccination strategy by which cytotoxic antigen-specific adaptive T(regs) can be elicited towards allergens involved in allergic asthma. Such a strategy could be of value in the treatment of allergic asthma.

Three-dimensional structure and IgE-binding properties of mature fully active Der p 1, a clinically relevant major allergen.

BACKGROUND: Der p 1 is a 25-kd allergen with cysteine protease activity. Sensitization to Der p 1 affects a large proportion of individuals with allergy, resulting in rhinitis, asthma, and/or atopic dermatitis. OBJECTIVE: We determined the Der p 1 crystallographic structure to understand the relationships among structure, function, and allergenicity. METHODS: Recombinant pro-Der p 1 was produced in Pichia pastoris and allowed to mature spontaneously before purification by a 2-step procedure. Protease activity was checked by using a fluorogenic peptide substrate. Allergenicity was analysed by IgE binding assays and basophil activation test. The determination of the 3-dimensional structure was obtained by X-ray crystallography at 1.9 A resolution. RESULTS: The recombinant protein is fully active and expresses an allergenicity equivalent to its natural counterpart. Der p 1 exhibits a cysteine protease fold typical of the papain family, has a magnesium binding site, and forms dimers with a large interface. The crystal lattice shows that the dimers are tightly packed in a compact double layer of proteins. Such an assembly likely exists in dry fecal pellets, the natural form of allergen exposure, and appears ideal to interact with cell surface and trigger allergic inflammation. CONCLUSION: We present here the 3-dimensional structural features of mature fully active Der p 1, one of the main allergens involved in human allergic diseases. This opens the possibility to evaluate the importance of enzymatic activity in pathology and possible new therapeutic interventions.

CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner.

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4(+)CD25(+) population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4(+) cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4(+) T cell line derived from BALB/c mice immunized with peptide 21-35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4(+)CD25(+) T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.

In vivo neutralization of a C2 domain-specific human anti-Factor VIII inhibitor by an anti-idiotypic antibody.

Factor VIII (FVIII) administration elicits specific inhibitory antibodies (Abs) in about 25% of patients with hemophilia A. The majority of such Abs reacts with FVIII C2 domain. mAbBO2C11 is a high-affinity human monoclonal antibody (mAb) directed toward the C2 domain, which is representative of a major class of human FVIII inhibitors. Anti-idiotypic Abs were raised to mAbBO2C11 to establish their neutralizing potential toward inhibitors. One mouse anti-idiotypic mAb, mAb14C12, specifically prevented mAbBO2C11 binding to FVIII C2 domain and fully neutralized mAbBO2C11 functional inhibitory properties. Modeling of the 3-D conformation of mAb14C12 VH and alignment with the 3-D structure of the C2 domain showed putative 31 surface-exposed amino acid residues either identical or homologous to the C2 domain. These included one C2 phospholipid-binding site, Leu2251-Leu2252, but not Met2199-Phe2200. Forty putative contact residues with mAbBO2C11 were identified. mAb14C12 dose-dependently neutralized mAbBO2C11 inhibitory activity in mice with hemophilia A reconstituted with human recombinant FVIII (rFVIII), allowing full expression of FVIII activity. It also neutralized in an immunoprecipitation assay approximately 50% of polyclonal anti-C2 Abs obtained from 3 of 6 unrelated patients. mAb14C12 is the first example of an anti-idiotypic Ab that fully restores FVIII activity in vivo in the presence of an anti-C2 inhibitor. The present results establish the in vitro and in vivo proof of concept for idiotype-mediated neutralization of a major class of FVIII inhibitors.