Microsoft word - our results year 3

Summary description of project context and objectives
The goals of a successful therapeutic approach to type 1 diabetes (T1D) are cessation of beta-cell
destruction, reversal of autoimmunity and preservation of surviving beta-cells allowing any natural
regenerative potential to be realised. These are crucial challenges for diabetes research. Any such
interventions to achieve beta-cell protection and restoration should realise these goals through modulation
of the immune system of the patient to a minimal degree in order to avoid severe disturbances of immune
surveillance mechanisms leading to intolerable side effects. The present state of our understanding of the
pathogenesis of T1D indicates that, in principle, therapeutic success is achievable and that interference in
the progressive loss of beta-cell mass in newly diagnosed T1D patients is within reach. This will require
innovative approaches that operate with a minimal degree of interference in the general functions of the
immune system. In order to move the field forward in this respect, we propose a series of studies that
represent a novel and integrated approach. Our studies, organized into six scientific work packages, plus a
training and an administrative work package, are designed as overlapping and complementary strategies
that run the full-length of the journey from bench to bedside, including innovative first-in-man studies that
aim to arrest autoimmunity in T1D with minimal or no immune suppression; rather we aim to harness its
extraordinary natural power for regulation, healing and regeneration.
The underlying concept for the therapeutic interventions to be developed in this proposal is the central role
of the immune system in T1D, but we also recognize the important role that the beta-cell adopts, as a
partner actively contributing to its own demise. This should undoubtedly be taken into account when
designing strategies for therapeutic interventions for beta-cell protection and restoration. The concept of a
key role for both the immune system and the beta-cell highlights the need for a “multiple hit” approach to
disease prevention. This will include both modulation and re-education of the immune system, boosting of
beta-cell defences against autoimmune damage and arresting the pro-inflammatory dialogue between
immune cells and beta-cells.
The specific objectives of the project NAIMIT are:
1. To explore novel immunomodulatory approaches using natural immunomodulators (e.g. antigen,
glucocorticoids and vitamin D derivatives) to modulate dendritic cell (DC) and T-lymphocyte function,
both in vitro and in vivo with the aim of inducing antigen-specific regulation.
2. To introduce novel immunomodulatory tools to induce antigen-specific tolerance: soluble monoclonal
TCRs (mTCRs) and L. lactis.
3. To gain insight into the mechanisms involved in immune-mediated beta-cell death aimed at harnessing
the beta-cell against autoimmune attack.
4. To unravel the means of communication between beta-cells and the immune system in order to interfere
with beta-cell destruction and propagation of the beta-cell destruction process.
5. To explore genetic variants responsible for the response to interventions allowing individualized
therapies.
6. To build a consortium spanning basic and clinical research to allow new and individualized therapeutic
approaches for clinical interventions in T1DM in the future.

WP1 – Re-educating antigen-presenting cells
The work in WP1 was designed to interfere with antigen-specific autoimmunity but to have minimal
impact on the global immune system. In this “bench to clinical assessment” work package, autologous DCs
are rendered tolerance-inducing in an antigen-specific manner. It was the aim to modulate DCs ex vivo
towards a tolerogenic phenotype using “natural” mediators (namely active vitamin D (1,25(OH)2D3 -
VitD) and/or glucocorticoids (GCs)). Tolerogenic DCs are being manipulated to orchestrate beta-cell-
specific tolerance, by pulsing them with beta-cell auto-antigenic peptide epitopes, such as proinsulin C19-
A3. The work package progresses favourably with respect to all objectives.
In the last year, we continued to elaborate on the characteristics of the DCs generated in the presence of
1,25(OH)2D3 - VitD as well as studying the effect of these DCs on T lymphocytes. As such, we
demonstrated that different types of Treg cells are being induced. Besides this basic scientific work, most
efforts were put on translating the knowledge to clinic, allowing as soon as possible the introduction of this
interesting tool into patients. For this, GMP-grade media and supplements have been introduced in the
protocols, SOPs validated, thus upgrading the tolerogenic DCs to a GMP cell product. Together with the
data showing that tolerogenic DCs can be induced from monocytes of T1D patients, we are encouraged and
on track to proceed with preparations of clinical-grade modulated DC-vaccine for the first-in-man study in
the next months.
WP2 – Restoring the T-cell balance
This WP aims to restore the T-cell balance through an antigen-specific route that avoids global immune
suppression. It builds upon the emerging programme of Peptide Immunotherapy (PIT), in which naturally
processed and presented peptide fragments from major beta-cell auto-antigens are administered
intradermally to promote the generation of islet-specific Tregs (IS-Tregs). Use of multiple peptides to
enhance the power and breadth of the approach will be a major asset. Furthermore, topical adjuvants such
as vitD, retinoic acid and GCs are being studied for their potential to enhance PIT. Significant progress has
been made on defining the changes in DCs in human skin following topical treatment with the vitD
analogue, calcipotriol alone or in combination with steroid (betamethasone) or a derivative of vitamin A
(retinoic acid). Topical treatment with betamethasone or betamethasone plus calcipotriol reduces levels of
proinflammatory cytokines in interstitial fluid with relative preservation of the regulatory (potential
tolerogenic) cytokine IL-10.
Further, 9 peptides from IA-2 and proinsulin identified by biochemical and in vitro testing have been
determined as candidates for development in a Multi-Pep cocktail. These have been synthesised in large
scale and have been combined at operational concentrations without precipitation. Building on NAIMIT,
additional extra-mural funding (Welcome Trust) has been obtained via a competitive award for the
chemical and toxicological testing of the cocktail, as well as GMP grade synthesis; through to preparation
for a Phase I study. Work commenced in 2012 on method development for: GMP-grade and clinical scale
of synthesis; solubility analyses for the 9 candidates; and method development for identification and
progress to date has been excellent. In preclinical models the early stages of development of a tolerance
model has been established, in which HLA-DR4 Tg mice are rendered “autoimmune” by immunization
with proinsulin. The autoimmunity can be prevented

by pre-treatment with single proinsulin peptide immunotherapy. Further, requirement for thymus-derived
Treg induction in the process of peptide immunotherapy is being studied. Finally, an extensive and
operational clinical network of biological samples from new-onset cases of T1DM is being established for
preclinical and clinical intervention studies, in order to prepare eventual interventions and proof of concept
in these patients.
WP3 – TCR-directed immunotherapy
In this WP the body’s own system of recognising antigens on the surface of cells, namely the T-cell
receptor (TCR), is being exploited by a very innovative technology, developed by the SME partner
Immunocore. Soluble mTCRs directed against specific beta-cell antigen epitopes in the context of the
common HLA-A2 (A*0201) molecule, an allele present in the majority of T1D patients, or its mouse
equivalent, have been engineered. These allow target-specific delivery of therapeutic agents. Different
pathways have been taken: first linking mTCRs directed against beta cell specific antigens specific for the
mouse system have been linked to IL4, IL13 and IL10 in order to perform proof of concept trials in
preclinical mouse models (NOD). These mTCRs have been synthesised and the first experiments in mice
using IL4 and IL13 fusions have been completed; IL10 constructs were too low in activity to merit in vivo
testing in mouse models and have thus been discontinued. Data on IL4 and IL13 are disappointing as
treatment with these TCRs could not delay diabetes recurrence in NOD mice. Second, major mTCR
engineering and intense collaborations between different NAIMIT beneficiaries have allowed SME
Immunocore to go in depth into the synthesis of human mTCRs directed against beta cell autoantigens. It
has become evident that all autoantigens identified until now, have very low binding affinity, thus making
easy synthesis of mTCRs a challenge. The last year has seen successful for engineering of the mTCR
specific for pre-proinsulin, overcoming significant technical barriers due to the low affinity of the wild type
receptor; this was achieved in part as a result of targeting mutations to the regions of the TCR making
direct contact with the target MHC-peptide, identified from a high resolution crystal structure and
additionally elongating other regions to bring them into contact with the MHC-peptide. Recently, pico-
Molar affinity was achieved, with a binding half-life of over 14 hrs, which is a significant achievement for
a sub-optimal autoimmune TCR. Thus this tool is ready for linking to eg. imaging molecules and will be
explored as described in the aims of the WP for imaging of human beta-cells.
WP4 – Mucosal intervention for tolerance restoration
Here focus is put on induction of mucosa-mediated tolerance to islet antigens. Orally administered antigen
encounters the gut associated lymphoid tissue (GALT), a well-developed immune network that not only
evolved to protect the host from ingested pathogens, but also developed the property of preventing the host
from reacting to ingested proteins. Modulation of immune-responses in GALT has been shown to be
relevant to prevent/delay autoimmune diabetes onset. Two distinct paths have been followed and we have
made significant progress in the two research branches of this WP. In the first branch, the potential of
probiotics administration as immunomodulator in T1D is being explored. We are performing in vitro
experiments aimed at characterizing the immunomodulatory effects of different probiotic bacterial strains.
In vivo studies in NOD mice showed that probiotic treatment significantly decreases islet expression of

proinflammatory cytokines and chemokines and, in parallel, determines an increased islet IL10 expression.
Second, we are exploring the therapeutic potential of an original tool introduced by beneficiary 12 (SME
ActoGeniX), in which recombinant L. lactis (ActoBiotics™) is a carrier for peptides, in association with
immunomodulatory molecules, allowing delivery of antigen to the GALT. Extensive experiments using L.
Lactis expressing human proinsulin in conjunction with IL10 have been carried out; demonstrating that
reversal of diabetes in newly diagnosed diabetic NOD mice can be reached in over 60% of cases, when
administered in combination with low, subtherapeutic doses of anti-CD3. This therapeutic effect is
combined with the induction of CD4+CD25+FoxP3+ regulatory T cells in the pancreatic draining lymph
nodes. Mechanistic studies on this regulatory T cell population highlighted their capacity of delaying
diabetes, as shown by co-transfer studies. Regulatory T cells were shown to migrate, accumulate and
proliferate locally in the islets upon combination therapy and suppress effector T cell responses in an
antigen-specific way. We hypothesize that antigen-specific tolerance initiates in the intestinal immune
system and then spreads to the periphery by T cells disseminating into pancreatic lymph nodes. More
recently, we demonstrated also that administration of human GAD65 or IA-2 along with IL10 was able to
reverse diabetes in 56% of newly diagnosed diabetic NOD mice under low-dose anti-CD3 induction
therapy. On the contrary, a similar therapeutic set-up with a mosaic protein comprising fragments of 3 beta-
cell specific antigens (GAD65, IA2 and proinsulin) - fusion was not effective at all in reversal of recent
onset diabetes. These results might imply that the CD4+ T cell epitopes on the different beta-cell antigens
may need to be appropriately processed to fill the MHC class II peptide-binding site and to be presented by
the professional antigen presenting cells. Clinical grade strains of the L.lactis have been prepared by
Actogenix and have been tested or are being tested in mice and negociations on the design of a clinical trial
in humans with type 1 diabetes are ongoing with different parties and international regulators.
WP5 – Beta-cell protection and restoration: Dialogue with the immune system
In this distinctive work package, we focus on the role of the beta-cell in its own destruction and specifically
on the way in which the immune system and the beta-cell communicate. We have obtained significant
insights in the gene networks induced by IL1# and TNF# as well as genes downstream of STAT-1 and
IRF-1 using microarray and proteomic techniques in mouse and rat models for T1D and, in recent months,
by RNA sequencing of human islets. Also in human islets we are performing proteomic 2D-DIGE analysis,
to investigate the pathways involved in cytokine-induced beta cell death as well as the anti-inflammatory
role of GLP-1 thereon. A role for two candidate genes for T1D, namely PTPN2 and MDA-5, in beta cell
responses to viral infections has been identified. Novel experiments allowed us to identify the specific beta
cell apoptosis pathways triggered following inhibition of PTPN2 or during a viral infection, namely
activation of the BH3-only protein BIM. This was the first evidence that candidate genes for T1D may act
at the beta cell level, modulating both beta cell apoptosis and the virus-induced dialog between beta cells
and the immune system. New observations, obtained by RNA sequencing, indicate that >60% of the known
candidate genes for T1D are expressed in human islets and have detectable changes in expression
following exposure to pro-inflammatory cytokines. Further, the impact of 1,25(OH)2D3 on C57Bl6 mouse
islets, exposed to inflammatory cytokines (IL1β and IFNg), is being studied by microarray analysis. A
major impact was seen on chemokine and cytokine expression. Follow-up experiments are in progress.

Finally, experiments in a rat model for type 1 diabetes (LEW.1AR1-iddm) have been started and many new
tools (miRNAs) have been designed allowing better analysis of the dialogue between the beta cell and the
immune system. New experiments, utilizing pancreatic material from type 1 diabetic patients suggest that
some markers of ER stress are up-regulated in islets from T1D patients, providing clinical support for our
experimental approaches. Finally, collaborative work inside NAIMIT, involving WPs 2 and 5, has
identified a novel role for the Th17 cells and the cytokine IL-17 in the dialog between beta cells and the
immune system, which contributes to trigger insulitis and beta cell loss.
WP6 – Pharmacogenetics: towards individualised therapies
This WP is built on the hypothesis that interventions should be individualised and tailored to the genetic
footprint of the disease in any individual patient. The purpose is to link up with WP1 and WP2, to explore
possible genetic signatures to predict responses of DCs and T-cells to VitD and GCs, depending on the
presence of polymorphisms in crucial genes in the signal transduction and metabolism of these steroids.
During the first year of NAIMIT focus for this WP was concentrated on organising the logistic network
between the partners involved in WP6 and in establishing standard operating procedures on how to proceed
with optimal sample collection, in order to provide samples to the ‘genetics’ partners of every patient and
control donor where DC and T cells are being isolated. SOPs were established and major efforts on
minimising blood volume were made. During year two and three different batches of blood samples from
healthy controls and T1D patients have been genotyped for HLA, VitD and GC polymorphisms. The
results are at present being linked to functional DC and T-cell parameters. In the meantime, further
characterisation of the vitamin D related genes that are involved in DC and T cell behaviour are being
studied with preliminary results for vitamin D pathway gene expression analysis of lymphocytes from
healthy controls in relation to genotypes. Vitamin D concentrations in 3,000 plasma samples from donors
from the Cambridge BioResource have been measured, so that we can recall individuals with very low and
optimal levels of vitamin D to assess the effects of vitamin D on the immune system using polychromatic
flow cytometry. Also immunophenotyping of individuals (flow cytometric analysis) in a trial in London
and Cambridge before and after vitamin D supplementation is ongoing.
Expected final results and potential impacts
The predicted impact of the present project is great, both on a scientific and a therapeutic level. We have
managed to execute this work with great energy, with all partners contributing substantially to the goals of
the project. The cell therapies, the antigen based approaches, the exploitation of the natural
immunomodulators and of great interest, the introduction of the new tools for tolerance induction, all hold
great promise. Research is progressing well in all work packages, and the first steps to the clinic are within
reach.

Source: http://naimit.eu/http://naimit.eu/wp-content/uploads/Our-results-year-3-dec-2012.pdf