Project Abstract
Type 1 diabetes (T1D) results from abnormal activation of self-reactive T effector lymphocytes (Teffs), which eliminate
pancreatic β cells, Suppression by regulatory T cells (Tregs) is a key mechanism limiting such aberrant autoreactivity.
People with T1D have quantitative and functional deficiencies of Tregs. Recent clinical trials of adoptive transfer of
autologous, ex vivo-expanded, polyclonal Tregs into T1D patients hinted that this may help preserve β cell function.
Our proposal targets enhancement and improvement of the current Treg therapies. We aim to create and validate a
novel, antigen-specific cellular therapy for T1D. Towards this goal, our team engineered Tregs bearing p cell-specific
Chimeric Antigen Receptors (CARs), to be tested herein. p cell-specificity is conferred by a single-chain variable fragment
(scFv) of an antibody recognizing unique human p cell marker NTPDase3. We hypothesize that β cell-specific CAR Tregs
will home to islets where CARs signaling will enable long-lasting antigen-dependent suppression in situ. We will test this
hypothesis in the following Specific Aims:
Aim 1. Test existing and newly generated, based on in silico predictions, B3s-CARs with range of binding affinities.
As binding of CARs to p cells that is either too strong or too weak is unlikely to provide adequate activation of CAR Tregs,
we will use computational modeling of our paratope sequence to design mutants with varying affinities to hNTPDase3. The
actual affinities of mutant scFvs will be compared by ELISA verified by surface plasmon resonance, to that of original
scFvB3s. Set of mutant scFvs ranging in their affinities will be built into CARs, transduced into T cells, which will be tested
in culture. CARs inducing, upon antigen exposure, activation, proliferation but not exhaustion ofT cells, will be prioritized.
Aim 2: Engineer β cell-specific CAR Tregs, testing CARs of varying affinities to optimize Treg function in culture
and in situ. We will produce and expand human Tregs bearing various CARs. We will then test them for activation,
proliferation, exhaustion, cytokine release, and the suppression of human islet-reactive Teffs in co-cultures with human β
cells and isolated islets. We will also test our CAR Tregs in situ on the live human pancreatic slices for migration to, and
retention within islets and for the local inhibition of human islet-reactive T cell clones.
Aim 3: Assess the function of islet-specific CAR Tregs in vivo in models of human islet transplants. We will evaluate
the kinetics/ dynamics of CAR Tregs horning and retention within human islets in mice transplanted with these islets in the
anterior chamber of the eye (ACE-platform). We will test the abilities of our CAR Tregs to restrain activity of human Teffs
and to preserve the transplanted islets in both allo- and auto-immune settings. Suppression of alloimmune attack will be
tested in NSG-MHcnun mice engrafted with PBLs and with islets originated from separate donors. Autoimmunity restrain
will be tested in the recently developed humanized HLA-DQ8-restrticed mice transplanted with HLA-matched human islets.
Completion of our project will confirm the feasibility of β cell-specific CAR Tregs, fully characterize them and evaluate
their therapeutic potency, and pave the road for future clinical trials of β cell-specific CAR Tregs in T1D.
