Enhancing Treg Therapeutic Efficacy in GVHD

Abstract: This renewal continues our optimization of regulatory T cell (Treg) therapy for creating transplant
tolerance to prevent graft-vs-host disease (GVHD). Our first-in-human phase I CD4 thymic Treg (tTreg) and CD4
inducible Treg (iTreg) trials that showed reduction but not elimination of GVHD. Importantly, relapse rates were
not improved and decreasing GVHD but not relapse is an insufficient outcome. In this renewal, we will build
on our exciting finding that Tregs can acquire super-suppressor function and by incorporating an anti-CD19 scFv
CAR (CAR19) increase their potency of killing. In other studies, we developed highly suppressive CAR19 CD8
iTregs that have an intrinsic killing mechanism. Comparing the relative efficacy of these cell types, alone or in
combination, will allow us to initiate clinical trials using one product simultaneously suppress GVHD and
paradoxically kill lymphoma, solving the 2 major alloHCT limitations. A commonality in the diverse
approaches and Treg subsets that acquire super-suppressor function is the presence of a striking metabolic
effects. We discovered that targeting selected cell surface or intracellular proteins led to mitochondria fusion,
high oxidative phosphorylation (OXPHOS) and super-suppressor Tregs. Polar metabolite analytics pointed to
key KEGG pathways [serine-glycine-one carbon network], linked by methionine and folate cycles, trans-
sulfuration for glutathione, and alanine-aspartate-L-glutamate. A net result is one-carbon (1C) methyl, formyl, or
methyl group transfer for biosynthesis, control of gene expression and damaging redox reactions. CAR19
expressing CD4 tTregs and human CD8 iTregs that are highly suppressive and cytolytic to hCD19+ targets will
be studied in GVHD/GVL models. Notably, whereas activated CAR19 CTLs can cause severe cytokine release
syndrome (CRS), our preliminary data indicate that CAR19 CD4 tTregs produce low inflammatory cytokines and
do not cause CRS. Aim 1A tests the hypothesis that metabolic reprogramming of Tregs depends on purine
synthesis, mitochondria fusion and OXPHOS, and glutathione for redox balance. Aim 1B exploits Tregs
with 4-1BB vs CD28 and CD2) signaling domains that affect metabolic reprogramming and Treg
persistence for optimal GVHD/GVL. Aim 2 is focused on the killing mechanisms that these Treg subsets use
to lyse targets. Toward that end, we recently discovered CTL supramolecular attack particles (SMAPs) as a new
mechanism for more sustained killing. SMAPs are cytotoxic core-shell particles that contain granzyme B and
perforin and are secreted after triggering the immunological synapse (IS), the contact signaling point for T cells
and APC. Identifying integrated IS signals for SMAP release will provide insights into Treg killing mechanisms,
aiding Aim 1. Supported lipid bilayers simulating the IS will assess CD2 as an IS signal integrator/amplifier of
killing by analyzing dual CAR Tregs. Our central hypotheses are that Tregs reprogrammed in aim 1 for 1C-,
purine- and glutathione- metabolism have enhanced suppression and SMAP release, triggered by CD2
integrated and amplified IS signals and optimal GVHD/GVL.

Grant Number: 5R01HL118979-12
NIH Institute/Center: NIH
Principal Investigator: Bruce Blazar