November 21, 2019

Crohn’s the thymus already expressing FOXP3, while induced Treg

Crohn’s
disease (CD) is an Inflammatory Bowel Disease (IBD) distinguished by
discontinuous excessive inflammation along the gastrointestinal tract (GI). Advanced
or untreated CD, commonly leads to scar formation or ulcers, further resulting
in intestinal stricture and bowel obstruction. 
In such cases, surgery is required to remove the affected region of
either small or large intestine.  Currently,
the most prevalent treatments against CD include anti-inflammatory drugs
(corticosteroids) and immunomodulators (anti-TNF), however efficient outcomes
appear to a proportion of patients. 
Additionally, CD approximately affects 2.5 million individuals of any
age in the Western world and has an accelerating increase in incidence in the
developing world.  The above evidence illustrates
the necessity of discovering novel therapies in order to bear a spectrum of
alternative treatments that can be implemented according to the individual. 

The
aetiology as well as pathogenesis of CD, and more generally of IBD is ambiguous
and complicated.  The most accepted
hypothesis suggests that the synergy between genetic and environmental factors
trigger disease onset by stimulating continuous expression of pro-inflammatory
cytokines.  Under normal conditions, the
intestinal mucosal barrier is essential for preventing invasion of commensal bacteria
in the epithelial cells.  This is
achieved through maintaining immune self-tolerance. However, the aforementioned
risk factors of CD cause imbalances of the gut microenvironment, leading to
increase of intestinal permeability which results in dysfunction of mucosal
barrier and subsequent breakdown of tolerance (Wang et al, 2016).  Therefore,
the breakdown of tolerance elicits excessive immune responses.  According to this, GWAS-studies indicated
that genetically susceptible individuals may carry specific genetic
polymorphisms related to bacteria recognition, further promoting tolerance
impairment. CD is considered a cell-mediated disease, as Th1 and Th17 responses
are predominantly associated with its pathogenesis, illustrated in Figure 1. 

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The
balance between Treg/Th17 cells is significant for mucosal homeostasis, through
sustaining the equilibrium among pro- and anti-inflammatory cytokines.  Dependent on the microenvironment, the pro-inflammatory
responses of Th17 cells are restricted by the activity of Tregs, and vice versa
(Ziegler and Buckner, 2009).  This is achieved in the presence of TGF?, a common mediator significant for Th17
and Treg differentiation.  Th17 cells are
thought to be originated from a subset of naïve T cell expressing the lectin
receptor, CD161 as well as the transcription factor ROR?t (Cosmi
et al, 2008).  Their differentiation depends
on the presence of the pro-inflammatory cytokines, IL-1?, IL-6, IL-21 and TGF?. Following activation, Th17 cells express
high levels pro-inflammatory of IL-17, IL-21, and IL-22 which have an essential
role in host defences against extracellular pathogens by triggering further
recruitment of innate immune cells (Galvez,
2014).  In contrast, regulatory T
cells (Tregs) are divided into two categories; natural and induced.  Natural Tregs (nTreg) arise from the thymus
already expressing FOXP3, while induced Treg (iTreg) develop from mature CD4+
effector T cells in the periphery.  Treg
differentiation in the periphery is achieved upon exposure to high
concentrations of TGF?.   FOXP3+ Treg’s main function involves regulation
of excessive immune responses as well as maintenance of self-tolerance to
prevent autoimmune disease development. 
This is achieved through TGF?
and IL-10 anti-inflammatory cytokine secretion at the site of inflammation (Yadav, Stephan and Bluestone, 2013).

 

During
CD, mucosal barrier dysfunction alters the Th17/Treg commitment towards Th17-induced
inflammatory response, and thus significantly eliminating iTreg within the
intestinal lamina propria.  As mentioned
before, even if these both subtypes exhibit contradictory functions, TGF? is required
for their differentiation.  At low
concentrations, TGF? synergizes with IL-6 and IL-6-dependent IL-21 to promote
IL-23 receptor expression, and thus Th17 differentiation.  On the other hand, at high concentrations of
TGF? and absence of IL-6 or IL-21, IL-23R is supressed resulting in inhibition
of ROR?t and induction of iTregs (Omenetti
and Pizzaro, 2015).  This suggests,
that Th17 cells can inhibit Tregs by the expression of IL-21, while Tregs through
IL-10 production (Raza et al, 2012).  However, in CD even if TGF? levels are elevated, the downstream signalling
pathway is destructed by the blockage of smad3 phosphorylation.  This is achieved by the up-regulation of a natural
intracellular inhibitor of smad signalling, known as smad7 which acts as a negative
feedback.  Interestingly, both iTreg and
Th17 cells illustrated of being instable with a degree of trans-differentiation
into other CD4+ subtypes.  To illustrate this,
a fraction of FOXP3+ Tregs showed high-degree of plasticity towards IL-17-producing
cells as well as loss of FOXP3 expression. 
This is caused due to the accumulation of Th17-generating cytokines, as
plasticity may be an adaptive mechanism (Ueno
et al, 2015).  Oppositely, a research
indicated that under homeostatic conditions, Th17 cells can be converted into CD4+
Foxp3- type1 Tregs (Tr1) in the presence of TGF? and aryl hydrocarbon receptor (AhR) (Gagliani et al, 2015).  As Tregs expressing IL-17 were found
up-regulated in IBD patients, trans-differentiation can be utilised as a
potential therapeutic target.

IL-35
is a novel anti-inflammatory cytokine, member of IL-12 family.  It is a heterodimer containing p35 and
Epstein-Bar virus-induced gene 3 (EBI3) subunits while the IL-35 receptor
requires the dimerization of gp130 and IL1R?2 domains.  A recent study, illustrated that this
cytokine is essential for Tregs to achieve their maximum regulatory activity,
both in vitro and in vivo. IL-35 was found to be mainly
produced by Tregs and B regulatory cells (Bregs) but not from effector cells.  Once IL-35 engagement to the receptor, STAT1
and STAT4 form a unique heterodimer which results in a continuous
feedback-loop, promoting IL-35 expression. 
Research has also observed expression of IL-35 in a population of IL-35-only
induced CD4+ Tregs, referred as iTr35 cells (Collison et al, 2010).  IL-35-induced cells were indicated of being
able to supress both CD4+ and CD8+ T cells proliferation, as well as their
differentiation into Th17 cells.  Ebi3
deficient mice have a significant increase in the production of IL-17.  This is achieved through Treg expansion and
increased production of IL-10. (Oslon,
Sullivan and Burlingham, 2013).  In
models of inflammatory bowel disease, IL-35 gene therapy and the adoptive
transfer of IL-35-expressing Tregs have been shown to cure colitis
symptoms.  According to the ability of
IL-35 of inducing the autologous Breg, IL-35+ Breg cells, as
well as, iTr35 can be utilised to eliminate excessive immune responses and
allow mucosal healing (Choi, Leung and
Bowlus, 2016).

All
the above indicate that cytokines are a key element underlying CD pathogenesis.
A treatment based on elevating TGF?
levels in the inflamed mucosa will be inefficient, as smad7 will interrupt its
signalling.  For this reason, an
alternative aspect must be identified to increase targeted-immunosuppression.  T cell redirected for universal-mediated
killing (TRUCKs) can be used to direct IL-35, specifically to the inflamed area
and expand regulatory cells without TGF?
signalling requirement.   Additionally,
introduction of anti-IL-6R monoclonal antibody can prevent Th17 differentiation
and plasticity, permitting further production of anti-inflammatory
cytokines.  

 

 

TRUCKs

TRUCK
is a fourth-generation chimeric antigen receptor (CAR)-redirected T cell which causes
the release of any transgenic product such as cytokines to promote accumulation
in a targeted tissue, following T cell activation.  Generally, CARs are consisted of an
ectodomain, a transmembrane domain, and an endodomain as shown in Figure 2.  The ectodomain is a variable portion of both
heavy and light chains of an immunoglobulin known as scFv.  The transmembrane domain, mostly CD28 is connected
to the artificial binding domain in order to provide stability to the
receptor.  Lastly, the endodomain is the
functional end of the receptor which is the CD3? component along with three
immunoreceptor tyrosine-based activation motifs (ITAMs), essential for T cell
activation.  In contrast to CARs, TRUCKs
in this case require the transfer of two trans-genes; CAR and IL-35.  To avoid transactivation of the cytokine
cassette by the CAR promoter, the two genes must be integrated at different
genomic sites.  Additionally, the nuclear
factor of the activated T cell (NFAT)-responsive expression cassette must be utilised
for inducing the expression of the cytokine.  

Leukapheresis
can be used to collect leukocytes from the patient by which CD4/CD8 composition
naïve T cells are separated through antibody bead markers.  T cell activation and expansion can be
achieved in vitro by either purifying
patient’s antigen presenting cells (APC) or using beads coated with
anti-CD3/anti-CD28 monoclonal antibodies. 
The combination with IL-2 growth factors, will further induce increase T
cell growth.  The delivery of foreign
genes into naive T cells can be achieved either using a viral or a non-viral
vector system.  The most commonly used
vectors are the genetically engineered retroviruses such as lentivirus.  Lentivirus vector system has the safer
integration site profile, as it has the lower-risk for insertional mutagenesis
caused by the integration of vector DNA into host cells.  Following T cell activation, the cells must
be infected by the recombinant virus vector encoding both the IL-35 and CARs.
As IL-35 is consisted of two subunits, the cytokine will be expressed in the
single chain format composed of covalently linked (p35-EBI3) chains.  Upon infection, the RNA will be
reverse-transcribed into DNA and permanently integrated into the genome of the
patient’s cells.  For TRUCK-T cell
culture, the CliniMACS bioreactor system can be used.   It is
a single device that can effectively enrich, activate, and expand the cells to
reach a clinically-approved threshold, significant process for transfusion into
the patient.  Transfusion can be carried
out through an intravenous infusion to the beforehand. 

The
main aim of TRUCKs is the accumulation of any cytokine to promote a second
immune response in a locally restricted manner, as the ideal outcome is to
cause the dampening of excessive inflammation by avoiding any systemic
cytotoxicity.  Several clinical trials
illustrated that direct administration of cytokines, usually result in cytokine
release syndrome accompanied by other adverse side effects. In this case, TRUCKs can be efficient even at low
levels. In comparison with tumour-infiltrating lymphocytes (TIL) IL-12 therapy,
the cell doses used for TRUCK-induced IL-12 were 50- to 100- fold lower.  Another advantage of TRUCKs, is the fact that
re-administration may not be required, since the targeted antigen will be continuously
released, further promoting T cell activation and differentiation (Chmielewski and Abken, 2015).  Integrin a4?7 and CCR9 are key homing receptors for
lymphocyte migration to gut mucosa.  The
induction of Vitamin A metabolite, known as Retinoic Acid (RA) can cause the
expression of these receptors, as a safety measure to prevent any ‘on-target
off-tumour’ TRUCK activation (Calisto et
al, 2011). 

 

 

 

 

IL-6 Inhibitor

Overexpression
of the pro-inflammatory cytokine IL-6 was found to be highly associated with CD
pathology, as it’s a major mediator involved in Th17 differentiation as well as
plasticity.  The IL-6 receptor (IL-6R) is
consisted by two chains; the IL-6 binding subunit and the membrane glycoprotein
gp130.  Additionally, IL-6 can achieve trans-signalling
through the presence of a naturally occurring soluble IL-6R (sIL-6R), even in
the absence of membrane IL-6R. 
Tocilizumab is a humanised anti-IL6R monoclonal antibody of lgG1 class
which inhibits signal transduction through blocking IL-6 attachment to both
membrane and soluble IL-6R (Tanaka,
Narazaki and Kishimoto, 2011).  Favourably,
the function of sgp130 is not altered by the inhibitory effects of tocilizumab,
as it is an essential subunit for IL-35R signalling (Hashizume and Mihara, 2009). A randomised trial of the drug was
executed which demonstrated ideal clinical responses to approximately 80% of
patients, suggesting its effectiveness.   However, endoscopic and histologic healing
was not observed, indicating the necessity for improvement (Ito et al, 2004).

Animal Model

To
investigate the possible effects of the drug, a suitable animal model is
required which predominantly represent characteristics of CD pathogenesis.  The chemically-induced colitis hapten reagent
2,4,6-trinitrobenzene sulfonic acid (TNBS) mouse model is commonly manipulated
for investigating immunologic aspects of the disease such as cytokine secretion
and potential immunotherapeutic effects. 
However, the pathogenic mechanism towards CD-like disease, remains
unclear.  The Th1 phenotype is the major
response which further promotes secretion of potent pro-inflammatory cytokines
to the colonic tissue, mostly TNF? and
IL-12.  Ideally, IL-17 was shown to be
essential for the development of acute colitis in TNBS-treated mice, as IL-17R-/-
mice treated with TNBS indicated mild inflammation, in contrast to the wild-type.  Thus, Th17 cells also demonstrated an
effector function in TNBS-inducing colitis. 
A research group observed that p19 (IL-23R subunit) deficient mice,
developed exacerbate colitis and suggested that Th17 can carry out both pro-
and anti-inflammatory responses.  However,
IL-23 was shown to be significant for activation of Group 3 Innate Lymphoid
Cell (ILC3) which further secrete the protective cytokine, IL-22.  This provides a possible explanation
regarding the   

Recently,
a study showed that TNBS-induced colitis mouse model with high salt diet (HSD),
established increased Th17 responses, Treg dysfunction, and thus intensify
immune responses (Wei, et al, 2017).  This can be used as a method to
strengthen mouse’s inflammatory responses, generating a more ultimate animal
model for drug administration.  Following
drug application into the TNBS mouse, the TRUCK must recognise a
tissue-specific antigen to prohibit ‘on-target off-antigen’ activation in
healthy tissues.  The regenerating
islet-derived type 4 (REG-4) is a protein constitutively produced by the
enteroendocrine cells in colon epithelial (Granlund,
et al, 2013).  Studies indicated that
REG-4 is highly expressed during colon inflammation and its associated with
poor prognosis in colorectal cancer (Que
et al, 2007).  The particular protein
can be found in the colon of both mice and humans, thus it can be considered a
suitable candidate for TRUCK-recognition.  

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