Immune monitoring to characterize T-cell responses of kidney transplant patients during co-stimulation blockade by belatacept

Belatacept represents a potential new treatment option for renal transplant
recipients which addresses the current unmet need for an immunosuppressive
treatment that provides short-term outcomes comparable to calcineurin inhibitor
(CNI)-based immunosuppressive regimens with the potential to avoid the renal,
cardiovascular, and metabolic toxicities of CNIs. In the two phase III studies
in renal transplant recipients of kidneys from standard or extended criteria
donors (the so-called BENEFIT and BENEFIT-EXT trials), belatacept was
comparable to ciclosporin (CsA) with regard to kidney allograft and patient
survival. In addition, belatacept treatment resulted in clinically meaningful
reductions in the proportions of patients with advanced renal dysfunction,
defined as chronic kidney disease (CKD) stage 4 or 5. While rates of acute
rejection (AR) were higher with belatacept than CsA (approximately 3-10%
higher), the rates of rejection that led to severe renal dysfunction or
graftloss were low.

Central to our understanding of the immune responses after transplantation is
the emerging role played by specific cell subsets, such as T-effector (Teff)
cells, T-regulatory cells (Tregs), B-cells, and dendritic cells. These cells
determine the occurrence of allograft rejection and tolerance by their ability
to migrate towards immunological hot-spots where they interact with each other
and orchestrate immune reactions. It is now possible to prevent allograft
rejection by inhibiting activation of Teff cells via costimulation blockade.
Inhibition of the T-cell costimulatory pathway CD28-B7 blocks T-cell activation
and promotes anergy or apoptosis. The modified cytotoxic T-lymphocyte antigen-4
(CTLA-4)-Ig belatacept (Nulojix®) indirectly blocks CD28 signaling and is
approved for the prevention of rejection after kidney transplantation. Two
recently completed phase III trials of belatacept have shown that it is a safe
and effective immunosuppressant that leads to a significantly better renal
function (GFR, glomerular filtration rate) as compared to a CsA-based regimen
in kidney transplant recipients. Acute rejection occurred at a higher frequency
among belatacept-treated patients, but at rates generally thought to be
clinically acceptable (17-22% at 12 months in the belatacept groups versus
7-14% in the CsA-groups). Moreover graft and patient survival where comparable
between belatacept and CsA-treated patients.

These clinical findings may be explained by laboratory observations. Cells that
down-regulate inflammatory immune activity, the FoxP3+CD25+ Tregs, are
dependent on costimulation for their homeostasis and function. Blockade of
costimulatory pathways not only suppresses Teff cells, but may also impair
activation of Tregs. This is possibly a dose-related phenomenon, with higher
concentrations of belatacept inhibiting both proinflammatory costimulation as
well as the negative signal imparted by CD86 in the CD28-B7 interaction.
Furthermore, any beneficial immunoregulatory response may have been compromised
by propensity of anti-CD25 induction therapy (utilized in the trial) to
temporarily reduce the number FoxP3+CD25+ Tregs in the circulation which appear
to develop more easily in patients receiving CNI-free immunosuppressive
regimens. Another explanation for the clinical finding of a higher incidence of
acute rejection in belatacept-treated patients may relate to memory T-cells.
Upon restimulation, memory T-cells express a rapid and robust response to the
transplanted organ and therefore represent a significant barrier to successful
transplantation. Unlike naïve T-cells, memory T-cells are activated relatively
independently of CD28-mediated costimulation, and are therefore less
susceptible to belatacept-based immunosuppression.

A unique feature of CD28 pathway is the activation of the NF-*B and NF-*B-
regulated genes, e.g. those of the pro-inflammatory cytokines IFN-γ, TNF-alpha,
and IL-2. Furthermore, CD28-mediated signals lead to the enhancement of several
T-cell functions, including survival and regulation of both cytotoxic and
humoral T-cell responses. In the presence of T-cell help, B-cells react to
protein antigens and differentiate into plasma cells. This T-B-cell contact
depends on the interaction with the co-stimulatory molecules CD28, CD154
(CD40Ligand), inducible costimulator (ICOS), and programmed death (PD)-1, and
the cytokine IL-21. Particularly, the follicular helper T-cell (Tfh cell)
subset of CD4+ T-cells is specialized in the provision of help to B-cells.
However, activated memory T-cells express other co-stimulatory molecules: CD137
(4-1BB) and CD154. Both are members of the TNF superfamily of molecules and
enhances after binding, T-cell proliferation, IL-2 secretion, survival,
cytolytic activity and B-cell activity. Recently, it has been shown that
antigen-specific T-cells are present in CD154-expressing CD4+ T-cells and CD137
CD8+ T-cells. A strong association between acute cellular rejection and the
frequency of peripheral allospecific CD154 memory T-cells was reported.
Alloactivated T-cells can be measured after an activation step, followed by a
brief incubation period (16-24h) with alloantigen, and subsequent staining for
CD137 and CD154.

Here, we postulate that the balance between the effect of belatacept on
regulatory T-cell function and the relative insensitivity of memory T-cells for
belatacept leads to donor-specific alloreactivity. We plan to study the
peripheral CD28-dependent intracellular signalling pathway, regulatory T-cell,
memory T-cell and Tfh functions. The proposed study will identify patients at
risk for rejection and will at the same time unravel the complex network of
cellular interactions during belatacept treatment.

Here, we postulate that the balance between the effect of belatacept on
regulatory T-cell function and the relative insensitivity of memory T-cells for
belatacept leads to donor-specific alloreactivity. We plan to study the
peripheral CD28-dependent intracellular signalling pathway, regulatory T-cell,
memory T-cell and Tfh functions. The proposed study will identify patients at
risk for rejection and will at the same time unravel the complex network of
cellular interactions during belatacept treatment. The primary aim of this
study, therefore is to characterize the function and phenotype of peripheral
T-cells that determine rejection and graft acceptance during belatacept therapy.

In addition, throughout the clinical study in all patients both immunologic,
pharmacokinetic and pharmacodynamic data will be collected. Pharmacokinetic
analyses will include the belatacept serum concentrations, as well as the
concentrations of neutralizing anti-belatacept antibodies. Based on the PK
data, the time-varying exposure to belatacept will be described. Belatacept
exposure-pharmacodynamic effect relationships will be established, and the
influence of covariates including bodyweight, age, and albumin concentration
will be studied. Of main interest will be the influence of belatacept serum
concentrations on saturation of CD80 and CD86 on lymphocytes (flowcytometry)
and on immunological tests (see description of PD and immunologic parameters).

The primary objectives of this study are:

1) To determine the presence, frequency, characteristics of effector CD4+ and
CD8+ T cells
2) To analyze T-B cell interaction during co-stimulation blockade
3) To assess belatacept concentrations and neutralizing anti-belatacept
antibodies (PK study), as well as the saturation and functional consequences of
CD80/CD86 receptor blockade by peripheral blood cells (PD study).

This will be an exploratory, hypothesis-generating, open-label,
active-controlled, randomized-controlled, clinical trial in which a total of n
= 40 consecutive, kidney transplant recipients (meeting the inclusion criteria)
will be treated with either a belatacept-based immunosuppressive regimen
(intervention group; protocol A) or our standard, Tac-based immunosuppressive
regimen (control group; protocol B) after providing written informed consent.
Patients will receive the routine clinical care and will be admitted to
hospital at the designated time points to receive their belatacept
intravenously in a day care setting. In addition, blood will be drawn to study
the above-described immunological parameters at day 4, and month 1, 3, 6 and 12
after transplantation. Patients randomized to the control arm will receive our
standard immunosuppressive regimen consisting of Tac in combination with MMF,
prednisolone, and basiliximab. Blood will be drawn at the same time points as
in the belatacept-treated patients to study the immunological endpoints of
interest. Follow-up will be 12 months (the first posttransplant year).
Treatment with belatacept will be continued after the first posttransplant year
provided that patients are willing and the drug is reimbursed. It is an
investigator-initiated study made possible by a grant from the manufacturer of
belatacept.

Patients in the Belatacept group (group A; experimental groep) will be treated
as follows:
Belatacept: 10 mg/kg intravenously on days 0, 4, 15, 30, 60, and 90 and 5 mg/kg
intravenously on month 4, 5, 6, 7, 8, 9, 10, 11, and 12.

In addition, all patients will receive Basiliximab (20 mg intravenously on days
0 and 4), mycophenolate mofetil (MMF; Cellcept®; starting dose of 1000 mg
b.i.d. aiming for predose concentrations of 1.5 - 3.0 mg/L), and prednisolone
(50 mg intravenously b.i.d. on days 0, 1, and 2, followed by 20 mg orally once
daily, which will be tapered subsequently to 5 mg per day at month 3 after
transplantation. Patients will continue to receive a low dose of prednisolone
throughout the first posttransplant year (minimum 2.5 mg per day).

Patients in the control group (protocol B) will receive our standard
immunosuppressive regimen consisting of tacrolimus (Prograf®) with a starting
dose of 0.2 mg/kg per day in two equally-divided doses aiming for predose
concentrations (C0) of 10-15 ng/mL (week 1-2), 8-12 ng/mL (week 3-4), and 5-10
ng/mL, thereafter. In addition, all patients will be treated with basiliximab,
MMF and prednisolone in the same dose as that of patients in the belatacept
group (see above).

In the second year of this study we want to investigate whether there are any
changes in the immune system caused by treatment with Belatacept. In the second
year of this study, after discontinuing belatacept and the conversion to
tacrolimus we want to investigate whether there are any changes in the immune
system. To monitor this, extra blood samples will be drawn at month 18 and
month 24 and whenever rejection is suspected.

Patients will receive belatacept (Nulojix®) or tacrolimus (Prograf®). In
addition, all patients -both in the experimental and control groups- will be
treated with mycophenolic acid, prednisolone and basiliximab

The firm Bristol Myers Squibb supplied Belatacept for one year. As the health
insurance does not reimburse this treatment in the Netherlands at this moment,
all patients in this study will be converted to once-daily, modified-release
Tacrolimus at time point month 12.

Clinical trials investigating belatacept have demonstrated that the incidence
of acute rejection is higher compared to classical, CNI-containing
immunosuppressive regimens (about 3-10% higher). This adverse effect has to be
balanced against the better renal function and more favourable cardio-vascular
profile that was obtained during belatacept therapy. The rejection episodes
occuring during belatacept treatment responded, in general, well to
anti-rejection therapy.

Second, the incidence of posttransplant lymphoproliferative disorder (PTLD) was
higher in the two phase III clinical trials among patients who received
belatacept as compared to the control group who received a CsA-based
immunosuppressive regimen. However, the higher incidence of PTLD occured mainly
in patients who were EBV-seronegative prior to transplantation. In the current
study, EBV-naïve patients will not be included, as mandated by the package
insert of belatacept.

In addition and unlike patients receiving the standard, tacrolimus-based
therapy, patients that will receive belatacept will be admitted to hospital for
intravenous administration of the drug. This will be done in daycare setting
and will in the early phase after transplantation coincide with the
hospitalization for the transplantation itself. These admissions will be short
(3 hrs) and coincide with scheduled visits to the outpatient clinic for routine
follow-up after transplantation. Apart from the admission for administration of
the drug, the insertion of an i.v. cannula, and the drawing of additional blood
for immunological monitoring purposes (explained above), the patients will
experience no other burdens.