Biomarker-guided IS weaning strategy in long-term (>3yrs) liver transplant recipients by identification of operationally tolerant patients

chart 1 Withdrawal of IS in operationally tolerant long-term liver transplant patients prospectively identified by biomarkers (intragraft molecular signature)

About 25-40% of liver transplant patients after year 3-5 post transplantation develop operational tolerance. However, in the majority (60-75%), standard immunosuppression has to be re-introduced because weaning failed and rejection and graft deterioration occurred.

A diagnostic test for detecting tolerance capable of identifying operationally tolerant recipients before an attempt at immunosuppression withdrawal is made, would radically change the long-term management of liver transplant recipients and would have a great beneficial impact in the well-being and quality of life.
Before this novel strategy can be applied to routine clinical practice, it needs to be validated within a large clinical prospective multicentre study.

The response to this need is the LIFT study (Study I multicentre biomarker-driven clinical trial), a “Prospective randomised marker-based trial to assess the clinical utility and safety of biomarker-guided immunosuppression withdrawal in liver transplantation”

The main objective of the trial is to determine whether a novel molecular test of tolerance can be employed to optimize immunosuppression withdrawal.

Targeted partial/complete weaning of standard IS in long-term stable kidney transplant patients characterised as low-responders and identified as putative "operationally tolerant" by recently developed biomarker panel

chart 2 Partial / complete withdrawal of IS in operationally tolerant long-term kidney transplant patients prospectively identified by biomarkers

Withdrawal of IS in long-term kidney transplant patients is even more challenging: from thousands of kidney transplant patients living in Europe or US, only some dozen are stably free of IS. Using the recent European transplant network structures (IOT/RISET), members of the consortium got access to a significant number of samples and data from those patients and could define discriminative immune markers. It is important to highlight that the kidney markers are distinct from those in liver recipients. These data suggest that biomarkers might be useful for targeted minimization of IS in stable long-term kidney transplant patients as well.

The WEANING trial (Study II France multicentre randomized parallel controlled trial) aims at improving the graft function in clinically selected highly stable patients following complete weaning of calcineurin inhibitor (CNI). Major strength of this study is the fact that it will be the first time that a withdrawal study will be placebo controlled double–blinded. As a secondary endpoint, the study targets to validate predefined biomarker profiles regarding their predictive value for safe CNI weaning.

The background of the study in UK (Study III) are two other successful projects: GAMBIT and Indices of Tolerance (IOT).
At first, they have analyzed the IOT gene list in samples from GAMBIT patients, obtaining excellent replication with high predictive accuracy and good test-retest stability.
The next essential step was to check the potential confounding effects of immunosuppressive drugs on the gene expression of the candidate ‘tolerance genes’, by comparing the gene expression between those on and off any given drug.
The subsequent strategy was to repeat the feature selection exercise in the initial array (Indices of Tolerance) using drug-independent gene expression. A set of 28 drug-independent genes was selected as optimal for prediction of tolerance with perfect predictive accuracy, from which 9 genes were selected as optimal to predict tolerance.
A proof-of-concept study has demonstrated that the signature remains stable when comparing samples from patients before and after steroid withdrawal.
The new signature defined is immunosuppression independent and is “tolerance specific” as it is significantly different from controls.

Data of both studies will be merged for ontology analysis to define the best final test format. It allows the development of safe weaning strategies in kidney transplant patients. It can also be used for monitoring novel therapies aiming to accelerate tolerance induction.

Biomarker-guided stratification into low/high responder after kidney transplantation

chart 3 Prevention of high-dose standard IS in low-responder kidney transplant patients identified by perioperative patient stratification

The current immunosuppressive therapy consists mainly from combination of three to four immunosuppressant agents. This therapy is not only costly, but leads also to many undesirable side effects, which not only limit its efficacy (impossibility to titrate to the required dose) but decrease also the patients adherence to therapy.
Minimizing immunosuppression (IS), e.g. monotherapy, as early as possible without losing control of acute/chronic rejections would be of great benefit and could reduce adverse effects and costs. However, this is only possible in a minority of patients yet. Therefore, a precise evaluation of the anti-donor alloimmune response in order to identify patients likely to accept the graft with no or very low IS would be of great value. The IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay has been shown in multiple reports to be capable of accurately assess the presence of highly alloreactive circulating memory/effector T-cells with donor-antigen specificity.

With the CELLIMIN study we are performing an International multicentre open label randomized non-inferiority Phase II clinical trial to demonstrate the utility and safety of the IFN-γ ELISPOT assay for the stratification of kidney transplant recipients into “low” and “high” IS regimens.
Pre-transplant patient stratification using the IFN-γ ELISPOT may help to accurately discriminate patients that may safely benefit from receiving low IS based on induction therapy with basiliximab and low doses TAC monotherapy, from others that should stay on higher IS such as the current standard triple-therapy regimen sustained on basiliximab/TAC/MMF/steroids.
This trial is a biomarker-driven study with the aim of assessing the feasibility of standard IS minimization. Thus, it does not investigate immunosuppressive drugs.

Shifting kidney transplant patients to low-responders suitable for early IS minimization

chart 4 Increasing the proportion of low-responder kidney transplant patients suitable to low-dose IS (monotherapy) by selective targeting of (allo)memory T cells

We designed an International multicentre open label single-arm Simon´s two-stage Phase II clinical trial (RIMINI) to provide evidence for efficacy and safety of the induction regimen with rATG and infliximab and a Go/no-Go rule for further clinical development.

rATG is a polyclonal antibody licensed for prevention of acute rejection in kidney transplantation that has been shown to be an effective tool to decrease T and B cell populations and by that allowing safe reduction/minimization of other immunosuppression. Reducing clonal size by rATG followed by low-dose immunosuppression revealed promising results but not in all patients.
Memory/effector T cells are a major challenge. Therefore, increasing the pool of low-responders by selective targeting effector/memory T cells would have a big advantage.

In a pilot trial (RISET) we could demonstrate that a novel protocol based on the combination of clonal size reduction (Campath-1) and selective targeting of very recently activated (allospecific) effector/memory T cells as well as acute inflammation (anti-TNF mAb) allows safe monotherapy (low-dose tacrolimus) as early as from day 3 post-transplant in all kidney transplant patients, even in patients with high donor-specific IFN-g ELISPOT. Two-year data showed excellent graft function and histology and exceptional low side effect profile.

The design of the study based on the promising pilot trial data had to be adapted to using rATG for initial reduction of clonal size as Alemtuzumab is not available longer. This change might be associated with some risk to reproduce the promising data. Therefore, the RIMINI trial is designed as Simon`s two-stage trial with interims analysis.

Validating set-1 biomarker tests ready for decision making for the on-site patient stratification, improving personalised IS, within the clinical trials described in WP1-3

chart 5 Scheme of test, sample, data, and QM logistics for set-1 biomarker tests that will be applied for patient stratification in biomarker-driven studies of WP 1&2

Validated biomarkers (set-1) – as decision-making markers applicable for guiding minimizing IS:

dot Molecular “tolerance signature” in liver biopsy of liver transplant patients
dot Molecular “tolerance signatures” in peripheral blood of kidney transplant patients
dot Donor-reactive IFN-g ELISPOT

The markers were selected, following on from previous studies performed in the EU funded projects IOT, RISET. The tests are well validated methodically.

Validating recently established set-2 biomarkers and implementing new biomarker candidates for improving personalized IS within the BIO-DrIM clinical trials

This task aims to validate the recently developed set-2 biomarkers in prospective IS minimization trials (WP1-3) and to explore new biomarker candidates.

chart 6 Scheme of test, sample, data, and QM logistics for set-2 biomarker tests that will be validated for patient stratification/monitoring in clinical trials of WP 1-3

The analytical platforms for analyzing the recently discovered and novel biomarkers are:

dot Quantitative RT-PCR (molecular signatures)
dot miRNA quantification (molecular signatures)
dot Multiparameter flowcytometry (immune cell subset analyses)
dot Ligand assays in urine, serum, cell culture supernatant (ELISA, Multiplex-Assays)
dot Cell-mediated allospecific memory (IFN-g/IL-2 two-color ELISPOT)

Methodically validated biomarkers (set-2A) – applicable for prospective validation concerning their clinical value:

dot Molecular “tolerance signature” in peripheral blood of liver and kidney transplant patients
dot Molecular signatures in urine of kidney transplant patients
dot Regulation in donor-reactive IFN-g ELISPOT
dot Toag-1 gene expression for measurement of quiet (tolerant) vs. activated cell-mediated immunity
dot Multiparameter flowcytometry for analyzing immune cell subsets in peripheral blood

The markers were selected, following on from previous studies (IOT, RISET). Additionally, HLA-antibodies is analyzed by routine HLA-antibody assays (Luminex/CDC).

New biomarkers (set-2B) – markers applicable for retrospective validation: e.g. DC subsets, T-cell subsets, Treg subsets, miRNAs.

Using our biomarker platforms, we will implement such markers into the trials of BIO-DrIM for exploring their putative value in retrospective analyses.

Analysing the health-economic impact of biomarker-guided personalized IS

Within the project we are delivering health-economic data demonstrating the usefulness of implementing biomarkers into the management of IS (personalized IS) concerning the cost/benefit ratio. We perform health-economic analyses by using Micro Costing. The method attempts to measure costs and benefits of service as accurately as possible, by including all fixed and variable costs of care at local prices, given the institutional structure within which service and care are being given. It will also take into account country-specific rules, such as fixed prices for diagnostic procedures (point system), drugs, and in/out-hospital service.

Studying the mechanisms behind successful weaning (regulation/effector balance)

The BIO-DrIM consortium is delivering data for better understanding the mechanisms behind successful minimizing IS and to identify putative new biomarkers.

In addition, we want to get deeper insights into memory T cells and their link to immunosuppression.

Disseminating the results to scientific, patient and public community (and developing commercialization strategies by partnering with SMEs/industries

Part of the work is dedicated to dissemination actions that will spread the knowledge and the results generated by the project to wider community of end users and to the scientific community. We are going to share our results with the different communities and we have high commitment in developing commercialization strategies (diagnostic products, drug and biomarker diagnostic combinatory products, novel targets of IS drugs) by partnering with SMEs / industry. Thus, a big effort is also devoted to implementation activities in support of the SMEs and industrial partners involved, for ensuring the translation of the results generated into market products with consequent important benefits and revenue growth. The academic members of the BIO-DrIM consortium are well experienced in translation and commercialization of novel diagnostic and therapeutic tools, as shown by successful spin-off companies and commercial products on the market.

By early interaction with the SME´s we want to accelerate the progress within the added value chain of product development by early preparation of the transfer to industry standard of production and Q&M as well as to regulatory challenges (EMA). Final goal is the development of promising tests to the IVD test format alone or in combination with approved drugs or with new ones.

A big attention is dedicated to the consortium management, with the final aim of delivering the successful progress of the project within the agreed time, cost and quality limits.


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