Clinical trial NCT04177095
Conditions: Kidney Transplant Rejection Immunosuppression
Sponsor: Massachusetts General Hospital
Start date: 2020-02-11; Completion date: 2023-06-01
Enrollment: 17 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
* To determine the utility of novel blood-based immune monitoring tools (Allosure and Trugraf) to facilitate belatacept monotherapy.
* To determine the percent of belatacept-treated renal transplant patients that can be safely converted to belatacept monotherapy.

Detailed description:
This study will examine whether renal transplant recipients treated with a belatacept-based immunosuppressive regimen can safely be weaned off all non-belatacept immunosuppression (mycophenolate, mTORi, prednisone) in a stepwise fashion. To this end, participants will undergo monthly "immune monitoring" using the Allosure (dd-cfDNA) and Trugraf (RNA profiling) blood tests to determine if they are in a state of immune quiescence. Only patients who are deemed to be immune quiescent, will continue to be weaned of non-belatacept immunosuppression.

Arms: arm 1: Renal transplant recipients treated with a combination of belatacept and any of the following: mycophenolate, sirolimus, everolimus and prednisone
Interventions: intervention 1: Allosure intervention 2: Immunosuppression reduction intervention 3: Trugraf
Intervention descriptions: intervention 1: Monthly monitoring of dd-cfDNA levels in blood intervention 2: Stepwise reduction in the dose, and ultimate discontinuation of, all non-belatacept immunosuppression (mycophenolate, sirolimus, everolimus, prednisone) guided by monitoring of Allosure and Trugraf results intervention 3: Monthly monitoring of Trugraf result

Protocol documents:
Prot_SAP_000.pdf: 
1 
 
Study protocol 
Immune monitoring to facilitate belatacept monotherapy 
1. Synopsis 
 
Title 
 
 
Immune monitoring to facilitate belatacept monotherapy   
Study design 
 
Prospective single-center, single-arm pilot study 
Study center 
 
Massachusetts General Hospital 
Principal investigator 
 
Dr Hannah Gilligan 
Co-investigators 
 
Dr Thomas Vanhove 
Objectives 
 
1. To determine the percent of belatacept-treated renal 
transplant patients that can be safely converted to 
belatacept monotherapy 
2. To determine the utility of novel immune monitoring 
tools to facilitate belatacept monotherapy 
Primary endpoint  
 
Occurrence of biopsy-proven acute rejection 
Secondary endpoints 
 
- 
Change in eGFR (CKD-EPI) 
- 
Appearance of proteinuria 
- 
Appearance of de novo donor-specific antibodies  
- 
Overall and death-censored graft survival 
Sample size 
 
20 
Summary of inclusion criteria 
 
- 
Age 18 years or older 
- 
Written informed consent 
- 
Single kidney transplant recipient (i.e. no combined 
organ transplants) 
- 
Treated with belatacept  
- 
At least 1 year after transplantation or after initiation 
of belatacept 
- 
Stable renal function  
- 
Blood biomarkers indicate immune quiescence  
- 
No history of biopsy-proven acute rejection  
- 
Absence of donor-specific antibodies (at any MFI) 
- 
No history of BK viremia in current allograft 


2 
 
- 
Spot urine protein/creatinine ratio < 0.5 g/g 
Maximum 
duration 
of 
follow-up 
per 
participant 
 
24 months after inclusion 
Protocol version 
 
Version 1.4 (10/25/2019) 
Version and date of protocol amendments 
 
N/A 
 
2. Background 
Belatacept is a selective costimulation blocker that is a non-nephrotoxic alternative to the calcineurin 
inhibitors cyclosporine and tacrolimus. In renal transplantation, belatacept has demonstrated similar 
graft and patient survival compared with cyclosporine, but is associated with superior renal function 
and lower rates of donor specific antibody (DSA) development.1,2 
Like calcineurin inhibitors, belatacept is generally prescribed as part of an immunosuppressive regimen 
that includes either mycophenolic acid (MMF) or and mTOR inhibitor (mTORi) and steroids. Clinical 
experience suggests that in a significant proportion of belatacept-treated patients, MMF, mTORi 
and/or steroids can be reduced or even stopped, likely because they have reached a state of partial 
immunological tolerance. However, true personalization of immunosuppressive therapy, i.e. delivering 
the minimum amount of immunosuppression necessary to prevent kidney graft rejection for any given 
patient, is not yet possible because of a lack of reliable tests to monitor the degree of alloimmune 
reactivity in a kidney graft. So-called protocol biopsies are useful in this respect, but are invasive and 
suffer from sampling bias and inter-observer variability. 
Several non-invasive immune monitoring tools have recently been developed that may aid in the 
personalization of immunosuppressive therapy. Two in particular are the focus of the current study. 
First, the presence of donor-derived cell-free DNA (dd-cfDNA) in the blood of kidney transplant 
recipients has been demonstrated to reflect graft injury.3–6 Very high values are a strong predictor of 
antibody-mediated rejection and low values (<1%) exclude active rejection with a negative predictive 
value of up to 98%.7 Second, a blood-based gene expression array (Trugraf™) can predict subclinical 
rejection in stable allografts with a negative predictive value of 78-88%.8  
Whether patients with both dd-cfDNA levels < 1% and a Trugraf profile compatible with a state of 
immunological quiescence can safely undergo a reduction in the intensity of their immunosuppressive 
regimen has not been explored. In the current study, this hypothesis will be tested in belatacept-
treated patients because they have a relatively high a priori likelihood of being in a state of 
immunological quiescence, which is also reflected by low rejection rates (<1%) after the first 
posttransplant year.9  
3. Trial objectives and Design 
3.1 Trial objectives  
- 
To determine the utility of novel blood-based immune monitoring tools (Allosure and Trugraf) to 
facilitate belatacept monotherapy. 


3 
 
- 
To determine the percent of belatacept-treated renal transplant patients that can be safely 
converted to belatacept monotherapy. 
 
3.2 Trial design 
Prospective single-center, single-arm interventional pilot study 
3.3 Primary endpoint at 24 months 
- 
Occurrence of biopsy-proven acute rejection 
3.4 Secondary endpoints at 24 months 
- 
Change in eGFR (CKD-EPI) 
- 
Appearance of proteinuria 
- 
Appearance of de novo donor-specific antibodies  
- 
Overall and death-censored graft survival 
4. Selection and withdrawal of subjects 
4.1  Inclusion criteria 
- 
Age minimum 18 years 
- 
Written informed consent 
- 
Single kidney transplant recipient (i.e. no combined organ transplants) 
- 
Treated with belatacept  
- 
At least 1 year after transplantation or after initiation of belatacept 
- 
Stable renal function (eGFR > 40 ml/min continuously during previous 6 months) 
- 
Blood biomarkers indicate immune quiescence (for Allosure this corresponds to dd-cfDNA < 1%; 
for Trugraf this corresponds to “TX” signature) 
- 
No history of BK viremia in current allograft 
 
4.2 Exclusion criteria 
- 
History of biopsy-proven acute rejection  
- 
Presence of donor-specific antibodies (at any MFI) 
- 
Spot urine protein/creatinine ratio > 0.5 g/g 
 
4.3 Selection of participants 
Belatacept-treated single kidney transplant recipients will be recruited from the outpatient clinic. 
Eligible patients are preselected through review of medical records. They are informed of the 
possibility to participate by a physician (the co-investigator, who is not their own nephrologist) at the 
time of an outpatient clinic. They will be given a copy of the informed consent and asked to call the co-
investigator if they are interested in participating.  


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4.4 Withdrawal of subjects 
Subjects will be withdrawn from the trial in case of: 
- 
Refusal of the subject to continue 
- 
For any other reason deemed necessary by the investigator and in the interest of the subject. 
In the following circumstances, subjects will continue to be monitored in the study but 
immunosuppression will not be weaned further: 
- 
Either one of the immune monitoring tests are positive at any point (Allosure > 1% or Trugraf 
signature compatible with rejection)  
- 
Occurrence of biopsy-proven acute rejection (Banff 2017 criteria) 
- 
Occurrence of new proteinuria > 0.5 g/g creatinine, confirmed on repeat urine sample after 2-4 
weeks 
- 
Occurrence of de novo donor-specific antibodies at any MFI 
- 
Decrease in eGFR > 20% from baseline without alternative explanation, confirmed on repeat 
urine sample after 2-4 weeks 
 
5. Trial Procedures 
5.1 By visit 
For all belatacept-treated patients, monthly visits in the outpatient clinic, where belatacept infusions 
are administered, are part of routine clinical care. Trial visits will coincide with these infusion visits, 
occurring monthly for the first year after inclusion. At study visits, subjects are questioned and 
examined by a physician and blood samples are drawn (see below). For patients who receive monthly 
belatacept infusions at home, they will have blood samples (clinical and research specimens)  drawn 
at their local Quest laboratory and the study physician will call them to assess for adverse events. If 
any adverse events are noted, a telemedicine or in-person visit with a transplant nephrologist will be 
scheduled at the discretion of the investigator. 
Reduction of immunosuppression occurs in a stepwise fashion, whereby each reduction is contingent 
on several factors: 
- 
Renal function is stable, defined as eGFR no more than 20% lower compared to value at 
inclusion 
- 
Both the Allosure and Trugraf tests indicate a state of immune quiescence. For the Allosure, 
this corresponds to a dd-cfDNA concentration of < 1%; for the Trugraf test, this corresponds 
to a “TX” signature.  
- 
No appearance of any de novo DSA (at any MFI) at any previous point during study 
- 
No proteinuria > 0.5 g/g creatinine on spot urine sample 
If, at any point, a patient does not fulfill all of the abovementioned criteria, immunosuppression will 
not be (further) reduced. In such cases, the principal investigator will decide whether to continue with 
the reduced-intensity immunosuppressive regimen or revert back to conventional triple therapy 
(belatacept + mycophenolate/mTORi + steroids).  


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Criteria for performing a kidney biopsy are: 
- 
Unexplained decrease in eGFR > 40% from baseline value 
- 
Occurrence of new proteinuria > 1.0 g/g creatinine on 2 separate random urine samples 
The principal investigator can decide to perform a kidney biopsy in patients who do not strictly meet 
the abovementioned criteria, if the probability of rejection is felt to be high, i.e. a combination of 
abnormalities such as new DSA and proteinuria < 1 g/g, or a very high (>3%) Allosure result combined 
with a mild (20%) decrease in eGFR. 
In case of a discrepancy between Allosure and Trugraf results (i.e. Allosure < 1% but Trugraf positive 
for rejection), immunosuppression will not be weaned further.  
 
Study timeline 
Time 
point 
Blood sample 
including immune 
monitoring 
Urine 
protein 
DSA 
monitoring 
Action if stable renal function, 
immune quiescence, no de novo 
DSA, no proteinuria > 0.5 g/g 
Inclusion Yes 
Yes 
Yes 
 
Month 1 
Yes 
Yes 
 
50 % reduction of MMF/mTORi 
dose 
Month 2 
Yes 
 
 
 
Month 3 
Yes 
 
Yes 
 
Month 4 
Yes 
Yes 
 
Stop MMF/mTORi 
Month 5 
Yes 
 
 
 
Month 6 
Yes 
 
Yes 
 
Month 7 
Yes 
Yes 
 
Reduction 
in 
prednisone 
to 
alternating 5mg – 2.5mg  
Month 8 
Yes 
 
 
Reduction 
in 
prednisone 
to 
2.5mg 
Month 9 
Yes 
 
Yes 
Reduction 
in 
prednisone 
to 
2.5mg – 0mg alternating 
Month 
10 
Yes 
 
 
Stop steroids 
Month 
11 
Yes 
 
 
 
Month 
12 
Yes 
Yes 
Yes 
 
 
 
From months 13 through 24, subjects will resume routine (standard of care) clinical monitoring, 
namely monthly laboratory analyses when they get their belatacept infusion. In addition to this, 
proteinuria and DSA will be checked at 24 months (which is not standard of care). 
5.2 Laboratory testing and sample shipping 
The following laboratory tests are performed at each study visit during the first 12 months: 


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- 
Routine laboratory analyses, including complete blood count, basic metabolic panel, total protein, 
fasting glucose. These analyses will be performed by the Central Laboratory of Massachusetts 
General Hospital or at the patient’s local Quest laboratory. 
- 
Two 10 ml Streck Cell-Free DNA BCT® whole blood tubes are drawn and stored at 7°C pending 
shipment. Streck tubes are shipped at ambient temperature (between 6° and 37°C) to CareDx using 
the shipping material and instructions provided by CareDx. 
- 
Two 2.5ml Paxgene® whole blood tubes are drawn and stored at -20°C pending shipment. These 
tubes are shipped on ice (-20°C) to Transplant Genomics. 
- 
One 10 ml EDTA and one 10 ml SST tube will be collected, centrifuged at 4°C for 10 minutes at 
3000 RPM. The resulting 5 ml of EDTA plasma and 5 ml of SST plasma will be stored at -80°C 
pending possible future analysis (i.e. biobanked). 
Donor-specific antibody screening occurs at 1, 4, 7, 10, 12 and 24 months. These analyses will be 
performed by the HLA laboratory of Massachusetts General Hospital. 
 
6. Statistics 
Normality will be tested using the Shapiro-Wilk test. Average eGFR and proteinuria at baseline and 24 
months will compared using paired samples t test. A 2-sided p-value < 0.05 will be considered 
statistically significant. All analyses will be performed using IBM SPSS Statistics version 23 (IBM, New 
York, NY). Figures will be generated using Graphpad Prism version 6 (Graphpad Prism, La Jolla, CA). 
7. Direct access to source data and document  
The investigator and the institution will permit trial-related monitoring, audits, EC review, and 
regulatory inspections (where appropriate) by providing direct access to source data and other 
documents (e.g. subjects’ case sheets, blood test reports, X-ray reports, histology reports etc).  
 
8. Ethics and regulatory approvals  
The trial will be conducted in compliance with the principles of the Declaration of Helsinki (2013), the 
principles of GCP and in accordance with all applicable regulatory requirements. This protocol and 
related documents will be submitted for review to the Ethics Committee. 
The Study can and will be conducted only on the basis of prior informed consent by the Subjects, or 
their legal representatives, to participate in the Study. The Participating Site will obtain a signed 
informed consent form (ICF) for all subjects prior to their enrollment and participation in the Study in 
compliance with all applicable laws, regulations and the approval of the (local) Ethics Committee, if 
required. The Participating Site will retain such ICFs in accordance with the requirements of all 
applicable regulatory agencies and laws. 
The Investigator and the Participating Site will treat all information and data relating to the Study 
disclosed to Investigator in this Study as confidential and will not disclose such information to any third 
parties or use such information for any purpose other than the performance of the Study. The 


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collection, processing and disclosure of personal data, such as patient health and medical information 
is subject to compliance with applicable personal data protection and the processing of personal data. 
 
9. Data Handling and Management  
The principal investigator is responsible for monitoring efficacy and safety every time a patient 
presents to the transplant clinic, which in principle is monthly for the duration of the study. An adverse 
event tracking log will be kept and unanticipated adverse events will be reported to Partners’ IRB 
within 5 working days, in accordance with PHRC guidelines for adverse event reporting.  
The PI is responsible for monitoring the validity and integrity of the data and adherence to the IRB-
approved protocol. 
Most of the information collected on subjects throughout this study is stored in the Electronic Medical 
Record (Epic) as part of routine clinical care. This includes vital parameters, laboratory results 
(including Allosure results, which are reported in Epic), reported adverse events and clinical findings 
on monthly outpatient visits. Relevant findings are extracted to an anonymized spreadsheet. Only the 
(co-)principal investigators will have access to the file that links patient names to anonymized 
identifiers. Paper informed consent documents are stored securely in the Department of Nephrology. 
10. Financial Aspects  
The study is financed by the companies CareDx and Transplant Genomics. 
11. References 
1. Durrbach A, Pestana JM, Pearson T et al. A phase III study of belatacept versus cyclosporine in 
kidney transplants from extended criteria donors (BENEFIT-EXT Study). Am. J. Transplant. 2010; 10: 
547–557. 
2. Vincenti F, Rostaing L, Grinyo J et al. Belatacept and Long-Term Outcomes in Kidney 
Transplantation. N. Engl. J. Med. 2016; 374: 333–343. 
3. Bloom RD, Bromberg JS, Poggio ED et al. Cell-Free DNA and Active Rejection in Kidney Allografts. J. 
Am. Soc. Nephrol. 2017; 28: 2221–2232. 
4. Whitlam JB, Ling L, Skene A et al. Diagnostic application of kidney allograft-derived absolute cell-
free DNA levels  during transplant dysfunction. Am. J. Transplant 2019; 19: 1037–1049. 
5. Huang E, Sethi S, Peng A et al. Early clinical experience using donor-derived cell-free DNA to detect 
rejection in kidney transplant recipients. Am. J. Transplant 2019; 19: 1663–1670. 
6. Gielis EM, Ledeganck KJ, Dendooven A et al. The use of plasma donor-derived, cell-free DNA to 
monitor acute rejection after kidney transplantation. Nephrol. Dial. Transplant 2019. 
7. Oellerich M, Shipkova M, Asendorf T et al. Absolute quantification of donor-derived cell-free DNA 
as a marker of rejection and graft injury in kidney transplantation: Results from a prospective 
observational study. Am. J. Transplant 2019. 


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8. Friedewald JJ, Kurian SM, Heilman RL et al. Development and clinical validity of a novel blood-
based molecular biomarker for  subclinical acute rejection following kidney transplant. Am. J. 
Transplant 2018.