Clinical trial NCT03025828
Conditions: Membranous Nephropathy
Keywords: Membranous nephropathy adrenocorticotropic hormone ACTH proteinuria glomerulonephritis
Sponsor: Icahn School of Medicine at Mount Sinai
Start date: 2018-03-19; Completion date: 2020-12-16
Enrollment: 5 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to evaluate the effect of adrenocorticotropic hormone (ACTH, Acthar) on the loss of proteins in the urine (proteinuria) in patients with membranous nephropathy. Acthar is a hormone that stimulates steroid production from small glands above the kidneys. It has direct protective effects on the kidney and is currently approved by the FDA to treat kidney disorders associated with proteins in the urine, but the mechanisms of action are not entirely understood and will be studied in the present trial.

Detailed description:
Patients with membranous nephropathy and nephrotic syndrome will be treated with ACTH for 6 months. Proteinuria remission at 12 months will be the primary endpoint. Different biomarkers including anti-PLA2R autoantibodies, circulating regulatory T cells, and autoreactive memory B cells will be serially measured to identify predictors of response to therapy.

Arms: arm 1: Acthar will be administered subcutaneously (SC) 80 units for the first week and then 80 units twice weekly
Interventions: intervention 1: ACTHar
Intervention descriptions: intervention 1: for 6 months

Protocol documents:
Prot_SAP_000.pdf: 
Changes in Autoreactive Memory B Cells as Biomarker of Response to 
Adrenocorticotropic Hormone in Patients With Membranous Nephropathy
PI: Paolo Cravedi, MD, PhD
NCT03025828
Date: July 16, 2018


 
Page 1 of 5 
  
 
 
Changes in Autoreactive Memory B Cells as Biomarker of Response to ACTH in Patients with 
Membranous Nephropathy 
1. Hypothesis: 
We hypothesize that adrenocorticotropic hormone (ACTH, Acthar) reduces proteinuria in patients with 
membranous nephropathy by inhibiting autoreactive memory B cells and by increasing regulatory T cells. 
Therefore, the number of circulating autoreactive B cells can be used as a biomarker of disease activity and 
response to Acthar therapy. 
 
2. Rationale for the study: 
Membranous nephropathy (MN) is an autoantibody-mediated glomerular disease and is the major cause of 
nephrotic syndrome (NS) in adults worldwide. The disease is caused by the formation of immune deposits on 
the outer aspect of the glomerular basement membrane. These immune deposits contain podocyte antigens 
and circulating antibodies specific to those antigens, resulting in complement activation, podocyte injury, 
proteinuria, and renal failure. In 2009, podocyte phospholipase A2 receptor (PLA2R) was reported as the 
antigenic target in over 70% of adult patients with MN 1. The disease shows a benign or indolent course in the 
majority of cases, but about 30-40% of affected patients progress to end-stage kidney disease (ESKD) 5-10 years 
after diagnosis 2.  
Treatment with steroids in association with immunosuppressants including alkylating agents and 
calcineurin inhibitors has been suggested for patients with persistent NS 3. However, these medications are 
burdened by substantial toxicity with an excess risk of serious adverse events. Starting from the evidence that 
B cells play a crucial role in the pathogenesis of the disease, both as precursors of autoantibody-producing cells 
and as antigen-presenting cells, we performed a series of prospective studies showing that anti-CD20 B-cell 
depleting monoclonal antibody rituximab safely reduces proteinuria in MN patients 4-7. About 30% of patients 
treated with rituximab or other immunosuppressive agents, however, do not respond to these therapies or 
relapse after initial response, possibly because these therapies (including rituximab) are only partially effective 
in inhibiting memory B cells, a B-cell subset important in autoantibody production. 
Adrenocorticopropic hormone (ACTH) has been recently proposed as an alternative therapy for MN, 
after the incidental finding by Berg et al. that this hormone had antiproteinuric effects in 14 MN patients 8. This 
observation was thereafter confirmed by other retrospective 9 and prospective 10 studies. ACTH is produced by 
the anterior pituitary gland and induces cortisol production by the adrenal glands. Its antiproteinuric effects, 
however, seem not to be mediated by the increased cortisol synthesis, since steroid therapy alone has been 
proven to be ineffective in inducing remission or delaying end-stage renal disease for MN 11. ACTH is a universal 
agonist for the five melanocortin receptors (MC1R-MC5R). MC1R has been implicated in mediating ACTH 
protective effects on podocytes by reducing cell oxidative stress, apoptosis, injury and loss in the proteinuric 
kidney disease models 12. The podocyte protective effects of ACTH are at the basis of its antiproteinuric effects 
in a wide range of glomerular diseases, including, besides MN, IgA nephropathy, focal segmental 
glomerulosclerosis (FSGS), lupus nephritis, and minimal change disease.  
More importantly in the context of MN, published data by others and our preliminary data presented below 
show that ACTH has also direct anti-inflammatory and immunomodulatory functions on T and B lymphocytes, 
including reduction of pro-inflammatory cytokines, nitric oxide, adhesion molecules, and production of anti-
inflammatory IL-10 13. In patients with West syndrome 14, ACTH administration resulted in helper T cell and total 
B cell inhibition. Evidence is also available that ACTH inhibits the recruitment of memory B cells in the cerebral 
fluid of patients with opsoclonus-myoclonus syndrome 15. In a mouse model of autoimmune encephalomyelitis 


 
Page 2 of 5 
16, ACTH inhibited autoreactive Th17 cells and promoted a significant increase in regulatory T cells (Treg), a T 
cell subset with  important  immune protective roles in autoimmune diseases, including MN 17. These immune 
modulating effects are consistent with the reported reduction of anti-PLA2R autoantibodies described in MN 
patients treated with ACTH 10. 
Building on this evidence, we hypothesize that the antiproteinuric effects of ACTH in MN are mediated by 
the inhibition of autoreactive memory B cells and the promotion of Treg. These unique effects, together with 
ACTH direct nephroprotective activity on podocytes, make ACTH a valuable option for MN patients, including 
those who do not respond or relapse after other therapies targeting different pathways involved in MN 
pathogenesis. The number of autoreactive memory B cells could also represent a biomarker of disease activity 
and response to ACTH therapy. 
To address our hypothesis, we designed a mechanistic prospective, open-label study to evaluate the effects 
of Acthar on autoreactive memory B cells and regulatory T cells in MN patients. This study is expected 1) to 
provide crucial knowledge on the mechanism of action of Acthar in MN, 2) identify autoreactive memory B cells 
as a biomarker of response to Acthar, and 3) collect additional information on the safety/efficacy of this 
treatment.  
 
3. Patient population: 
This mechanistic, open-label study will include patients with histological diagnosis of primary membranous 
nephropathy (with renal biopsy performed no longer than 3 years prior) and persistent (>3months) proteinuria 
>3.5g/24h despite at least 3 months of ACE inhibitor or angiotensin receptor blocker therapy. The study will 
include both subjects naïve to previous treatments and subjects who received previous immunosuppressive 
treatment with steroids + alkylating agents (for at least 6 months), calcineurin inhibitors +/- steroids (6-12 
months), or rituximab (6 months). 
Inclusion criteria 
- 
Age 18 to 70 years 
- 
Free of immunosuppression for at least 3 months  
- 
Capability of understanding the purpose of the study 
- 
Written informed consent 
 
Exclusion criteria 
- 
eGFR < 30ml/min/1.73m2 
- 
Kidney Transplant 
- 
Secondary MN (defined on the basis of clinical criteria) 
- 
Type 1, uncontrolled Type 2 diabetes mellitus glycosylated hemoglobin (HbA1c) >8% (prior diagnosis of 
gestational diabetes mellitus is not an exclusion) 
- 
History of previous use of Acthar for treatment of nephrotic syndrome 
- 
Prior sensitivity to Acthar or other porcine protein products 
- 
Contraindication to Acthar per Prescribing Information 
- 
Planned treatment with live or live attenuated vaccines once enrolled in the study 
- 
More than three previous treatment regiments  
- 
Participation to other clinical trials over the previous 12 months 
- 
History of cancer, except carcinoma in situ and treated basal and squamous cell carcinomas 
- 
Pregnancy 
- 
Lactation 
- 
Current substance abuse 
- 
Any clinically relevant condition that might affect study participation and/or study results 
 
 


 
Page 3 of 5 
4. Study design 
 
Research strategy 
Clinical study design 
This will be a prospective-cohort study in which all eligible patients will receive Acthar therapy. After inclusion, 
patients will undergo monthly evaluations of clinical parameters (body weight and BP), biochemistry, 
proteinuria and serum creatinine up to month 3, then at month 6, 9, and 12 after treatment start. At baseline 
and at 6 and 12 months we will also collect blood (30ml) to 
measure circulating anti-PLA2R reactive memory B cells, Treg 
numbers, and anti-PLA2R autoantibodies (Fig ).  
Fig . Study design. 
Treatment. 
ACTH 
(Acthar) 
will 
be 
administered 
subcutaneously (SC) at the dose of 80 units for the first week 
and then 80 units SC twice weekly to complete 6 months of 
therapy, with a cumulative drug exposure of 3,920 units. 
- Data handling: A password-protected and coded, electronic database will be created to include patient 
demographics, physical and laboratory results including BP, serum creatinine, immunosuppressive drug doses 
and trough levels. Acthar doses will be also recorded at the time of each blood withdrawal, to monitor response 
to Acthar therapy. 
Ex vivo studies 
- T and B cells. PBMC will be isolated by Ficoll gradient centrifugation. Flow cytometry assays will be performed 
on fresh cells. We will use standardized multicolor flow cytometry to phenotype CD4+ and CD8+ T cells and B 
cells, including regulatory (CD127loFoxP3+) , naïve (CD45RA+CD27+), central memory (CD45RO+CD27+), effector 
memory (CD45RO+CD27-) and T cells, naïve B cells (CD19+CD27-IgD+), non-class-switched memory B cells 
(CD19+CD27+IgD+), class-switched memory B cells (CD19+CD27+IgD-IgM-CD20+CD38+/-), and CD3+CD4+CXCR5+ 
and follicular helper T cells. Appropriate isotype matched monoclonal antibodies will be used as controls. 
Phenotyping will be performed using 3-color flow cytometry on a Canto analyzer (Becton Dickinson, Franklin 
Lakes, NJ).  
MC1R surface expression on T and B cells will be also assessed using AMNIS Imaging Flow Cytometer MKII (EMD 
Millipore, Darmstadt, Germany). PBMC from age- and gender-matched healthy subjects will be used as controls.  
- Anti-PLA2R B cells and antibodies. Anti-PLA2R reactive B cells will be measured according to the protocol 
described in the preliminary data. Anti-PLA2R circulating autoantibodies in the serum will be measured by ELISA 
using the EURIMMUNE kit (EA 1254-9601 G). 
- MC1R polymorphisms. The human MC1R gene is highly polymorphic, with >35 genetic variants identified at 
present. We will perform MC1R genotyping on our cohort of patients to start testing the hypothesis that 
different genotypes are associated with different responses to Acthar therapy. We will also compare changes in 
immunological parameters (Treg, Teff, anti-PLA2R B cells and antibodies) after Acthar therapy according to the 
MCR1 polymorphisms. 
We have standardized both flow cytometry assessments 18 for use in clinical trials. We will perform all assays 
under good laboratory practice (GLP) compliance; previous work shows high reproducibility. We have published 
experience in performing serial measurements of Teff and Treg numbers and function in dialysis patients 19, 
which documents the feasibility of the proposed study. 
Clinical parameters 
Biochemistry 
Serum creatinine 
Proteinuria 
PBMC 
Anti-PLA2R B cells 
Circulating Treg 
Anti-PLA2R Ab 
Acthar 
0 
3 
6 
12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
1 
X 
X 
X 
X 
2 
X 
X 
X 
X 


 
Page 4 of 5 
5. Primary endpoint: 
- 
Primary endpoint of the study will be the change in proteinuria from baseline (before Acthar therapy) to 
12 months after therapy 
 
Secondary endpoints will include both clinical and mechanistic endpoints. 
Secondary clinical endpoints: 
- 
Complete (proteinuria reduction <500mg/24h) or partial (proteinuria reduction <3.5g/24h but 
>500mg/24h and >50% reduction than baseline, with stable renal function) remission at 12 months after 
inclusion  
- 
Time to complete or partial proteinuria remission  
- 
24h proteinuria at 6 and 12 months after inclusion 
- 
Serum albumin at 6 and 12 months after inclusion 
- 
Estimated GFR at 6 and 12 months after inclusion 
- 
Safety variables 
 
Secondary mechanistic endpoints: 
- 
Number of anti-PLA2R memory B cells at baseline and 3, 6, and 12 months after inclusion 
- 
Number of circulating CD4+CD25+CD127intFoxP3+ Tregs at baseline and at 3, 6, and 12 months after 
inclusion 
- 
Levels of anti-PLA2R antibodies at baseline and 3, 6, and 12 months after inclusion 
- 
Changes in T cell subsets after ACTH treatment 
- 
Changes in MC1R in T cells and B cells at baseline. 
  
Analysis plan and statistics: The clinical analyses will primarily evaluate the change in proteinuria and its 
relationship with autoreactive Bmem after Acthar therapy and secondarily they will assess rate of complete or 
partial remission changes in serum albumin, and eGFR over the 12-month follow-up period. Mechanistic, ex vivo 
studies will measure changes in total Tregs after Acthar therapy and their relationship with changes in 
proteinuria and anti-PLA2R Abs.  
Analyses will be performed initially in an intention-to-treat fashion. Thereafter, to exclude patients who stopped 
Acthar therapy during the follow-up period, we will consider in secondary analyses only those subjects who will 
have received the entire course of Acthar treatment. A p value of <0.05 will be considered statistically significant. 
We have access to appropriate biostatistical support through the Icahn School of Medicine to assist with these 
analyses (http://icahn.mssm.edu/research/grant-application-resource-center/statistical-support). 
 
Sample size estimation. This will be a pilot, explorative study and the sample size will not be calculated on the 
basis of the expected treatment effect. However, according to previous reports, we expect that approximately 
70% of patients will reach complete or partial remission during the first year of follow-up and that 20 subjects 
will be enough to detect as statistically significant a change in proteinuria between baseline and 12 months 20. 
The same number of patients will be enough to detect a significant difference in PLA2R antibodies 10. To account 
for possible drop-outs, we will include 25 patients. The data generated from this study will form the basis for 
the sample-size estimation of a larger prospective study testing the utility of anti-PLA2R memory B cells as a 
biomarker for response to Acthar therapy in MN patients. 
 
 
 
 
References 


 
Page 5 of 5 
 
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