Peptide GAM immunoadsorption in anti‐PLA2R positive autoimmune membranous nephropathy. The PRISM trial

1 INTRODUCTION

Membranous nephropathy (MN) is a significant cause of nephrotic syndrome worldwide, in which the majority of patients present with autoimmune pathophysiology associated with the anti-PLA2R autoantibody.1-6 Current standard of care is immunosuppression, using either rituximab or cyclosporine in the United States, or modified Ponticelli regime or rituximab in Europe. All are essentially empiric regimes, in use for over 25 years, with few RCT studies until recently.7-9 While these have improved mortality and renal outcomes, they can be associated with significant side-effects.10

Since the anti-PLA2R antibody was discovered in 2009, there has been a dramatic improvement in our understanding of the underlying disease pathology.1 The antibody is now known to be of a predominantly IgG4 subclass, although the other IgG subclasses are often represented, albeit to a lesser extent.11 An increasing body of circumstantial evidence suggests that not only is it a highly sensitive biomarker, but is also likely pathogenic in nature.11-14 Higher antibody burden is associated with poorer prognosis with reduced incidence of remission and increased rates of renal dysfunction.11, 15-17

Immunoadsorption (IA) is an extracorporeal therapy for the efficient removal of antibodies without the need for factor replacement, and a minimal side-effect profile.

With its highly efficient removal of all IgG subclasses, IA presents a unique opportunity to not only investigate its efficacy as a novel treatment for autoimmune MN, but to also model the immune system in response to rapid removal of the antibody. It has previously been used in nephrotic syndrome, including four patients with MN, which showed a significant reduction in proteinuria, although this returned to baseline after only 1 month. However, this study was in the pre-anti-PLA2R era and limited with no long-term data on outcomes.18 Here we have carried out a pilot study to assess the validity of IA as a therapy in anti-PLA2R–associated MN and to further understand the disease pathogenesis using kinetic modeling of the immune system in response to the rapid removal of the pathogenic antibody in the absence of immunosuppression.

2 METHODS

This was a multicenter, open-label single-arm prospective clinical trial carried out across the Northwest of England (NCT03255447).19 All study visits were carried out at the National Institute for Health Research (NIHR)/Wellcome Trust Clinical Research Facility, Manchester University Hospitals. The trial was conducted according to the principles of the Declaration of Helsinki. Full ethical approval received from the NHS Health Research Authority Research Ethics Committee (16/NW/0560) and all study sites had local Research and Development approval. All patients provided written informed consent before any study procedures were carried out.

2.1 Primary and secondary outcomes

The primary outcome was anti-PLA2R titer at week 2 and its profile during the study period. Secondary outcomes were focused on the safety and tolerability of immunoadsorption in anti-PLA2R positive MN. Other secondary outcomes included change in proteinuria and renal function as measured by urinary protein:creatinine ratio (uPCR) and estimated glomerular filtration rate (MDRD-eGFR), respectively, and serum albumin, total immunoglobulin levels, and urinary C5b-9:creatinine at weeks 12, 24, and 52. Adverse events (AEs) and serious adverse events (SAEs) were based on physician assessment. SAEs included any adverse event requiring hospital admission, prolonged hospital stay, or intravenous antibiotic therapy outside of the protocol. Quality of life assessed using the EuroQol five dimensions questionnaires (EQ5D) completed by patients at baseline and at weeks 12, 24, and 52.20

2.2 Inclusion criteria Newly diagnosed or relapsing adult patients (above 18 years old) with renal biopsy confirmed membranous nephropathy within the last 3 years.

Persistent active disease despite 6 months supportive care using Renin-Angiotensin inhibitors.

Active disease was defined as uPCR >300 mg/mmol or 24-hour urinary protein of >3.5 g/1.73m2; and patients with serum anti-PLA2R titers above 170 u/mL. Disease severity that in the physicians' view warranted treatment prior to completion of 6 months supportive care. Up to date Haemophilus and Pneumococcal vaccinations. Able to provide informed consent. 2.3 Exclusion criteria

Patients were excluded if there was any evidence of secondary membranous nephropathy, eGFR of less than 20 mL/min, received any treatment with steroids or immunosuppression (including but not limited to cyclophosphamide, Mycophenolate mofetil or azathioprine) or Biologics (including but not limited to Rituximab or belimumab) within 6 months of screening. Patients must also not have had therapeutic plasma exchange within 28 days of screening or previous renal transplantation. Patients could also be excluded if they had a comorbidity, which in their physicians' view would preclude the patient from treatment with immunoadsorption or if they are pregnant at the time of screening.

2.4 Filtration/adsorption device

Peptide GAM immunoadsorption (GLOBAFFIN Fresenius Medical Care Deutschland GmbH) was chosen for this study as it specifically removes IgG, in particular IgG1, IgG2, and IgG4 (and to a lesser extent IgG3).11, 21, 22 This system consists of the Art Universal and ADAsorb. The Art Universal (Fresenius Medical Care) Hemadsorption System is intended for performing adsorption treatments or plasma fractionation for the selective or semiselective elimination of undesired components in the patient's blood or plasma.

The ADAsorb (Medicap Clinic GmbH) is a secondary system for controlling and monitoring adsorbers for extracorporeal apheresis. The system uses microprocessors to monitor all predetermined adsorption and desorption parameters of the adsorbers. It is operated in conjunction with a plasma separation system (Table S4, Supporting Information).

This is a dual column system allowing for continuous immunoadsorption to the required plasma volume clearance. While one column is actively involved in immunoglobulin adsorption, the other is being desorbed. Once the active adsorption column has become saturated, the plasma flow is diverted to the newly refreshed column, allowing for the saturated column to start the desorption process. Peptide GAM immunoadsorption specifically removes IgG, and in particular IgG1, IgG2, and IgG4 (and to a lesser extent IgG3) and has been chosen for this study as anti-PLA2R antibodies have been shown to be IgG with a predominant IgG4 subclass.11, 21, 22

2.5 Immunoadsorption protocol and monitoring

Each patient underwent daily immunoadsorption for 5 days via a femoral vascath (Double lumen blood access catheter; Medcomp, Harleysville, PA), aiming for pump speeds of 100 to 150 mL/min. The multiracial visual inspection catheter tool observation record (MR VICTOR) score was used daily to assess for signs of infection.23 If patients were unable to complete 5 days consecutively, they were allowed to complete five sessions within 7 days. If, however, the treatment was deferred for more than 48 hours, the patient would need to have an extra session to ensure the adequate removal of antibody. In this case, the patient would receive six sessions in 8 days.

All patients at the start of treatment will have biochemical and clinical evidence of nephrotic syndrome including oedema. For this reason, to accurately determine a patient's plasma volume, all patients had bioimpedance measurement (BCM) at screening. The BCM-derived normohydration weight (ideal weight [IW]) was then used to calculate the plasma volume using Kaplan formulae; estimated plasma volume = (0.065 × IW(kg)) × (1 − Haematocrit).24 To ensure adequate antibody clearance, we aimed to treat 2.5 plasma volumes daily with a maximum treatment of 1.5 L plasma/h. Anticoagulation was provided using a combination of heparin and citrate sodium with continuous IV calcium replacement as per local guidelines throughout the treatment sessions (10 mL 10% calcium gluconate for every 2 L of plasma treated). Serum calcium levels were assessed at the beginning, mid-point based on plasma volume, and end of therapy.

Therapy would be stopped in the case of allergic reaction to the column, extracorporeal therapy, or any other adverse or serious adverse event deemed by the safety committee to warrant withdrawal of treatment.

2.6 Laboratory measurements

All samples were tested using the Manchester Enzyme-Linked immunosorbent assay (ELISA) for anti-PLA2R antibodies with the normal level taken as <40 U/mL.11 All patients had full tissue typing on day 1 pretreatment. Patients were stratified by the presence (heterozygous or homozygous) or absence of the HLA-DQA1*05 high-risk allele.25 Absolute urinary C5b-9 was tested using BD Biosciences OptEIA Human C5b-9 ELISA kit. Urinary C5b-9:creatinine ratio was calculated by dividing absolute urinary C5b-9 by paired urinary creatinine.

2.7 Statistical analysis

Descriptive statistics and survival analysis used to evaluate the primary and secondary outcome measures. Wilcoxon signed-rank test used for nonparametric paired comparisons and Mann-Whitney U test for unpaired groupwise comparisons. Friedman's test with Kendall's W used for one-way repeated measure analysis and performed on complete cases only. Patients censored following treatment failure or death. If a patient required rescue immunosuppression as judged by the clinical team, this was recorded as a treatment failure, and subsequent data censored. Within-patient correlations for urinary C5b-9:creatinine ratio were calculated using repeated measures correlation. All analysis carried out using the R statistical program version 3.6.1.26

3 RESULTS

Thirteen patients were screened and consented. One patient went into spontaneous remission prior to the start of treatment, with a normalization of anti-PLA2R antibody, and was removed from the study.

Ten patients were naïve to steroids and immunosuppression prior to treatment, and two had failed cyclophosphamide-based regimens. PRISM07 completed the full cyclophosphamide-based course more than 6 months prior to screening and remained with persistent nephrotic syndrome, worsening proteinuria and antibody level. PRISM04 was unable to complete the course due to leucopenia and sepsis more than 1 year prior to screening (Tables 1 and S1).

TABLE 1. Demographics for each patient Patient PRISM01 PRISM02 PRISM03 PRISM04 PRISM05 PRISM06 PRISM07 PRISM09 PRISM10 PRISM11 PRISM12 PRISM13 Mean SD Age at dx 50 73 73 79 53 67 62 42 72 86 36 67 63 15.09 Age at IA 52 73 73 81 53 67 64 42 72 86 37 68 64 15.07 Sex M M M M M M M M M F M M Ethnicity White British Asian White British White British White British White British Black White British White British White British White British Asian Previous IS Nil Nil Nil Cyclo Nil Nil Cyclo Nil Nil Nil Nil Nil HTN Yes Yes Yes Yes Yes No No No Yes Yes Yes No DM No No No No No No No No No Yes Yes No ACEi Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ARB No Yes No No No No No No No No No No Statin Yes Yes Yes No Yes Yes Yes No No Yes Yes Yes BMI 56.8 29.8 23.7 29.4 28.5 30.9 25.5 32.7 24.5 21.6 36.6 26.1 31 9.27 anti-PLA2R 222 1006 159 125 105 679 2940 799 1341 >3000 396 726 773 823.01 eGFR 50 48 82 27 57 45 29 44 24 32 77 67 49 19.3 uPCR 636 571 1379 972 598 1290 462 1133 548 1624 1051 844 926 377.57 Albumin 22 22 23 18 22 21 19 18 25 13 13 23 20 3.85 Total PV treated 11.1 10.8 11.1 10.9 11.8 11.5 11.4 11.8 12 12.2 12.5 12.5 12 0.59 HLA-DQA1 status 01:01 01:03 01:02 01:03 01:03 03:01 05:01 05:01 03:02 02:01 05:01 05:01 03:01 05:01 04:01 05:05/09/11 03:01 05:01 05:01/05/09/11 05:01/05/09/11 05:01 05:01 05:01/05/09/11 05:05 Alternative IS No Yes No No No No No No No No No Yes Died No No No No No No No No No Yes No No Abbreviations: Age at dx, age at diagnosis; age at IA, age at start of treatment; previous IS, previous immunosuppression; cyclo, cyclophosphamide; HTN, hypertension; DM, diabetes mellitus; ACEi, angiotensin-converting-enzyme inhibitor; ARB, angiotensin-receptor blocker; BMI, body mass index (kg/m2); eGFR, estimated glomerular filtration rate (mL/min/1.73 m2); uPCR, urinary protein:creatinine ratio (mg/mmol); total PV treated, total plasma volumes treated (liters); alternative IS, alternative immunosuppression required following immunoadsorption. 3.1 Treatments delivered

All 12 patients (11 male and 1 female) completed five sessions of immunoadsorption successfully within 7 days. Two patients missed one treatment during therapy week due to mechanical faults with the machine, and both had their fifth session on the Saturday (day 6) as per protocol. Two patients required rescue immunosuppression and were censored for follow-up. PRISM02 had increasing antibody levels, proteinuria, and worsening renal function so commenced on Prednisolone and Cyclophosphamide. PRISM13 was started on Prednisolone and Ciclosporin by his parent team due to ongoing nephrotic range proteinuria despite a falling antibody titer.

3.2 Primary outcome: Serum anti-PLA2R

Median anti-PLA2R pretreatment on day 1 was 702.50 U/mL (IQR 206.25-1089.75), reducing to 91.00 U/mL (IQR 31.00-120.50) at the end of the treatment week, equating to an 87% reduction in median antibody titer associated with a 93% reduction in total IgG levels (Table 2).

TABLE 2. Serum antibody and immunoglobulin (Ig) reduction, and percentage change, compared to baseline during treatment week. Median (IQR) Serum anti-PLA2R Serum anti-PLA2R change Serum Ig Serum Ig change Day 1 Pretreatment 702.50 (206.25–1089.75) 0.00 (0.00-0.00) 4.85 (3.67-5.54) 0.00 (0.00-0.00) Post-treatment 285.50 (82.50-817.00) 42.56 (30.46-60.16) 1.11 (0.84-1.38) 77.60 (75.37-80.29) Day 2 Pretreatment 473.00 (146.50-984.00) 20.95 (14.54-38.79) 2.04 (1.63-2.44) 62.14 (58.77-63.61) Post-treatment 274.00 (80.50-469.75) 68.39 (58.02-80.95) 0.52 (0.42-0.67) 88.70 (87.75-91.46) Day 3 Pretreatment 262.50 (120.75-1028.75) 35.14 (16.05-41.92) 1.41 (1.24-1.67) 73.89 (68.00-74.45) Post-treatment 132.00 (50.25-242.25) 74.78 (72.92-78.63) 0.47 (0.35-0.69) 90.64 (86.13-92.45) Day 4 Pretreatment 234.00 (82.00-461.00) 54.77 (41.18-57.89) 1.10 (0.93-1.24) 75.67 (74.46-78.96) Post-treatment 84.00 (37.75-238.50) 81.46 (75.64-85.31) 0.36 (0.30-0.48) 91.74 (90.99-93.73) Day 5 Pretreatment 252.00 (91.50-515.50) 52.70 (41.12-62.50) 1.03 (0.86-1.22) 79.44 (77.92-81.04) Post-treatment 91.00 (31.00-120.50) 86.60 (83.06-91.55) 0.34 (0.23-0.41) 92.85 (91.62-94.63)

At week two there was a significant rise in anti-PLA2R level with all but one patient experiencing an increase in their level; P-value .023 (see Figure 1 and Table 3). Following this initial rise, there was a general decline in antibody level over the follow-up period, with median titer of 539 U/mL (IQR 281.75-3000.00), 370 U/mL (IQR 240.00-2265.00), and 165 U/mL (IQR 110.00-269.00) at weeks 12, 24, and 52, respectively. There was a statistically significant difference in median anti-PLA2R levels across follow-up timepoints; P-value .017, Kendall's W 0.335 (Table 3 and Figure 2).

image

Serum anti-PLA2R titer measured at week 1 pretreatment, week 2 post-treatment, and the end of follow-up at week 52, presented for each patient individually. Significance tested using paired Wilcoxon signed-rank test

TABLE 3. Results table. All values median (IQR). Friedman's and Kendall's W tests on complete cases only Pretreatment Week 2 Week 12 Week 24 Week 52 Friedman's Kendalls W n 12 12 12 11 9 P-value Wt Anti-PLA2R 702.50 [206.25, 1089.75] 1045.00 [685.75, 3000.00] 539.00 [281.75, 3000.00] 370.00 [240.00, 2265.00] 165.00 [110.00, 269.00] .017 0.335 uPCR 677.00 [526.50, 1012.00] 680.00 [455.50, 1024.25] 671.00 [581.00, 1001.75] 678.50 [478.00, 928.25] 630.00 [361.00, 895.50] .829 0.053 eGFR 46.50 [31.25, 59.50] 44.00 [30.00, 56.00] 42.00 [26.00, 62.00] 42.00 [32.00, 56.50] 44.00 [22.00, 66.00] .676 0.073 Albumin 20.50 [19.00, 21.25] 19.00 [16.25, 20.25] 21.50 [19.25, 25.25] 23.00 [20.00, 24.50] 25.00 [23.00, 28.00] <.001 0.613 IgG 4.85 [3.67, 5.54] 2.21 [1.89, 3.45] 5.72 [3.51, 7.20] 5.31 [3.68, 6.62] 7.44 [5.79, 8.14] <.001 0.672 C5b-9:creatinine 170.48 [13.44, 342.88] 224.48 [104.16, 566.63] 78.92 [40.58, 282.28] 141.28 [23.31, 253.60] 93.86 [43.22, 166.99] .602 0.098 EQ-5D utility 1.00 [0.84, 1.00] 1.00 [0.82, 1.00] 1.00 [0.84, 1.00] 1.00 [0.79, 1.00] 1.00 [0.85, 1.00] .696 0.062 EQ-5D VAS 70.00 [65.00, 80.00] 70.00 [63.25, 76.00] 76.50 [68.12, 84.50] 77.00 [67.00, 87.50] 80.00 [65.00, 90.00] .283 0.158 Abbreviations: anti-PLA2R, serum anti-PLA2R antibody titer (U/mL); uPCR, urinary protein:cr

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