Obinutuzumab is effective for refractory primary membranous nephropathy

Abstract

Purpose

Primary membranous nephropathy has been recognized as an autoimmune disease since the discovery of anti-phospholipase A2 receptor (PLA2R) antibody. Anti-CD20 monoclonal antibodies are currently recommended as an effective therapeutic option. Among these, obinutuzumab (OBZ), a novel humanized type II anti-CD20 monoclonal antibody, has shown promise. However, studies evaluating the efficacy of OBZ in treating refractory membranous nephropathy remain limited. This study aims to evaluate the therapeutic outcomes of OBZ in patients with refractory membranous nephropathy.

Methods

We retrospectively analyzed 28 cases of refractory membranous nephropathy patients treated with OBZ at our center, with a minimum follow-up of 3 months. These patients had previously failed or relapsed after treatment with glucocorticoid plus cyclophosphamide, calcineurin inhibitors (CNIs), and/ or rituximab. The primary outcomes were complete remission (CR, proteinuria < 0.3 g/d) or partial mission (proteinuria < 3.5 g/d or with 50% reduction from baseline).

Result

During a median follow-up of 13.2 months, 9 patients (32.1%) achieved CR, 16 patients (57.2%) achieved PR, resulting in an overall response rate of 89.3%. Three patients (10.7%) showed no response to OBZ treatment. Circulating CD19 B-cell count decreased < 5 cells/mL in 82.9% patients within 2 weeks post-infusion. Serum anti-PLA2R concentrations decreased to ≤ 2 RU/mL in 23 of 28 (82.1%) patients. Following OBZ administration, there was a significant reduction in 24-hour urine protein levels (median: 5.60 [IQR, 3.61–8.32] g/d at baseline vs. 0.51 [IQR, 0.16–1.33] g/d at the last visit, p < 0.05) and a significant increase in serum albumin levels (28.08 ± 7.03 g/L at baseline vs. 40.61 ± 5.89 g/L, p < 0.05). No serious adverse events were observed.

Conclusion

This study demonstrates that OBZ was effective and well-tolerated in patients with refractory membranous nephropathy. These findings highlight its potential role in this population and warrant further validation in larger, multi-center studies.

Introduction

Primary membranous nephropathy (PMN) is a common histopathological subtype of primary nephrotic syndrome in Chinese adults. The typical pathological characteristics include thickening of glomerular basement membrane due to subepithelial immune complex deposition, primarily involving IgG and complement C3. Over the past decades, the incidence of pMN has been increasing in China [1]. PMN has been recognized as an autoimmune disease since the discovery of anti-phospholipase A2 receptor (PLA2R) antibodies and Thrombospondin type-1 domain-containing 7 A (THSD7A) [2, 3]. B-cells play a pivotal role in the pathogenesis of pMN. B cells depletion to reduce antibody production has become a cornerstone of MN treatment. In addition to traditional supportive and immune-suppressive therapies, rituximab (RTX), a first generation of chimeric monoclonal antibody targeting the B cell surface antigen CD20, has been successfully employed in the treatment of MN and is recommended by the 2021 KDIGO Guidelines [4]. However, the overall clinical remission rate of RTX is approximately 60%, with 40% of patients showing no response and a high recurrence rate [5].

Obinutuzumab (OBZ), a novel humanized type II anti-CD20 monoclonal antibody, has demonstrated superior B-cell cytotoxicity compared to RTX in vitro studies [6]. OBZ has been glycoengineered with an afucosylated Fc [7] facilitating its high potency in NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), and It also demonstrates slower internalization and reduced efficiency in complement recruitment. Accumulating evidence suggests that OBZ-based immunochemotherapy and maintenance therapy are superior to RTX-based therapy in the treatment of lymphoma [8, 9]. In the field of kidney disease, OBZ has been reported as a safe and effective therapy for patients with anti-neutrophil cytoplasmic antibodies(ANCA)-associated vasculitis who have refractory disease or a history of anaphylaxis to RTX [10]. In a randomized controlled study of proliferative lupus nephritis, OBZ treatment group exhibited better renal function improvement and proteinuria remission [11].

Although clinical reports on the use of OBZ in the treatment of refractory MN are limited, the therapeutic outcomes are encouraging [12–14]. Hu XF et al. reported that OBZ was associated with higher odds of clinical remission compared with RTX at 12 months in patients with MN [15]. In this study, we retrospectively analyzed the efficacy and safety of OBZ in refractory MN patients at our center, aiming to provide additional evidence for clinical practice.

Population and methods

Patients

This retrospective study included refractory MN patients who received at least two administrations of OBZ during hospitalization at this tertiary referral hospital between January 2022 and December 2024. The inclusion criteria of refractory MN included: (i) treatment resistance, defined as the persistence of nephrotic syndrome, or a decrease in 24-hour proteinuria to 2–3.5 g/d without an increase in serum albumin to normal and subsequent rise in 24-h proteinuria, with a duration greater than one year after the initiation of GC + CTX or 6 months after receiving CNIs ± GC; (ii) dependence on CNIs, defined as relapse during the CNIs tapering or within 6 months after CNIs withdrawal; (iii) relapse occurring within one year after GC + CTX discontinuation; (iv) failed RTX therapy was defined as no response (NR) or no partial remission (PR) of urinary protein quantification during a follow-up period of at least 6 months under a regimen of a cumulative dose of 2000 mg of RTX within one month. Exclusion criteria were: i), patients with other glomerulonephritis; ii), patients who received OBZ as first-line treatment; iii), patients with inadequate follow up time(less than 3 months); iv), patients lost to follow up.

Immunosuppressive therapy

All enrolled MN patients were administrated with OBZ. OBZ was administered at a dosage of 1000 mg per infusion. The interval between the first and second infusions was typically 2 to 3 weeks. A third infusion was administered if a patient did not respond within 6 months after the initial two infusions, experienced a relapse, or had B-lymphocyte counts repopulated to over 5 cells/mL. Prior to OBZ infusion, systematic premedication were given to patients to prevent infusion-related reactions, including oral ibuprofen 300 mg, intravenous injection of dexamethasone 20 mg, and intramuscular injection of promethazine 25 mg. OBZ was diluted in 500 mL normal saline, and infused at at an initial rate of 10 mL/h for the first 30 min, with the rate progressively increased every 30 min to a maximum of 200 mL/h. Safety outcomes, including serious adverse events and nonserious adverse events, were collected during OBZ administration as well as throughout the follow-up period. Serious adverse events included additional hospitalizations and life- or organ-threatening events. Nonserious adverse events included infusion-related reactions, hypogammaglobulinemia, or other adverse reactions.

Follow up and outcomes

All enrolled patients attended outpatient clinical visits every 1–3 months for physical examination and laboratory tests including 24-hour proteinuria, serum albumin, serum creatinine and anti-PLA2R antibody levels and B-lymphocyte counts.

Clinical endpoint definitions mainly included proteinuria remission and renal function deterioration. Proteinuria status was defined according to the the 2021 KDIGO guidelines as follows: no response(NR) to treatment, no improvement in 24-hour urinary protein quantification; partial remission (PR), urinary protein 0.3–3.5 g/day or a reduction to less than half of the baseline value, with improvement in serum albumin concentration and stable eGFR; Complete remission (CR), urinary protein < 0.3 g/day. Renal function was assessed based on serum creatinine levels, with renal function deterioration defined as a 30% increase in serum creatinine from baseline.

Statistical analysis

Descriptive statistics were used to summarize the demographic and clinical characteristics of the study participants. Continuous variables are presented as the mean ± standard deviation (SD) for normally distributed data or as the median with interquartile range (IQR) for non-normally distributed data. Statistical analyses were conducted using SPSS (version 21.0 IBM, NY, USA) and GraphPadPrism (version10.0; San Diego, California, USA).

Result

Clinical characteristics

A total of 53 pMN patients received OBZ in Shandong provincial Qianfoshan hospital nephrology department between January 2022 to December 2024. After excluding non-refractory pMN (N = 14), or use of other immunosuppressants(N = 3), or inadequate follow-up time (N = 8), 28 refractory MN patients were comprehensively analyzed (Fig. 1). All these patients had experienced treatment failure or relapse with glucocorticoid plus cyclophosphamide, CNIs, and/ or RTX.

Fig. 1.

Flow diagram of patient selection

The baseline clinical characteristics of MN patients were listed in Table 1. Among the 28 patients, males predominated, accounting for 64.3%. The median follow-up time was 12 months (interquartile range (IQR), 6.75-18.00 months). The median duration of prior therapy was 30 months (IQR, 12-57months). The mean ages at the initiation of OBZ treatment were 48.11 ± 13.88 years. At the time of enrollment, the median level of urinary protein was 5.60 (IQR, 3.61–8.32) g/d, serum albumin was 28.08 ± 7.03 g/L, serum creatinine was 79.5 (IQR, 58.0-88.75)umol/L, eGFR was 93.92 ± 31.39 ml/min/1.73m2. Serum levels of anti-PLA2R antibodies were 21.95(IQR, 2.24-227.03)RU/mL, the percentage of PLA2R-negative MN patients was 35.8%. Before initiation of OBZ, 10(35.7%) patients had failed RTX therapy, 23(82.1%) patients had failed CNIs±glucocorticoid therapy and 4(14.3%) patients had failed glucocorticoid ± CTX therapy. All patients were prescribed with angiotensin receptor blocker (ARB) or Angiotensin Converting Enzyme Inhibitor(ACEI). Among the 10 patients who failed RXT treatment, two achieved CR, seven achieved PR and one showed no response after OBZ treatment.

Table 1.

Baseline characteristics of the refractory MN patients

Characteristics	Total (n = 28)
Age(y)	48.11 ± 13.88
Gender male(n,%)	18(64.30%)
Body mass index, kg/m2	25.62 ± 3.70
Kidney biopsy(n,%)	26(92.86%)
Systolic blood pressure, mmHg	136.67 ± 21.26
Duration of prior therapy, months	30(12–57)
Laboratory tests
Serum creatinine, umol/L	79.5(58.0-88.75)
eGFR, ml/min/1.73m2	93.92 ± 31.39
Albumin, g/L	28.08 ± 7.03
Total cholesterol, mmol/L	6.38 ± 1.63
Triglycerides, mmol/L	2.24 ± 1.14
Low density lipoprotein, mmol/L	3.43 ± 1.38
Anti-PLA2R antibody, RU/mL	21.95(2.24-227.03)
Anti-PLA2R positivity	18(64.2%)
24-h urine protein, g/d	5.60(3.61–8.32)
Previous immunosuppressive drugs
Rituximab	10(35.7%)
CNIs ± GC	23(82.1%)
GC ± CTX	4(14.3%)
Concomitant diseases
Hypertension	9(32.1%)
Diabetes mellitus	6(21.4%)

Refractory MN included both primary refractory MN and relapsing disease. Table 2 depicts the characteristics of two groups at three stages (pre-prior therapy, pre-OBZ, post-OBZ therapy). Compared with the primary refractory MN group, relapsing patients were younger (61.67 ± 7.03 years vs. 44.41 ± 12.84 years, p = 0.04). No significant differences were observed in other parameters, such as gender, anti-PLA2R-antibody positive rate, 24-hour proteinuria, serum albumin, or proteinuria remission rate post-OBZ treatment.

Table 2.

Characteristics of primary refractory and relapse iMN patients

Parameters	Groups
	Primary refractory MN	Relapse MN	P values
Number of patients	6	22
Clinical baselines
Age(y)	61.67 ± 7.03	44.41 ± 12.84	0.04
Gender male(n,%)	4(66.70%)	14(63.60%)	0.72
Pre-prior therapy
Anti-PLA2R antibody positivity(n,%)	5(83.30%)	16(72.70%)	0.50
24-h urine protein (g/d)	5.65(3.99,10.61)	7.47(4.05,10.54)	0.69
Albumin, g/L	21.55 ± 1.68	22.69 ± 4.45	0.34
Pre-OBZ therapy
Anti-PLA2R antibody positivity(n,%)	5(83.30%)	13(59.10%)	0.24
24-h urine protein (g/d)	6.19(5.43,12.21)	5.42(3.02,7.57)	0.19
Post-OBZ therapy
24-h urine protein (g/d)	6.19(5.43,12.21)	5.42(3.02,7.57)	0.19
Clinical outcomes
Number of PR	4(66.70%)	12(54.50%)	0.47
Number of CR	1(16.70%)	8(36.40%)	0.23
Number of NR	1(16.70%)	2(9.10%)	0.41

Among the 28 patients, 25 (89.3%) achieved CR (32.1%) or PR (57.2%) during the follow-up period (Fig. 2), while 3 patients showed no-response to OBZ therapy. The median time to remission was 4.0 (IQR, 1.0–6.0) months. By the 6-month follow-up, 16 patients (57.2%) achieved clinical remission of PR, while the CR rate was 0%. By the end of follow-up, 9 patients (32.1%) achieved clinical remission of CR, while the PR rate was 57.2%. At the 3-month, 6-month, 9-month, and 12-month follow-ups, proteinuria levels were 2.81 g/24 h (IQR, 1.33–4.54 g/24 h), 1.99 g/24 h (IQR, 0.86–3.55 g/24 h), 1.98 g/24 h (IQR, 0.29–3.76 g/24 h), and 0.51 g/24 h (IQR, 0.16–1.33 g/24 h), respectively. Corresponding albumin levels were 34.74 ± 7.18 g/L, 37.19 ± 7.72 g/L, 38.56 ± 7.28 g/L, and 40.61 ± 5.89 g/L, respectively (Fig. 3). By day 15 post-second OBZ administration, 70.6% of pMN patients experienced a reduction in proteinuria from baseline. Based on B-lymphocyte count repopulation exceeding 5 cells/mL or relapse of 24-hour proteinuria, 12 patients (42.86%) received a third OBZ dose. Among the 16 patients who did not receive the third dose, 5 achieved CR, 1 showed no response, and the others achieved PR. Compared with those not receiving the third dose, the treatment group showed no significant differences in CR rate, PR rate, or NR rate (p = 0.89, 0.45, and 0.37, respectively)(Table 3).

Fig. 2.

Kaplan-Meier curve for clinical remission after OBZ administration

Fig. 3.

Changes of 24-hour proteinuria and serum albumin after administration of OBZ during follow-up. Changes of 24-hour proteinuria (A) and serum albumin (B) after administration of obinutuzumab, * represents P < 0.05

Table 3.

Patient clinical baselines and outcomes grouped by the third OBZ dose

Parameters	Groups
	With the 3rd OBZ	Without the 3rd OBZ	P values
Number of patients	12	16
Clinical baselines
Age(y)	43.16 ± 13.16	51.81 ± 13.61	0.10
Gender male(n,%)	9(75.00%)	9(56.30%)	0.13
Systolic blood pressure, mmHg	133.75 ± 15.42	138.69 ± 24.91	0.55
Serum creatinine, umol/L	82.5(63.0–98.0)	75.0(57.0-83.75)	0.26
eGFR, ml/min/1.73m2	93.19 ± 27.49	94.48 ± 34.90	0.92
Albumin, g/L	28.59 ± 8.42	27.70 ± 6.06	0.75
Anti-PLA2R antibody, RU/mL	63.96(7.30-353.51)	10.07(2.03-129.96)	0.28
24-h urine protein (g/d)	5.73(2.50–8.36)	5.53(4.38–8.32)	0.98
Clinical outcomes
Number of PR	6(50.00%)	10(62.50%)	0.45
Number of CR	4(33.30%)	5(31.30%)	0.89
Number of NR	2(16.70%)	1(6.3%)	0.37

No deterioration of renal function was observed in any of the patients. All patients maintained stable renal function during follow-up. One patient exhibited a significant decrease in serum creatinine from baseline.

Immunological parameters

Data of CD19 + lymphocytes were available for 15 patients at baseline, among whom 1 patient had undetectable CD19 + lymphocyte counts due to prior RTX therapy. B lymphocytes counts dropped below 2 in 23 (82%) patients after the second OBZ dose. During the 6-month follow-up period, 26 patients maintained persistently low levels of B lymphocyte counts, except for one patient who showed no response and another who only achieved partial remission (PR) of proteinuria. All 23 patients with high anti-PLA2R titers exhibited a significant decrease following the second dose of OBZ (Fig. 4). In six patients, urine protein quantification worsened as peripheral blood B-lymphocyte counts rebounded. However, in two other patients, despite a rebound in peripheral blood B-lymphocyte counts, urine protein quantification did not worsen. Notably, none of these six patients exhibited an increase in anti-PLA2R antibody levels.

Fig. 4.

Changes in peripheral CD19 + B lymphocytes and serum anti-PLA2R antibody levels after OBZ administration during follow-up. Changes of peripheral CD19 + B lymphocytes (A) and serum anti-PLA2R antibody levels (B) after administration of obinutuzumab, * represents P < 0.05

Safety Outcomes

Two patients experienced symptoms of dizziness and mild dyspnea during the first administration of the medication, while one patient reported chills. These symptoms were alleviated following the administration of 5 mg dexamethasone. During the follow-up period, nine patients contracted COVID-19, presenting primarily with fever and pharyngeal pain, without lung involvement. Only one patient experienced a relapse attributed to COVID-19 infection.

Discussion

Currently, the primary treatment options for patients with moderate to high-risk pMN include corticosteroids combined with cyclophosphamide (CYC), corticosteroids combined with calcineurin inhibitors (CNI), or RTX. However, the proteinuria remission rates vary significantly, and these therapeutic regimens require further optimization. The CNI regimen achieves a response rate of approximately 50%-70% within 6 months, with 42.6% of patients achieving PR and 31.3% achieving CR [16, 17]. The CYC regimen demonstrates a 78.9% efficacy rate within 6 months, with 53.5% achieving PR and 25.4% achieving CR [17]. RTX has not shown superiority over CNI or CYC in inducing remission, with approximately 60% of patients achieving remission [5]. In this study, we evaluated the efficacy of OBZ in the pMN patients refractory to these conventional therapies.

Our findings revealed that 89.3% of patients treated with OBZ achieved PR or CR of proteinuria, a significantly higher remission rate compared to other treatments. The overall response rate to OBZ in patients who had failed prior RTX therapy was 90% in our study. One patient, whose anti-PLA2R titers did not significantly decrease and whose B cells were not completely depleted after RTX treatment, experienced a rapid decrease in anti-PLA2R titers and B cell depletion, achieving partial remission following OBZ treatment. This result aligns with findings from recent case reports [14, 18]. A study by Xu et al. reported that 80% of patients refractory to RTX achieved clinical remission after OBZ treatment.

Both RTX and OBZ are anti-CD20 monoclonal antibodies. RTX, a type I anti-CD20 monoclonal antibody, induces B-cell depletion and is recommended for the treatment of pMN and other autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [19, 20]. However, 40% of pMN patients fail to respond to RTX. Possible explanations include the elimination of RTX in the urine in patients with albuminuria [21], as well as internalization and destruction of RTX by target B cells and the presence of anti-rituximab antibodies, which collectively reduce RTX levels [22–26].To address the issue of internalization, OBZ has been designed to block the internalization site by binding to an overlapping epitope of CD20 in a different orientation. Further studies are needed to determine whether OBZ is excreted in the urine.

In our study, we found that OBZ not only demonstrated a high response rate in pMN, but also achieved this response within a relatively short time, consistent with the findings of Su X et al. [27]. Anti-PLA2R antibody titers significantly decreased, and peripheral B cell counts were almost completely depleted by day 15 following the second dose of OBZ. This was accompanied by an increase in serum albumin levels and a reduction in urinary protein levels. Marinov AD and Reddy V et al. reported that OBZ exhibited stronger B-cell depletion and natural killer cell activation in patients with lupus and in lupus mouse models, respectively [28, 29]. Similar to our foundings, Similar to our findings, 82% of CD19 + B cells were depleted within two weeks. Compared to RTX, OBZ exerts its therapeutic effects primarily through mechanisms independent of complement-mediated activity [30].

The optimal therapeutic dose and administration intervals for OBZ have not yet been established, necessitating further clinical studies for validation. In our study, no significant differences in proteinuria remission rates were observed between the two-dose and three-dose OBZ groups, warranting further investigation. Among the three patients who did not achieve proteinuria remission, one had a long-standing history of diabetes, another discontinued medication for one year due to pregnancy planning, and the third exhibited negative serum anti-PLA2R antibody levels. Approximately 70% of MN cases are associated with positive anti-PLA2R antibodies [1], while about 2% are positive for thrombospondin type-1 domain-containing 7 A (THSD7A) [31]. Of the 28 enrolled patients, 10 were anti-PLA2R antibody-negative, and no other autoantibodies were detected. We speculate that other B-cell-mediated immune responses may persist in PLA2R-negative MN patients, requiring further clinical validation. To evaluate the efficacy of OBZ in different subgroups, we analyzed the characteristics of primary refractory MN and relapsing patients across three stages: pre-prior therapy, pre-OBZ therapy, and post-OBZ therapy. Our findings revealed that relapsing patients were younger than those with primary refractory disease, suggesting that younger patients may exhibit more complex immune dysregulation which needs further deeply exploration.

Several limitations of this study should be noted. First, it is a retrospective analysis with a small sample size conducted at a single center. Second, the follow-up period was relatively short, leaving the long-term outcomes and relapse rates of OBZ therapy uncertain. Third, missing data may weaken the reliability of the conclusions. Therefore, larger randomized controlled trials with extended follow-up periods are necessary to validate these findings.

OBZ demonstrated favorable clinical tolerance and safety. Only three patients experienced mild symptoms during intravenous administration. During the follow-up period, all patients experienced the COVID-19 outbreak in our country at the end of 2022, with nine patients contracting COVID-19. The primary symptoms included fever and pharyngeal pain, without lung involvement. No other infections were reported. Only one patient experienced a relapse during the COVID-19 infection. Notably, after recovering from COVID-19, the urinary protein levels of this patient rapidly decreased to less than 500 mg.

This study highlights the efficacy and safety of OBZ in patients with refractory pMN treated at our center. Despite being a retrospective study with a small sample size and short follow-up duration, OBZ demonstrated high efficiency and rapid response, underscoring its potential as a treatment option for pMN.

Author contributions

N Zh prepared the manuscript. P Ch, Bb W, Xq T, Lj T, J Ch and Zs W treated the patients and collected data. N Zh and Xq T conduct statistical analysis of the data. Y Zh supervised the publication. All authors have read and approved the manuscript.

Funding

This study was supported by grants from the Natural Science Foundation of Shandong Province (ZR2021MH282 to Na Zhao).

Data availability

The data used during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval

This study protocol was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Shandong First Medical University, approval number #S347, which was conducted in accordance with the Declaration of Helsinki. All participants were provided with written and oral explanations prior to participation. Informed consent to participate was obtained from all of the participants in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.