Bortezomib for antibody-mediated rejection of kidney transplant in youth: associations with donor-specific antibody

Abstract

Background:

Antibody mediated rejection is one of the most significant risk factors for allograft dysfunction and failure in children and adolescents with kidney transplants, yet optimal treatment remains unidentified. To date, there are mixed findings regarding the use of Bortezomib, a plasma cell apoptosis inducer, as an adjunct therapy in the treatment of antibody mediated rejection.

Methods:

In a retrospective single center study, we reviewed the efficacy and tolerability of bortezomib as adjunct therapy for treatment-refractory antibody mediated rejection.

Results:

Six patients with a median age of 14.6 years (range 6.9–20.1 years) received bortezomib at a mean of 71 months (range 15–83 months) post kidney transplant. Four patients experienced decline in estimated glomerular filtration rate (eGFR) from 4–42%. One patient started bortezomib while on hemodialysis and did not recover graft function, and another patient progressed to hemodialysis six months after receiving bortezomib. Although DSA did not completely resolve, there was a statistically significant decline in DSA MFI pre and 12-months post-BZ (p=0.012, paired t-test) for the subjects who were not on dialysis at the time of bortezomib. Chronic Allograft Damage Index (CADI) score of ≥ 3 was seen in all six subjects at their biopsy prior to therapy. No adverse effects were reported.

Conclusions:

Bortezomib was well tolerated and resulted in improvements in MFI of DSA among 4 pediatric subjects without allograft failure, although no effects were observed on eGFR trajectory. Further studies are needed to clarify whether earlier intervention with bortezomib could prevent renal failure progression.

Graphical Abstract

Bortezomib was well tolerated as an adjunctive therapy in four pediatric kidney transplant patients with refractory antibody mediated rejection and persistent donor specific antibodies and resulted in an overall statistically significant reduction in DSA MFI.

Introduction:

Antibody mediated rejection (AMR) is classified by histologic evidence of acute and/or chronic injury associated with evidence of current/recent antibody interaction with the vascular endothelium and serologic evidence of donor-specific antibodies (DSA) to human leukocyte antigen (HLA) or non-HLA antigens.¹ AMR continues to be the largest risk factor for graft dysfunction and loss in pediatric kidney transplant recipients.^2–5 Prompt recognition and treatment is imperative for graft survival; though optimal treatment remains unknown.

Current management strategies for AMR focus mainly on targeting HLA associated antibody mediated rejection pathogenesis with varying success (intravenous immunoglobulin; IVIG), plasmapheresis, T-cell therapies (anti-thymocyte globulin) and B-cell therapies (rituximab).⁶ Consensus guidelines exist regarding utilization of these therapies based on timeline of AMR presentation (early, <30 days posttransplant, or late, ≥30 days) and whether AMR is active or chronic, though the focus is on adult patients.⁷ There are other non-HLA antibodies that have been reported to cause AMR in kidney transplant recipients,⁸ such as anti-angiotensin II type 1 receptor (AT1R) antibodies. For the last decade, there have been increasing reports of bortezomib use (Velcade^®; Millenium Pharmaceuticals, Inc., Cambridge, MA) in solid organ transplant patients as an adjunct for the management of AMR.^9–15 Bortezomib functions as a proteasome inhibitor, targeting plasma cells (such as those that produce donor specific antibodies; DSA) and inducing apoptosis with downstream improvements in allograft function.^10,16

Several small, early studies and case reports in the adult literature suggested promising results of bortezomib in patients with AMR; demonstrating rejection reversal, marked and prolonged reductions in DSA levels and improved renal allograft function⁹ when used with other anti-rejection therapies.^6,9,10 Ultimately, a larger randomized control trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA¹¹ (Table A2).

In the pediatric literature, the number of cases reported remain limited, though consistently align with the largest retrospective cohort study yet published to our knowledge: all data shows a trend towards lowering DSAs with mixed effects on estimated glomerular filtration rate (eGFR) (Table A2).^12–15 Since 2019, we have used bortezomib as a second-line agent in kidney transplants with AMR who have failed other therapies, such as IVIG, rituximab, and plasmapheresis (PP). Patients with concurrent AMR and acute cellular rejection (ACR) have also received thymoglobulin. In this study, we aim to add to the growing body of literature investigating the efficacy and tolerability of bortezomib as an adjunctive therapy in pediatric kidney transplant patients with refractory AMR and AT1R AMR.

Methods:

This is a single center retrospective review of all pediatric kidney transplant patients at a large academic center in the northeast region of the United States (Children’s Hospital of Philadelphia) who received bortezomib (BZ) for biopsy-proven, treatment refractory antibody mediated rejection (per Banff 2017 criteria) from March 2019 to November 2022. Patients were selected if they were noted to have persistent donor specific antibodies (DSA) or persistent anti-angiotensin II type 1 receptor (AT1R) antibodies (positive when ≥10 U/mL at a 1:100 dilution) and persistent kidney dysfunction without notable improvement after first line treatments for acute rejection. Six patients met inclusion criteria. Demographic and clinical data were manually abstracted for each patient by two independent reviewers and cross-verified for accuracy.

Kidney biopsy was obtained within 6 months prior to initiation of BZ and histologic findings were used to calculate the Chronic Allograft Damage Index (CADI) score, which has been utilized as a surrogate marker for long-term graft survival¹⁷ and in this study served as an additional prognostic indicator to highlight patients at increased risk for graft loss. The CADI score is composed of six histologic parameters: vascular intimal sclerosis (cv), tubular atrophy (ct), interstitial fibrosis (ci), interstitial inflammation (i), mesangial matrix increase (mm), glomerulosclerosis (g) and each parameter is scored 0–3, therefore CADI maximum score is 18, with 0–1 considered “low,” ≥ 2 to 3.9 considered “elevated”, and ≥ 4 considered “high”.¹⁸ Studies have shown a CADI score of ≥ 2 is indicative of chronic allograft damage and associated with increased risk of allograft loss.^17,19,20

Patients received bortezomib, 4 doses at 1.3 mg/m² per dose on days 1, 5, 8 and 12, followed by an additional cycle (4 doses) at the same interval one month later per published protocols.^21,22 No pre-medications were routinely given as they are not recommended per manufacturer’s package insert. Acetaminophen and diphenhydramine, as well as anaphylaxis protocol, were ordered “as needed” though no patient required them.

DSAs were obtained at baseline and followed at months 1, 3, and 12 after completion of therapy or until completion of follow-up. We defined “immune dominant DSA” as the DSA with maximum MFI. Serum creatinine, age, and height were checked with each infusion, then every 1–3 months thereafter, and eGFR was calculated using the Chronic Kidney Disease in Children under 25 years of age (CKiD U25) creatinine-based equation at each time point.²³ Where appropriate, AT1R antibody level was checked at baseline and after completion of cycle 1 and cycle 2.

Charts were also abstracted for recent or concurrent anti-rejection therapies, adverse events associated with the administration of bortezomib, such as infusion or hypersensitivity reactions; presence of peripheral neuropathy, bone marrow suppression (thrombocytopenia <50,000, neutropenia), thrombotic thrombocytopenic purpura, hepatic dysfunction, gastrointestinal symptoms (nausea, vomiting, diarrhea or constipation), pulmonary toxicity, development of posterior reversible encephalopathy, and infections (such as Herpes Simplex virus, Cytomegalovirus, Epstein Barr, and BK virus), and graft survival at the end of follow-up.

Primary outcomes of interest were: 1) graft failure and 2) antibody resolution, defined as reduction in DSA MFI to <1500 by Luminex^® assay or reduction in AT1R to normal on respective assay (Hopkins Lab, UCLA Lab) at 12 months post-completion of therapy. Secondary outcomes were: 3) >50% reduction in DSA MFI between pre-BZ and 12 months post-BZ, and 4) a clinically significant change in eGFR, defined as ≥ 20% improvement from baseline at 12 months post-completion of therapy. As an exploratory outcome, the DSA relative intensity scale “DSA-RIS” was calculated for each subject at 1, 3 and 12 months post-bortezomib as per prior literature.^24–26

Statistical analyses:

Pre- and post-Bortezomib DSA MFI value and pre- and post-Bortezomib eGFR were compared within patients using paired t-test. All analyses were performed using STATA version 17.0 (StataCorp LLC, College Station, TX) with 2-sided hypothesis testing and p-value of < 0.05 for significance.

Results:

Six patients received bortezomib for treatment-refractory AMR. Patient demographics are outlined in Table 1. This was the primary kidney transplant for all patients. Patient 1 had been hemodialysis dependent for 3 weeks at initiation of BZ and received one full cycle (4 doses) as salvage therapy without return of graft function. The remaining five patients received two cycles of BZ. Median time from kidney transplant to initiation of BZ was 71 months (range: 15–83 months). For patients without AT1R AMR, median time from onset of first de novo HLA-DSA to first BZ dose was 34 months (range: 2.6 – 64.6 months). Two patients were found to have non-HLA-DSA positivity to AT1R antibody. Patient 1 received BZ one month after they were found to be AT1R antibody positive, and patient 5 was noted to be AT1R antibody positive 1.6 months after receiving the first BZ dose. Both patients (1 and 5) were started on losartan for AT1R positivity, patient 1 developed hyperkalemia and losartan was shortly after discontinued.

Table 1.

Patient demographics and clinical characteristics.

Patient	Age at Transplant	Sex	Primary Disease	Donor status	Antigen Mismatch (A-B-DR)	Antigen Mismatch (DQA1 -DQB1)	Months to BZ (*)	Biopsy impression prior to BZ	C4d	AT1R Ab	Prior Rejection Therapy
	<2 years post-transplant to bortezomib
1	5.6	F	Cloacal anomaly, renal dysplasia	Deceased	2-2-1	2-1	15	No evidence of AMR, Banff 1B TCR	N	Y	ATG (6 mg/kg) + MP, then IVIG (4 g/kg) + rituximab (750 mg/m2), concurrent PP
2	16.0	M	FSGS	Living	1-1-1	1-1	19	Active AMR, acute TCR	Y	N	ATG (6 mg/kg) then IVIG (2 g/kg) + rituximab (375 mg/m2) + PP
	≥ 2 years post-transplant to bortezomib
3	10.7	F	FSGS	Deceased	1-2-2	2-2	64	Chronic active AMR	Y	N	IVIG (4 g/kg) + rituximab (750 mg/m2)
4	7.3	F	DHUS	Deceased	1-0-1	2-2	78	Chronic active AMR, Banff 1A TCR	Y	N	IVIG (4 g/kg) + rituximab (750 mg/m2)
5	13.3	M	Cortical Necrosis	Deceased	2-2-1	0-0	81	Chronic active AMR	Y	Y	Rituximab (1050 mg/m2) prior, concurrent IVIG (2 g/kg)
6	6.4	M	ARPKD	Living	0-1-1	1-1	83	Chronic active AMR, borderline ACR	Y	N	IVIG (4 g/kg) + rituximab (750 mg/m2)

^(*)Indicates time between kidney transplant and first BZ dose. (BZ: bortezomib; ATG: anti-thymocyte globulin; AMR: antibody mediated rejection; TCR: T-cell mediated rejection; FSGS: focal segmental glomerulosclerosis; DHUS: diarrhea-associated hemolytic uremic syndrome; ARPKD: autosomal recessive polycystic kidney disease; IVIG: intravenous immunoglobulin; MP: methylprednisolone pulse; PP: plasmapheresis)

Histology:

Baseline and relevant clinical variables are outlined in Table 1. Five patients (patients 1, 2, 3, 4 and 6) had biopsy proven active or chronic active AMR and positive C4d staining, one of which also had elevated AT1R antibody (patient 1). Patient 5 had no evidence of AMR with negative C4d, but active TCR and presence of AT1R antibody. All patients had a CADI score ≥ 3 (mean 5.5, SD 1.89, range 3–9) at the time of BZ initiation.

Prior rejection therapy:

All patients received prior rejection therapy as outlined in Table 1. Patients with acute TCR received a total of 6 mg/kg of thymoglobulin over 4 doses. All patients received prior IVIG and rituximab. Two patients received plasmapheresis (patients 1 and 2), one of which (patient 1) was concurrent with BZ therapy. Patient 5 received concurrent IVIG during BZ.

Allograft function and survival:

Of the five remaining patients who were not dialysis dependent at initiation of BZ treatment, only one patient (patient 2) had graft failure at 6 months post-BZ and was started on hemodialysis, Table 2. There was an overall decline in eGFR pre and 12 months post-treatment for all patients, though this was not statistically significant (p=0.09, paired t-test), Figure 1.

Table 2.

Primary and secondary outcomes for six patients post bortezomib (CADI: Chronic Allograft Damage Index)

Patient	Biopsy impression	CADI Score	GFR Change (pre/post)	Graft failure	DSA or AT1R Resolved?
1	No evidence of AMR, Banff 1B TCR, AT1R ab	3	--	--	Yes (AT1R)
2	Active AMR, acute TCR	4	Graft failure	Yes	No
3	Chronic active AMR	6	↓13%	No	No
4	Chronic active AMR, Banff 1A TCR	6	↓42%	No	Partial
5	Chronic active AMR, AT1R ab	5	↓4%	No	No (AT1R)
6	Suspicious chronic active AMR, borderline ACR	9	↓33%	No	Partial

Figure 1.

eGFR change over time from baseline to 12 months post-bortezomib

(Patient 1 on hemodialysis at time of bortezomib, remained dialysis dependent with no return of graft function. Patient 2 had graft failure 6 months post-bortezomib requiring hemodialysis)

Antibody response:

For patients without graft failure (patient 3, 4, 5 and 6), there was a statistically significant decline in MFI DSA pre and 12-months post-BZ (p=0.012, paired t-test) without resolution in immune-dominant DSA (DSA with the highest MFI value) for any patient (Figure A1). Two patients (patients 4 and 6) showed resolution in non-immune dominant DSA. When examining DSA-RIS, three subjects (patients 3, 4 and 5) showed improvement in 6, 10 and 7 points, respectively over 12 months post-bortezomib. Only patient 2 did not show improved DSA-RIS. Patient 1 had resolution of AT1R antibody three months after completion of BZ which remained negative at 12 months post. Patient 5 had shown improvement to “borderline” AT1R at three months post, but this increased to positive by 12 months after completion of BZ. No changes in blood pressure were observed in AT1R positive patients after BZ.

Adverse events:

Patient 2 had graft failure and was transitioned to an adult outpatient hemodialysis unit six months after completion of BZ, thus adverse events after this time point are unknown. For the remaining patients, no adverse events were reported during therapy or at 24 months of follow-up.

Discussion:

In this small, single center study, we review the efficacy and tolerability of bortezomib as an adjunctive therapy in six pediatric kidney transplant patients with treatment-refractory AMR. This is the first pediatric study to our knowledge to report outcomes of bortezomib use in pediatric patients with AT1R-antibody associated AMR.

Overall, bortezomib was well tolerated without adverse events. Consistent with prior studies, Bortezomib may have been associated with stabilization in eGFR,^9,11–15 as there was no significant change in eGFR pre- and post-bortezomib, Table 2. One patient (patient 3) suffered graft loss in the setting of overt non-adherence with refusal to take baseline immunosuppression and participate in clinic visits and laboratory monitoring. Otherwise, there were no additional graft losses in the 12 months following bortezomib therapy.

Bortezomib was associated with a statistically significant reduction in the MFI of all detectable DSAs for each patient but without resolution in any immune-dominant DSA. This overall significant reduction of DSAs corroborate the findings in the pediatric literature where studies have shown >25–50% reduction in MFI of immune-dominant DSAs.^12–15 In our study even though some immune dominant DSA’s had a reduction of up to 75% (patient 4), MFI was still quite high at >5000 12 months post-therapy, Figure A1. The clinical relevance of persistent DSAs in this case is unclear in the setting of stable GFR. These findings are inconsistent with prior studies which have demonstrated modest improvement in eGFR despite persistent DSAs.¹³

In our patients with AT1R-antibody associated AMR, one patient experienced prolonged resolution of AT1R antibody within three months after initiation of BZ therapy. However, she remained hemodialysis dependent without recovery of allograft function. Contrarily, patient 5 had persistent AT1R antibody at 12 months post-therapy, but his eGFR was largely unchanged (decreased by 4%). One prior adult study showed complete resolution of AT1R antibodies when bortezomib was used as part of a multimodal regimen in a patient with mixed anti-HLA and anti-AT1R mediated rejection with complete resolution of AT1R antibodies after bortezomib therapy.²⁷ Bortezomib may have beneficial effects by modulating plasma cells and thus decreasing production of AT1R antibodies.²⁸ To our knowledge, this is the first study to report outcomes in two pediatric patients with AT1R antibody mediated AMR treated with bortezomib. Another notable strength of this study is the heterogeneity of the patients included, with a spectrum of age and primary disease pathology.

This study has several limitations. First, given that all patients received recent or concurrent anti-rejection therapies around the time of bortezomib administration, it is unclear if the effects observed are related to bortezomib, other therapies, or both. Additionally, the timeline from kidney transplant to bortezomib use was highly variable, at <2 years for two patients and 5–7 years for the remaining four patients. The efficacy of early use of bortezomib as anti-rejection therapy has not been widely studied, but Kolonko et al. demonstrated non-inferiority and acceptable safety of bortezomib in conjunction with plasmapheresis as a first line anti-rejection approach in 13 adult kidney transplant patients with early (median 19 days) AMR,²⁹ which is a promising area for future exploration, as the use of plasmapheresis prior to bortezomib therapy is reported in the majority of pediatric cases in the literature, even though not all in same time frame as Kolonko et al.^12–15,29 At bortezomib initiation, all patients already had a CADI score ≥ 3, and 4/6 had a score ≥ 5, indicating a significantly increased risk of graft loss independent of anti-rejection therapies.¹⁷ At the time of this study, routine protocol biopsies were not performed at our institution on all patients post-kidney transplant without clinical indication for biopsy (rising Cr, de novo DSA). Furthermore, the patients in this study did not have follow-up biopsies after completion of bortezomib therapy to assess for histologic response and change in CADI score. It is possible that earlier incorporation of protocol biopsies with close attention to the CADI score can serve as a way to risk-stratify patients at increased risk for decreased graft survival and allow for earlier incorporation of anti-rejection therapies, including bortezomib. Another limitation to acknowledge is related to DSA monitoring, which is semi-quantitative and varies depending on the saturation of specific antigens on a particular batch of Luminex beads. We calculated the DSA-RIS to try to mitigate the potential internal assay variability, but this approach may not complete capture the clinical significance. Additionally, our sample was small with limited racial/ethnic representation and so it may not be generalizable.

In conclusion, this study adds to the nascent but growing literature examining the use of bortezomib in kidney transplanted youth. Bortezomib was well tolerated in our small study population without adverse effects and may have assisted in stabilization of eGFR and an overall reduction in DSA MFI. More research is necessary to explore bortezomib as an adjunctive therapy in pediatric kidney transplant patients with both HLA- and non-HLA (AT1R) antibody mediated rejection and the use of the CADI score to improve long-term graft survival in this high-risk population.

Abbreviations:

ACR

acute cellular rejection

AMR

antibody mediated rejection

AT1R

angiotensin II type 1 receptor

BZ

bortezomib

CADI

chronic allograft damage index

DSA

donor specific antibody

GFR

glomerular filtration rate

HLA

human leukocyte antigen

MFI

mean fluorescence intensity

Appendix

Figure A1.

Change in DSA MFI at bortezomib administration (baseline) and trend over 12-month period.

Table A1.

Donor Specific Antibodies (DSA) during bortezomib administration (baseline) and trend over 12-month period

Patient	Donor Specific Antibody	Baseline	1 month post	3 months post	12 months post	DSA Change (pre/post)
1 †	AT1R antibody †	29.8		<10	<10	--
2	DQA1*01:01 DQB1*05:01	18,951	17,984	19,769	--	↑4%
3	DQA1*03:01 DQB1*03:02	7,338	5,693	5,131	3,495	↓52%
	DRB1*04:01	7,555	5,364	4,760	2,756	↓64%
4	DQA1*04:01 DQB1*04:02	20, 931	18,403	15,150	12,047	↓42%
	DQA1*05:03 DQB1*03:01	20,141	13,104	13,205	5,027	↓75%
	DRB1*01:03	5,364	3,545	3,361	1,371	↓74%
	DPA1*01:03 DPB1*03:01	0	0	2,354	341	‡ New
5 †	AT1R antibody †	>40		13	29	--
6	DR103/DRB101:03	1,795	882	330	206	↓89%
	DQ5/DQA*01:01 DQB1*05:01	15,282	9,093	8,573	5,676	↓63%

^†indicates subjects with AT1R antibody,

^‡indicates de-novo DSA post initiation of bortezomib

Table A2:

Prior studies reporting use of Bortezomib (BZ) in kidney transplantation in children and adults.

Author, year	Study Design	Clinical indication for BZ	Concurrent treatment	Bortezomib regimen	Outcome
Pediatrics
Kizilbash et al., Pediatr Transplant, 2017.14	Retrospective cohort, 33 patients, 9 pediatric kidney transplant centers.	94% of patients had C4d+ and peritubular capillaritis, 45% had glomerulitis. 79% had concurrent ACR.	Varied regimens: IVIG (90%), PP (78%), rituximab (78%), IVIG, PP and rituximab (60%)	Regimens varied across centers. 21/33 patients received four doses at 1.3 mg/m2/dose. Mean cumulative dose: 6.2 mg/m2, median: 5.2 mg/m2.	eGFR: improved or stabilized in 61% (3m) and 36% (12m) post-BZ. DSA: 56% of patients showed ≥ 25% reduction in MFI of iDSA (1–3 m post first dose).
Pearl et al., Pediatr Nephrol, 2016.13	Case series, 7 patients (ages 5–19, median 15 y)	Refractory C4d+ AMR	PP (days 1, 4, 8, 15, 17, 19, 22) immediately prior to BZ, rituximab (day 1), IVIG (day 22)	1.3 mg/m2/dose for four doses (days 1,4, 8, 15).	eGFR: graft loss (1/7), improvement from mean of 42 to mean of 53 mL/min/1.73m2 12m post-BZ DSA: 4/7 had class 1 DSA with 50% improvement at 12m post, 7/7 with class 2 DSA, 1/7 with resolution at 12m post Histology: 5/7 improved (1/7 with early AMR and complete resolution)
Roberti & Vyas, Pediatr Transplant, 2015.12	Case series, 2 pediatric patients	Resistant acute AMR, persistent DSAs.	IV MP pulse with IVIG and PP 3x/week for 10 treatments.	1.3 mg/m2/dose IV (after PP) on (days 1, 4, 7, 10).	eGFR: improvement/preservation of normal renal function DSA: persistent >50% reduction in DSAs (6m post)
Twombley et al., Pediatr Transplant, 2013.15	Case series, 3 adolescent Hispanic males (2 received BZ)	Acute AMR	PP, IVIG	1.3 mg/m2/dose for four doses	eGFR: improvement in eGFR (2y post) DSA: >50% reduction in DSA (6m post)
Adult
Eskandary et al., JASN, 2018.11	Prospective, placebo controlled RCT; 44 patients	All DSA positive with AMR morphology	Stepwise increase in MMF (up to 1000 mg/d) or enteric-coated MPA (up to 720 mg/d), if tolerated.	1.3 mg/m2/dose IV on days 1, 4, 8, and 11, x2 cycles	eGFR: no significant difference at 24m, no difference in 2-year graft survival DSA: no significant difference at 24m Histology: no significant change in morphologic or molecular rejection in follow-up biopsy (24m)
Everly et al., Transplantation, 2008.9 (First report of BZ use)	Case series, 6 patients (ages 28–56)	Mixed AMR and ACR	Varied regimens: PP, IVIG, rituximab	1.3 mg/m2/dose for four doses (two patients required a second cycle of four doses)	eGFR: graft failure (1/6), all others with improved Cr at 7d – 4m post treatment DSA: >50% reduction in iDSA at 5m Histology: improved inflammation and improvement in AMR or ACR at 8–26 d post-BZ

(ACR: acute cellular rejection; AMR: antibody mediated rejection; BZ: bortezomib; iDSA: immunodominant donor specific antibody; IVIG: intravenous immunoglobulin; MMF: mycophenolate mofetil; MP: methylprednisolone pulse; MPA: mycophenolic acid; PP: plasmapheresis; RCT: randomized controlled trial)

Data statement:

The data that supports the findings of this study are available in the supplementary material of this article