Obinutuzumab Effectively Depletes Key B-cell Subsets in Blood and Tissue in End-stage Renal Disease Patients

Abstract

The THEORY study evaluated the effects of single and multiple doses of obinutuzumab, a type 2 anti-CD20 antibody that induces antibody-dependent cell-mediated cytotoxicity and direct cell death, in combination with standard of care in patients with end-stage renal disease.

Methods: We measured B-cell subsets and protein biomarkers of B-cell activity in peripheral blood before and after obinutuzumab administration in THEORY patients, and B-cell subsets in lymph nodes in THEORY patients and an untreated comparator cohort.

Results: Obinutuzumab treatment resulted in a rapid loss of B-cell subsets (including naive B, memory B, double-negative, immunoglobulin D⁺ transitional cells, and plasmablasts/plasma cells) in peripheral blood and tissue. This loss of B cells was associated with increased B cell-activating factor and decreased CXCL13 levels in circulation.

Conclusions: Our data further characterize the mechanistic profile of obinutuzumab and suggest that it may elicit greater efficacy in indications such as lupus where B-cell targeting therapeutics are limited by the resistance of pathogenic tissue B cells to depletion.

FIGURE 1.

Multiple doses of obinutuzumab elicited a rapid and sustained depletion of B-cell subsets in peripheral blood in ESRD patients. B-cell subsets (black = cohort 1, blue = cohort 2) were measured in patients before and after doses of obinutuzumab (naive, memory, switched memory, unswitched memory, immunoglobulin D⁺ transitional, double negative, and plasmablasts/plasma cells are A–G for cohort 1 and H–N for cohort 2, respectively). One cohort 2 patient (noted here) had antidrug antibodies that reduced exposure and resulted in return of detectable B cells in circulation by wk 24. Baseline samples were drawn preinfusion. ESRD, end-stage renal disease.

FIGURE 2.

B cells in lymph nodes are lower in patients treated with obinutuzumab vs untreated comparators. Lymph nodes collected during kidney transplant, for both study and comparator nodes, were bisected; half was subjected to cell dissociation and stained for flow cytometry. CI, confidence interval.

FIGURE 3.

Immunohistochemistry confirms that B cells, not T cells, are decreased in patients treated with obinutuzumab. Lymph nodes collected during kidney transplant, for both study and comparator nodes, were bisected; half was subjected to cell dissociation, formalin-fixed and paraffin-embedded, and then stained via immunohistochemistry for the B-cell marker CD79a and the T-cell marker CD3. A, plot of B cells and T cells per analyzed tissue area (patient averages shown). Bars represent mean and 95% confidence interval. B, Representative B-cell staining in obinutuzumab-treated and comparator patients’ nodes. C, Representative T-cell staining in obinutuzumab-treated and comparator patients’ nodes. CD, cluster of differentiation; DAPI, 4′,6-diamidino-2-phenylindole.

FIGURE 4.

BAFF increased in patients treated with obinutuzumab. Serum samples were collected from patients before and after dosing with obinutuzumab and BAFF measured by ELISA ([A] all patients, [B] patients who went to transplant, [C] patients with detectable B cells in tissue; black = cohort 1, blue = cohort 2). Samples collected on days of obinutuzumab administration were collected predose. BAFF, B cell–activating factor; BL, baseline; ELISA, enzyme-linked immunosorbent assay; TP, time of kidney transplant; W, week.

FIGURE 5.

CXCL13 decreased in patients treated with obinutuzumab. Serum samples were collected from patients before and after dosing with obinutuzumab and CXCL13 measured by ELISA ([A] all patients, [B] patients who went to transplant, [C] patients with detectable B cells in tissue; black = cohort 1, blue = cohort 2). Samples collected on days of obinutuzumab administration were collected predose. BL, baseline; CXCL13, C-X-C motif chemokine ligand 13; ELISA, enzyme-linked immunosorbent assay; TP, time of kidney transplant; W, week.