Desensitization With Proteasome Inhibition and Costimulation Blockade Modulates the Xenoreactive Humoral Response in Nonhuman Primate Xenotransplantation

Abstract

Introduction: "Delayed" antibody-mediated xenograft rejection is one of the most important obstacles to clinical application of pig organ xenografts. The aim of this study was to assess the impact of a structured desensitization regimen including proteasome inhibition and next-generation costimulation blockade on xenoreactive antibodies.

Methods: Rhesus macaques with moderate-high pre-treatment xenoreactive antibody titers (N = 2) were selected. Recipients received twice-weekly carfilzomib (20 mg/m²), anti-CD154 (20 mg/kg) every other week, and CD4 and CD20 lymphocyte cell depletion. Bone marrow was acquired to assess plasma cell depletion in response to proteasome inhibition. A flow cytometry-based xenoreactive crossmatch assay was performed to assess levels of circulating xenoreactive antibodies.

Results: The desensitization regimen resulted in a >50% depletion of CD38+CD27+ bone marrow plasma cells; these changes were progressive over the duration of the desensitization treatment period. The desensitization strategy and plasma cell depletion resulted in a progressive reduction in anti-pig IgG antibodies. Following xenotransplantation, both desensitized recipients demonstrated superior graft survival to a highly xenoreactive recipient (MST 30 days vs. 6 days), but neither desensitized recipient experienced prolonged graft survival.

Conclusions: A structured desensitization regimen including proteasome inhibition and costimulation blockade results in plasma cell depletion and resultant reduction in circulating xenoreactive anti-pig IgG antibodies, with a modest improvement in xenograft survival. This desensitization regimen has promise for pig-to-NHP xenotransplant models.

Keywords: antibody; proteasome inhibition; xenotransplantation.

FIGURE 1

Proteasome inhibitor‐based desensitization protocol dosing regimen. Moderate‐high xenoreactive rhesus macaques received an 8‐week desensitization protocol including twice weekly carfilzomib, biweekly costimulation blockade (CD154 mAb), and CD4 and CD20 depleting antibody therapy every 4 weeks. Carfilzomib was continued for 4 weeks post‐transplant.

FIGURE 2

Bone marrow plasma cell sampling. Recipients on the desensitization protocol received protocol percutaneous humerus bone marrow biopsies every4 weeks during therapy. (A) Both recipients demonstrated a reduction in CD19⁺CD20⁻CD38⁺CD27⁺IgM⁻IgD⁻ plasma cells during the course of their desensitization, which rebounded in RM2 after cessation of carfilzomib treatment. (B) Bone marrow biopsies demonstrated reduction in CD19⁺, CD27⁺, and CD38⁺ cells using viSNE analysis.

FIGURE 3

Xenoreactive IgG and IgM antibody titers. Recipients demonstrated a progressive reduction in both IgG and IgM xenoreactive antibody titers throughout the course of the pre‐transplant desensitization regimen. RM2 demonstrated rebound IgG after cessation of carfilzomib therapy.

FIGURE 4

Xenograft survival and histology. (A) Desensitized recipients exhibited improved graft survival relative to highly xenoreactive controls (MST 30 vs. 6 days). (B) Histology at time of rejection revealed features consistent with ABMR in the desensitized recipients, while low‐titer controls had normal kidney architecture and physiology.