Immunotherapy targeting B cells and long-lived plasma cells effectively eliminates pre-existing donor-specific allo-antibodies

Abstract

Pre-existing anti-human leukocyte antigen (HLA) allo-antibodies constitute a major barrier to transplantation. Current desensitization approaches fail due to ineffective depletion of allo-specific memory B cells (Bmems) and long-lived plasma cells (LLPCs). We evaluate the efficacy of chimeric antigen receptor (CAR) T cells targeting CD19 and B cell maturation antigen (BCMA) to eliminate allo-antibodies in a skin pre-sensitized murine model of islet allo-transplantation. We find that treatment of allo-sensitized hosts with CAR T cells targeting Bmems and LLPCs eliminates donor-specific allo-antibodies (DSAs) and mitigates hyperacute rejection of subsequent islet allografts. We then assess the clinical efficacy of the CAR T therapy for desensitization in patients with multiple myeloma (MM) with pre-existing HLA allo-antibodies who were treated with the combination of CART-BCMA and CART-19 (ClinicalTrials.gov: NCT03549442) and observe clinically meaningful allo-antibody reduction. These findings provide logical rationale for clinical evaluation of CAR T-based immunotherapy in highly sensitized candidates to promote successful transplantation.

Keywords: BCMA; CAR T cells; CD19; TACI; allo-antibodies; desensitization; immunotherapy; long-lived plasma cells; rejection; transplantation.

Graphical abstract

Figure 1

Allo-specific clones reside in the B cell and both CD19⁺ and CD19⁻ PC compartments B6 mice (n = 3/group) were sensitized two serial skin grafts from B6 (isograft control) or BALB/c (allograft) donors. (A) Two weeks after rejection of the second skin graft, B cells, CD19⁺ PCs, and CD19⁻ PCs were sorted. (B) Sorted cells were analyzed by ELISPOT for secretion of IgG specific to BALB/c MHC (H2-K^d, H2-D^d, I-A^d). PCs were assessed directly after sorting, while B cells were activated for 4 days prior to ELISPOT. Representative wells are shown. (C) Allo-specific cells in each cell compartment as a percentage of plated cells. Error bars indicate mean ± SD. ∗p < 0.05, ∗∗∗p < 0.005.

Figure 2

Combination CAR T cells effectively deplete B cells and PCs (A) Schematic of CARs targeting murine CD19 and BCMA. (B) Expression of indicated CARs in murine T cells. (C) CAR T cell-mediated cytotoxicity of K562 cells expressing murine CD19 (mCD19) or murine BCMA (mBCMA). (D) Schematic of in vivo CAR T cell evaluation. (E–G) Total B cells (E and F) and PCs (G) were measured 14 days after CAR T infusion (n = 3–5/group). (H and I) Evaluation of PC subsets in spleen and bone marrow by gating on CD138⁺TACI⁺ PCs shown in (G) (n = 3–5/group). (J) IgG-specific PCs were measured by ELISPOT 14 days after CAR T infusion (n = 6–9/group). Error bars indicate mean ± SD. Statistical comparisons performed using Mann-Whitney U test, and only significant differences are indicated (∗p < 0.05). All experiments are representative of 3 or more independent replicates.

Figure 3

Combination CAR T cells eliminate pre-existing DSAs and promote transplant (A) Schematic of mouse model evaluating CAR T cells for desensitization (n = 5–7/group). (B) Representative longitudinal flow crossmatch. Baseline represents the post-sensitization, pre-CART time point. (C) DSAs by flow crossmatch based on T cell flow crossmatch (post-CART MFIs are normalized to the baseline pre-CART MFI). (D) DSAs across groups at week 13. (E and F) Longitudinal (E) and week 13 (F) ELISA-based measurement of serum H2-D^d-specific IgG. Results are representative of 3 replicates. Dotted line indicates nonsensitized mice. (G) Individual blood glucose following test islet allograft (representative of 2 independent experiments). (H) Kaplan-Meier analysis of islet allograft survival including control nonsensitized mice without T cell treatment and with and without ATG. (I) Correlation of pre-transplant DSA and islet allograft survival. Spearman’s rank correlation is −0.68 (95% confidence interval [CI]: −0.51, −0.83) with a corresponding p = 0.0026. (J) DSAs at time of islet allograft rejection or experimental endpoint (day 60). All error bars indicate mean ± SD. Statistical comparisons were performed using ANOVA and Tukey’s test (C and E), Mann-Whitney U test (D, F, and J), and log-rank test using Benjamini-Hochberg correction (E), and only significant differences are indicated (∗p < 0.05). In (C), ∗p < 0.05 between Comb-CART vs. NTD (bottom) and Combo-CART vs. CART19.

Figure 4

Reconstitution of the humoral immune compartment following CAR T treatment (A and B) Total circulating B cells at the indicated time points post-CAR T injection (A) and at the time of islet rejection or experiment endpoint (B) from the experiment in Figure 2 (n = 5–7/group). (C) Total serum IgG was measured at the indicated times relative to CAR T transfer from the experiment in Figure 2. (D) Individual Ig isotypes and subclasses were measured 13 weeks post-CAR T transfer. Error bars indicate mean ± SD. All statistical comparisons performed using ANOVA and Tukey’s test (A and B) and Mann-Whitney U test, and only significant differences are indicated (∗p < 0.05). In (A), ∗p < 0.05 between Comb-CART vs. NTD (bottom) and CART19 vs. NTD (top). In (C), ∗p < 0.05 between Comb-CART vs. NTD (bottom), CART19 vs. NTD (middle), and CART19 vs. Combo-CART (top). Results are representative of two independent experiments.

Figure 5

Clinical desensitization after treatment with B cell- and PC-targeted CAR T cells (A) Schematic of clinical study and CAR T assignment (CART-BCMA and/or CART-19) for the indicated five subjects. (B) Swimmer’s plot of multiple myeloma response. (C–F) Longitudinal measurement of (C) CAR T cells, (D) B cells, and (E) soluble BCMA (sBCMA) (F) allo-antibodies. Dotted lines in (F) indicate the laboratory threshold for assignment of unacceptable antigens. PD, progressive disease; MRD, minimal residual disease; PR, partial response; VGPR, very good partial response; CR, complete response; sCR, stringent complete response.