No abstract

Background To date, in‐depth analysis of leukapheresis products as starting material for CAR T‐cell manufacturing, specifically Tisagenlecleucel production, are scarce. In this study, we report on lymphapheresis data for production of Tisagenlecleucel for elderly and pretreated lymphoma patients. Study Design and Methods Spectra Optia from Terumo BCT, Lakewood, CO, was employed for apheresis using the cMNC program. Apheresis success was defined as meeting a target total nucleated cell (TNC) count of ≥2 × 109, a CD3‐positive lymphocyte count of ≥1 × 109 and an overall viability of ≥70% in the lymphapheresis product. Results Twenty‐three patients (age 37–77 years) and 24 apheresis runs were evaluated. The median CD3‐positive lymphocyte count in peripheral blood at the beginning of apheresis was 565 cells/μl (range: 70–1345 cells/μl). Circulating lymphoma cells were detected in one patient prior to apheresis. Target criteria were met in 21 of 23 patients. The median TNC count in the apheresate was 11.2 × 109 (range: 2.9 × 109–47.4 × 109). The median CD3‐positive lymphocyte count in the apheresate was 2.55 × 109 (range: 0.370 × 109–6.915 × 109), which resulted in a median collection efficiency for CD3‐positive lymphocytes of 63.7% (range: 9.56%–93.6%). No adverse events associated with the apheresis process were observed. Conclusions Lymphapheresis with the Spectra Optia cMNC program provided a sufficient quantity of CD3‐positive lymphocytes for CAR T‐cell manufacturing for the majority of patients despite their heavy pretreatment and advanced age. Moreover, we are the first to advocate early pre‐emptive lymphocyte collection in DLBCL‐NOS patients intended to undergo treatment with Tisagenlecleucel.

The immunomodulatory fusion protein abatacept has recently been investigated for the treatment of steroid-refractory chronic graft-versus-host disease (cGvHD) in a phase 1 clinical trial. We analyzed the safety and efficacy of abatacept for cGvHD therapy in a retrospective study with 15 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) and received abatacept for cGvHD with a median age of 49 years. Grading was performed as part of the clinical routine according to the National Institute of Health’s (NIH) consensus criteria at initiation of abatacept and 1, 3, 6, 9 and 12 months thereafter. The median time of follow-up was 191 days (range 55–393 days). Best overall response rate (ORR) was 40%. In particular, patients with bronchiolitis obliterans syndrome showed significant clinical improvement and durable responses following abatacept treatment with a response rate of 89% based on improvement in lung severity score (n = 6) or stabilized lung function (n = 4) or both (n = 3). Infectious complications CTCAE °III or higher were observed in 3/15 patients. None of the patients relapsed from the underlying malignancy. Thus, abatacept appears to be a promising treatment option for cGvHD, in particular for patients with lung involvement. However, further evaluation within a phase 2 clinical trial is required.

2976 Graft-versus-host disease (GVHD) and infectious complications are main causes of non-relapse mortality after allogeneic stem cell transplantation (SCT). Impaired immune function after SCT is usually attributed to the immunosuppressive medication applied for GVHD prophylaxis or therapy. Using a major histocompatibility complex (MHC)–mismatched murine model of GVHD (C57BL/6→BALB/c), we now examined the influence of GVHD on B cell immunity after SCT in the absence of pharmacologic immunosuppression. Lethally irradiated BALB/c (H-2d) recipients were transplanted with T cell-depleted bone marrow (TCD BM; 2.5×106) from C57BL/6 (H-2b) donors and parallel groups received CD4+CD25− conventional donor T cells (Tconv; 0.25 × 106) 2d later. Mice that received TCD BM alone (n =10) did not develop GVHD and showed a rapid and complete reconstitution of B cells in peripheral blood (PB) (25 ± 7% CD19+ B cells at d21; 55 ± 5% at d100). Mice that received additional donor Tconv cells (n =12) developed severe GVHD and completely lacked donor and host B cells in PB until their early death or throughout the observation period of 100d (p<0.001). Animals that were protected from severe GVHD by the co-infusion of donor CD4+CD25+Foxp3+ regulatory T cells (Treg; 0.25×106 Treg transplanted together with TCD BM; 2d later transfusion of 0.25 × 106 Tconv) showed a delayed, but finally full reconstitution of their B cell compartment in PB (9 ± 12% CD19+ B cells at d21; 42 ± 17% at d100). Similarly, animals without GVHD after TCD BMT and animals protected from GVHD by co-transplanted Treg cells showed a complete reconstitution of their B cell compartment in spleen and BM at d100 (spleen: 26±4, 7×106 and 31 ± 9.3×106 CD19+ B cells, respectively; BM: 2, 2 ± 0, 3×106 and 2.9 ± 0.9×106 B cells, respectively). In contrast, B cells were not only undetectable in peripheral lymphoid organs in animals with severe GVHD but also in the BM, suggesting that B cell precursors were affected. To examine whether GVHD solely impedes B cell regeneration or actively contributes to B cell eradication, GVHD was induced after B cell reconstitution at d21 after TCD BMT by donor lymphocyte infusions (DLI). Within 1wk after the transfer of 8×106 or 12×106 donor CD4+ lymphocytes, a significant reduction of B cells in PB was detected (from 30.3 ± 5.2% to 10 ± 6.9% and 36.3 ± 9.2% to 5.9 ± 1.3%, respectively; n =4). Thus, GVHD not only affected B cell reconstitution, but even eradicated stem cell-derived B cells that were syngeneic to the GVHD-inducing T cells, suggesting that GVHD-induced inflammation contributed to B cell depletion. To examine the influence of GVHD on precursor cells, serial transplants were performed. Yet, TCD BM from both, animals with and without GVHD, reconstituted their B cell compartment upon secondary transplantation (n =18; 33.1 ± 14.8% vs. 32.4 ± 17% at d100), thereby proving that the stem cell compartment was not affected. Next, we examined the effect of GVHD on precursor cells. Multipotent BM precursors (lin−, Sca-1+, c-kit+ [LSK]) were not significantly different in GVHD animals (TCD BM plus Tconv; n =12) as compared to controls (TCD BM only; n =10; 3.5×103 ± 2.8×103 vs. 5.8×103 ± 2.5×103, respectively). However, common lymphoid precursors (CLP; Lin−, FLt3+, CD127+) in the BM were significantly reduced in animals with GVHD (0.3×103 ± 0.17×103) as compared to transplant recipients without GVHD (4.4×103 ± 2.2×103, p<0.001). These results suggest that the dysregulated production of pro-inflammatory cytokines during GVHD is toxic for early B cell precursors and/or that the alloresponse destroys the BM niche for developing B cells. As IFN- γ and TNF are known to be elevated in GVHD and to impair B lymphopoiesis even in a non-transplant setting, we generated mixed chimeras using BM from wt and cytokine receptor deficient animals. Yet, a selective B cell reconstitution from receptor deficient BM was not observed in GVHD, suggesting that neither of these cytokines is exclusively responsible for its toxic effects on B cell precursors. Taken together, our results show that GVHD not solely affects immune reconstitution by the well known destruction of secondary lymphoid organs, but it disturbs early lymphoid progenitors in the BM through inflammatory, but not necessarily allo-specific immune responses. Disclosures: No relevant conflicts of interest to declare.