Department of Cellular Therapeutics (GMP), German Red Cross Blood Service BaWü-He, Institute Frankfurt, Frankfurt, Germany.
Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt, Germany.
J Transl Med. 2023 Nov 25;21(1):849. doi: 10.1186/s12967-023-04738-8.
In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαβ/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαβ/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy.
We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA or of TCRαβ and CD19 cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2-6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry.
Ten sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA resp. TCRαβ and CD19 cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45 and HSPCs was > 70% and in line with expectation.
The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαβ/CD19-depleted products included HSPCs, TCRγδ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.
在体外或体内耗竭同种反应性 T 细胞可以促进单倍体相合造血干细胞移植（HSCT）。因此，报道了使用 TCRαβ/CD19 耗尽的造血干细胞/祖细胞（HSPC）移植物进行非常满意的移植结果。目前，在 CliniMACS Plus 上进行的半自动制造工艺虽然强大，但仍需要大量的手工劳动才能完成。为了推进和进一步促进大规模细胞处理，在 CliniMACS Prodigy 上建立了一个新的 TCRαβ/CD19 耗尽模块，该模块结合了之前描述的 CD45RA 耗尽模块（用于作为同种反应性减弱的供体淋巴细胞输注）。
我们评估了来自 G-CSF 动员志愿者供体的六个单采产品，这些产品由 Prodigy 自动分割，每个部分分别耗尽 CD45RA 或 TCRαβ 和 CD19 细胞。我们研究了两种产品的关键质量属性。使用流式细胞术评估两种产品的 HSPCs 和成熟亚群的恢复情况，以及靶向细胞的耗竭效率。通过流式细胞术评估在 2-6°C 下储存 48 小时后以及冻融对产品活力和白细胞（WBC）和 HSPC 恢复的影响。
完成了 10 个连续的自动处理过程，除了安装管道设置之外，几乎不需要手动操作。CD45RA 或 TCRαβ 和 CD19 细胞的耗竭效率与之前的报道相当，两种产品均实现了 4 个对数级别的靶向细胞耗竭。HSPC 产品保留了 TCRγδ 和 NK 细胞。单采产品 48 小时储存与预期的 HSPC 适度损失相关，但耗竭仍有效。耗尽的产品在单采后至少 72 小时内保持稳定，干细胞活力>90%。冻融导致 NK 细胞丢失；冻融后活 CD45 和 HSPC 的恢复>70%，符合预期。
密闭、GMP 兼容的工艺从同一动员的单采产品中生成两种单独的药物产品。CD45RA 耗尽的产品含有功能性记忆 T 细胞，而 TCRαβ/CD19 耗尽的产品包含 HSPCs、TCRγδ 和 NK 细胞。两种产品都有望有效耗尽移植物抗宿主病（GVHD）反应，同时提供免疫监测，支持单倍体相合 HSCT。
