摘要：

5445 We developed a proprietary cell therapy process to selectively expand Vγ9Vδ2 (Tgd) lymphocytes in vitro with BrHPP (a Tgd agonist) and IL-2, following short term culture of White Blood Cells (WBC). We started from an in vitro small scale (24 well plate) assay, based on 1.5 million unfractionated PBMC stimulated with a single dose of BrHPP at day 0 and cultured in the presence of IL2. This small scale assay reproducibly produced more than 35 million cells in about 3 weeks, among which more than 60% T gd (from PBMC with about 1% Tgd at start). From this basis, we set up a large scale amplification of greatly enriched cells therapeutic grade cell product. Materials and methods : We investigated several cell culture parameters i) culture media, ii) secured Fetal Calf Sera (FCS) versus secured Human sera, iii) source of WBC iv) a suitable closed culture system, v) freezing procedures vi) culture conditions adapted to closed culture system, vii) culture maintenance frequency and viii) final cell harvesting and conditioning procedure. The read outs used were Vd2 enrichment (flow cytometry), cell amplification & viability, and functionality (cytotoxicity and TNF release assay) Results : Optimization of the process led to the following parameters : mononuclear cells are isolated by leukapheresis, frozen in FCS+DMSO and stored in liquid nitrogen. Large scale cultures are initiated from 0.6 billion cells in 3 liter cell culture bags with RPMI+FCS+IL-2+BrHPP and maintained twice a week for 15 days in RPMI+FCS+IL-2. The process allows to obtain about 9 billion cells among which more than 80% of Tgd for individual known as responder to BrHPP (healthy volunteer or patient). The final cell suspension is washed and harvested automatically with help of the CytoMate™ (Nexell), finally formulated in 4% Human Albumin and packaged at 10 to 100 million cells/ml. The cell product is stored at 4°C and remains viable as well as functional up to 8 hours. Conclusion : INNACELL GD process meets criteria for the therapeutic use i) large quantity of cells is reproducibly produced (total cell amplification of about 14 and Tgd specific amplification greater than 1000), ii) specificity of the expansion (more than 80% of Tgd lymphocytes), iii) security of the system (closed cultures in bags), iv) recovery yield of the cell concentration step (about 90%) for individuals known to be responder to the molecule BrHPP. Further development is ongoing to optimize storage conditions of the final cell product for shipment. This INNACELL GD® cell product is currently under a phase I clinical trial in patients with MRCC.

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