Clinical-grade myeloma Ag pre-loaded DC vaccines retain potency after cryopreservation
Clinical-grade myeloma Ag pre-loaded DC vaccines retain potency after cryopreservation
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DOI：
10.1080/14653240510027235
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发表时间：
2005-09-01
期刊：
影响因子：
4.5
通讯作者：
van Rhee, F
中科院分区：
文献类型：
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作者：
Szmania, S;Yi, Q;van Rhee, F
骨髓瘤Ag负载的成熟DC疫苗的使用是一种有吸引力的免疫策略，该疫苗以单次使用的等分试样冷冻保存。在这项研究中，我们调查了保留的表型，活力和效力的DC疫苗冻融后。方法塑料粘附单核细胞，来自稳态白细胞分离，培养在无血清培养基中含有GM-CSF和IL-4。在第6天用骨髓瘤裂解物（ML）或独特型（Id）Ag和钥孔血蓝蛋白（KLH）加载DC，在第7天用CD 40-配体诱导成熟，并在第9天冷冻保存。17个临床规模的培养DC产量，回收率和免疫表型进行了评估后，效力进行了验证，异基因混合淋巴细胞培养和Ag presentation assays.Results我们生产了88个单独的疫苗从17个临床规模的文化。收获时的中位DC产量为131 × 10（6）（范围37- 375 × 10（6）），解冻后活DC的中位回收率为69%（范围11-100%）。我们证实了DC的活力（7AAD（-））、表型（CD 14（-）、CD 83（+）/CD 40（+）、CD 83（+）/CD 80（+）、CD 83（+）/CD 86（+）、CD 83（+）/CD 54（+）、HLA-DR++）以及DC在解冻后呈递Ag和刺激同种异体T细胞的能力。冷冻保存不干扰DC活性，允许时间进行严格的质量控制（QC）和灵活的结内接种时间表，并减少了制备多种疫苗的时间。
Background The use of myeloma Ag-loaded mature DC vaccines, cryopreserved in single-use aliquots, is an attractive immunotherapeutic strategy. In this study we investigated the retention of phenotype, viability and potency of DC vaccines after freezing and thawing.Methods Plastic-adherent monocytes, derived from a steady-state leukapheresis, were cultured in serum-free media containing GM-CSF and IL-4. DC were loaded on day 6 with myeloma lysate (ML) or idiotype (Id) Ag and keyhole limpet hemocyanin (KLH), induced to mature on day 7 with CD40-ligand and cryopreserved on day 9. Seventeen clinical-scale cultures were evaluated for DC yield, recovery and immunophenotype after potency was validated with allogeneic mixed lymphocyte culture and Ag presentation assays.Results We produced 88 individual vaccines from 17 clinical-scale cultures. Median DC yield at harvest was 131x10(6) (range 37-375x10(6)) and median recovery of viable DC after thawing was 69% (range 11-100%). We confirmed viability (7AAD(-)), phenotype (CD14(-), CD83(+)/CD40(+), CD83(+)/CD80(+), CD83(+)/CD86(+), CD83(+)/CD54(+), HLA-DR++) and the ability of the DC to present Ag and stimulate allogeneic T cells post-thawing.Discussion We have validated a serum-free culture system for the production of DC. Cryopreservation did not interfere with DC activity, allowed time for rigorous quality control (QC) and flexible scheduling of intranodal vaccination, and reduced the time to prepare multiple vaccines.