一种新型自体NK细胞扩增培养方法的建立和初步临床应用

A new method of autologous NK cell culture and expansion for clinical application

目的应用一种新型的自体NK细胞扩增培养方法,并进行初步的临床试验。方法采集患者静脉血30ml,用Ficoll法分离外周血单核细胞,用NKGM培养体系培养两周,收集细胞后回输患者。实验全程在GMP实验室进行,并做好质量控制。结果通过14 d的细胞培养,淋巴细胞总数扩增了约200～300倍,其中NK细胞和CD8+T细胞均扩增了约500倍,最终NK细胞和CD8+T细胞的比例约为36%～53%、38%～51%。对1例处于化疗后部分缓解的肺癌且伴多发转移的患者进行两个疗程的细胞治疗,患者血清肿瘤标志物糖类抗原(CA)19-9呈进行性降低。而且在NK细胞输注后的2～6周,仍可检测出患者外周血淋巴细胞中NK细胞的比例高达约31%。结论 NKGM培养体系可通过两周的培养使外周血单核细胞扩增约250倍,而且NK细胞的纯度可高达53%。同时,该细胞疗法具有很好的安全性和便捷性,应用于肿瘤患者可通过维持外周血NK细胞的高比例而发挥积极的临床疗效。

Objective Adopt a new method of autologous NK cell culture and expansion,and use it for preliminary clinical trials.Methods Isolate peripheral blood mononuclear cells with Ficoll reagent after pumping 30 milliliter blood from the patient.Then,use the NKGM reagent to culture and expand the lymphocytes.Two weeks later,collect the cells for cytotherapy.All steps are performed in GMP lab with quality control.Results After culture for 14 days,the total number of lymphocytes expanded about 200～300 times,while both NK cell and CD8+ T cell expanded about 500 times.The final percentage of NK cell was about 36%～53%,while the CD8+ T cell's was about 38%～51%.With two courses of cytotherapy,the serum tumor marker CA19-9 of the cancer patient,who received partial remission after chemotherapy,decreased gradually.Interestingly,the percentage of NK cells in PBMC could still keep up to about 31% in 2～6 weeks after NK cells infusion.Conclusions The total number of lymphocytes could expand about 250 times after 14 days culture,and the percentage of NK cells was up to 53%.Furthermore,it's very safe and convenient to apply this culture system for cytotherapy,and the percentage of NK cells in PBMC could maintain a high level for a month after NK cells infusion.Whether it plays an active role in cancer immunotherapy needs to be further studied.