Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201, USA.
J Exp Clin Cancer Res. 2010 Oct 11;29(1):134. doi: 10.1186/1756-9966-29-134.
The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i) if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii) their ability to kill allogeneic and autologous tumor targets by direct cytotoxicity and by antibody-mediated cellular cytotoxicity and (iii) defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing.
Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated.
Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors.
These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical evidence necessary to support clinical trials utilizing autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy in select solid tumors.
自体自然杀伤（NK）细胞作为一种有效治疗实体瘤的方法已经被广泛研究。然而，目前的应用受到了以下因素的限制：（i）外周血中 NK 细胞数量较少；（ii）大规模生产符合良好生产规范（GMP）的细胞存在困难；（iii）需要激活 NK 细胞以诱导 NK 细胞介导的杀伤；（iv）自体抑制性受体-配体相互作用的限制。为了解决这些问题，我们确定了以下内容：（i）是否可以从 PBMC 和通过切向流离心分离得到的 GMP 合规细胞部分中大量扩增 NK 细胞；（ii）通过直接细胞毒性和抗体介导的细胞毒性杀伤同种异体和自体肿瘤靶细胞的能力；（iii）定义介导肿瘤靶细胞杀伤的 NK 细胞特异性受体-配体相互作用。
人 NK 细胞在 14 天内进行扩增。我们研究了扩增效率、扩增前后 NK 受体谱、NK 特异性配体的表达、对同种异体和自体肿瘤靶细胞的细胞毒性、添加嵌合 EGFR 单克隆抗体前后的情况。
细胞扩增使 NK 细胞受体谱向激活方向转变，并导致对各种同种异体肿瘤细胞系和自体胃癌细胞的杀伤，同时对正常 PBMC 无杀伤作用。阻断研究证实，自体细胞毒性是通过多种激活受体-配体相互作用建立的。重要的是，扩增的 NK 细胞还通过目前用于治疗某些实体瘤患者的抗体在自体和同种异体环境中介导 ADCC。
这些数据表明，大量的细胞毒性 NK 细胞可以从 PBMC 和通过 GMP 合规的切向流离心分离得到的富含淋巴细胞的部分中产生，为利用自体扩增 NK 细胞进行临床试验提供了必要的临床前证据，这些 NK 细胞可直接使用，也可与单克隆抗体联合用于未来某些实体瘤的细胞免疫治疗。
