University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN, USA.
Department for Hematology and Oncology, Medicine Department 2, University Hospital of Tuebingen, Tuebingen, 72076, Germany.
Target Oncol. 2016 Jun;11(3):353-61. doi: 10.1007/s11523-015-0391-8.
Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse, and express the well-known surface marker CD133.
DNA fragments from two fully humanized single chain fragment variable (scFv) antibodies recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigens to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated a unique antibody that recognized the molecular framework and was species cross-reactive.
In vitro chromium-51 ((51)Cr) release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5 % CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug.
These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133 and that this anti-CD133 scFv may be useful in this bispecific platform or perhaps in the design of more complex trispecific molecules for carcinoma therapy.
自然杀伤 (NK) 细胞是一种强有力的细胞毒性淋巴细胞，在肿瘤免疫监视和控制中发挥着关键作用。癌症干细胞 (CSC) 启动并维持肿瘤细胞的生长，介导耐药性癌症的复发，并表达众所周知的表面标记 CD133。
两个完全人源化单链片段可变 (scFv) 抗体的 DNA 片段，分别识别 NK 细胞上的 CD16 和 CSC 上的 CD133，通过基因拼接形成一种新型药物 16×133 BiKE，同时识别这两种抗原以促进免疫突触的形成。抗 CD133 是通过融合蛋白制备的，该融合蛋白是通过融合编码 CD133 的两个细胞外结构域的 DNA 片段制成的。用该融合蛋白免疫小鼠产生了一种独特的抗体，该抗体识别分子框架并具有种间交叉反应性。
在高和低效应器：靶标比的体外铬-51 ((51)Cr) 释放细胞毒性测定中，该异二聚体生物药物能够显著增强 NK 细胞对人结肠直肠癌细胞系 Caco-2 的杀伤作用，该细胞系已知过度表达 CD133。该药物对 CD133 的肿瘤相关抗原特异性甚至增强了 NK 细胞对 NK 耐药的人类 Burkitt 淋巴瘤 Daudi 细胞系的细胞毒性，该细胞系表面表达的 CD133 少于 5%。流式细胞术分析显示，在药物存在的情况下，与 Caco-2 靶细胞共培养时，NK 细胞脱颗粒和干扰素-γ产生增加。
这些研究表明，可以使用靶向 CD133 的双特异性抗体有效地招募先天免疫系统来杀死 CSC，并且这种抗 CD133 scFv 可能在该双特异性平台中有用，或者在设计用于癌症治疗的更复杂的三特异性分子中有用。
