Protein Interactions Group, CCRNP, NCI-Frederick, NIH, 1050 Boyle Street, Frederick, MD 21702, USA.
Arthritis Res Ther. 2011 Apr 8;13(2):R59. doi: 10.1186/ar3312.
Folate receptor beta (FRβ) is only detectable in placenta and limited to some hematopoietic cells of myeloid lineage in healthy people. Studies have indicated that FRβ is over-expressed in activated macrophages in autoimmune diseases and some cancer cells. In this study we aimed to develop an FRβ-specific human monoclonal antibody (mAb) that could be used as a therapeutic agent to treat rheumatoid arthritis and other autoimmune diseases, as well as FRβ positive cancers.
Functional recombinant FRβ protein was produced in insect cells and used as antigen to isolate a mAb, m909, from a human naïve Fab phage display library. Binding of Fab and IgG1 m909 to FRβ was measured by ELISA, surface plasmon resonance, immune fluorescence staining, and flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with FRβ positive CHO cells as target cells and isolated peripheral blood monocytes as effector cells in an in vitro assay.
Fab m909 bound with relatively high affinity (equilibrium dissociation constant 57 nM) to FRβ. The IgG1 m909 showed much higher (femtomolar) avidity as measured by ELISA, and it bound to FRβ positive cells in a dose-dependent manner, but not to parental FRβ negative cells. m909 did not compete with folate for the binding to FRβ on cells. m909 was not only able to select FRβ positive, activated macrophages from synovial fluid cells of arthritis patients as efficiently as folate, but also able to mediate ADCC in FRβ positive cells.
Unlike folate-drug conjugates, m909 selectively binds to FRβ, does not recognize FRα, and has at least one effector function. m909 alone has potential to eliminate FRβ positive cells. Because m909 does not compete with folate for receptor binding, it can be used with folate-drug conjugates in a combination therapy. m909 can also be a valuable research reagent.
叶酸受体β（FRβ）仅在胎盘组织中检测到，在健康人群中仅限于某些髓系造血细胞。研究表明，FRβ在自身免疫性疾病和某些癌细胞中的激活巨噬细胞中过度表达。在这项研究中，我们旨在开发一种 FRβ 特异性人源单克隆抗体（mAb），可作为治疗剂用于治疗类风湿关节炎和其他自身免疫性疾病，以及 FRβ 阳性癌症。
功能性重组 FRβ 蛋白在昆虫细胞中产生，并用作抗原从人类原始 Fab 噬菌体展示文库中分离出 mAb m909。通过 ELISA、表面等离子体共振、免疫荧光染色和流式细胞术测量 Fab 和 IgG1 m909 与 FRβ 的结合。用 FRβ 阳性 CHO 细胞作为靶细胞，分离的外周血单核细胞作为效应细胞，在体外测定中评估抗体依赖性细胞介导的细胞毒性（ADCC）。
Fab m909 与 FRβ 具有相对较高的亲和力（平衡解离常数 57 nM）。ELISA 测量结果表明，IgG1 m909 的亲和力更高（飞摩尔），并且以剂量依赖性方式与 FRβ 阳性细胞结合，但与亲本 FRβ 阴性细胞不结合。m909 与细胞上的 FRβ 结合不与叶酸竞争。m909 不仅能够像叶酸一样从关节炎患者的滑膜液细胞中有效选择 FRβ 阳性、激活的巨噬细胞，而且能够在 FRβ 阳性细胞中介导 ADCC。
与叶酸药物缀合物不同，m909 选择性地与 FRβ 结合，不识别 FRα，并且具有至少一种效应功能。m909 单独具有消除 FRβ 阳性细胞的潜力。由于 m909 与受体结合不与叶酸竞争，因此它可以与叶酸药物缀合物联合用于联合治疗。m909 也可以作为有价值的研究试剂。
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