Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein.

In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112−150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize “hot spot” residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope.

Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies.

EGF-CFC超家族的创始成员Human Cripto-1（Cripto-1）是胚胎发育和致癌过程中许多过程的关键调节剂。Cripto-1在正常的成人组织中几乎不存在，甚至不存在，而在各种肿瘤中却异常地重新表达。CFC结构域介导的Cripto-1功能的阻断被认为是抑制该蛋白致瘤活性的一种有前途的治疗干预点。

在这项工作中，我们报告了针对人类蛋白质的合成折叠CFC [112-150]结构域的鼠单克隆抗体的产生和表征。通过减法ELISA分析，筛选出克隆特异性识别CFC域上“热点”残基的能力，这对于与激活素I型受体（ALK4）和GRP78的相互作用至关重要。在选定的抗体上，SPR和表位作图研究证实了它们的特异性，并揭示了识别仅在构象表位上发生。

此外，FACS分析已经证实1B4抗体能够识别一组人类癌细胞中的膜锚定和可溶性天然Cripto-1蛋白。最后，我们通过体外细胞信号分析和细胞周期分析评估了其功能作用。这些发现表明，选定的抗CFC mAb具有中和蛋白质致癌活性的潜力，可以用作适合作为Cripto-1过表达的癌细胞和组织的肿瘤归巢剂并克服常规癌症中耐药性的治疗方法分子。疗法。