公开(公告)号：EP1505076A2

公开(公告)日：2005-02-09

申请号：EP03720119.1

申请日：2003-04-28

申请人： CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA (CIGB)

发明人： Gavilondo Cowley, Jorge Victor ,  Ayala Vila, Marta, C/ 186, No. 3117 Apto 3D e/ ,  Freyre Almeida Freya, de los Milagros ,  Acevedo Castro, Boris E., Calle Segunda ,  Bell Garcia, Hanssel, C/62 00906, 2do piso, apt 16 ,  Roque Navarro, Lourdes Tatiana ,  GonzaLez Lopez, Luis Javier, Calle 186, 003115 ,  Cremata Alvarez, José Alberto, Ave 31 No. 18207 ,  Montesino Segui, Raquel, Calle 184 No. 3112

IPC分类号： C07K16/30 ,  A61K51/10

CPC分类号： C07K14/22 ,  C07K16/3007 ,  C07K2317/622 ,  C07K2317/626

摘要： The invention refers to mono- and divalent (diabody) single chain Fv (scFv) antibody fragments, obtained by recombinant DNA techniques from the anti-carcinoembryonic antigen (CEA) monoclonal antibody (Mab) CB/ior-CEA.1. This antibody has high affinity for CEA and is employed in the diagnosis and follow-up of human colorectal tumors. As the original Mab, the monovalent fragment and the diabody exhibit high affinity for human CEA and recognize an epitope dependent of carbohydrate conservation. The monovalent scFv fragment and the diabody have affinity constants for CEA of (5.0 ± 0.4) x 10 9 L mol -1 and (2.8 ± 0.3) x 10 10 L mol -1 , respectively. These two fragments do not show cross reactivity with human normal cells and tissues, exception made of the normal colonic mucosa, where CEA is occasionally present. The fragments can be produced through the expression in recombinant microorganisms, starting from the cloning of the encoding variable region nucleic acid sequences obtained from the hybridoma that produces Mab CB/ior-CEA.1. As the original Mab, the monovalent scFv and the diabody have the ability to identify in vivo cells that produce human CEA and grow as tumors in mice. The monovalent scFv and the diabody have a molecular size 5 and 2.5 times lower, respectively, than the mouse Mab, and do not have Fc domains, fact this that confers them the potential to better penetrate tissues in vivo and to be less immunogenic in man.

摘要翻译： 本发明涉及通过来自抗癌胚抗原（CEA）单克隆抗体（Mab）CB / i-CEA.1的重组DNA技术获得的单价和二价（双抗体）单链Fv（scFv）抗体片段。 该抗体对CEA具有高亲和力，用于诊断和追踪人结肠直肠肿瘤。 作为原始的Mab，单价片段和双抗体对人CEA表现出高亲和力，并识别依赖于碳水化合物保护的表位。 单价scFv片段和双抗体对（5.0 +/- 0.4）×10 -9 L mol -1和（2.8 +/- 0.3）×10 10 L mol -1的CEA具有亲和力常数 ， 分别。 这两个片段不显示与人正常细胞和组织的交叉反应性，例外是正常结肠粘膜，其中CEA偶尔存在。 通过从产生Mab CB / i-CEA.1的杂交瘤获得的编码可变区核酸序列的克隆开始，可以通过重组微生物中的表达产生片段。 作为原始的Mab，单价scFv和双抗体具有鉴定产生人CEA的体内细胞并在小鼠中作为肿瘤生长的能力。 单价scFv和双抗体分别具有比小鼠Mab低5和2.5倍的分子量，并且不具有Fc结构域，事实上，这赋予它们在体内更好地渗透组织的潜力并且在人中具有较少的免疫原性 。