Lee Yie Chia, Boehm Mark K, Chester Kerry A, Begent Richard H J, Perkins Stephen J
Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, University College London, Gower Street, London WC1E 6BT, UK.
J Mol Biol. 2002 Jun 28;320(1):107-27. doi: 10.1016/S0022-2836(02)00403-5.
MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for seven other scFv molecules have shown that, while the contact residues for symmetric back-to-back dimer formation in MFE-23 are not fully conserved, in principle, back-to-back contacts can be formed in these too. This offers possibilities for the creation of other forms of scFv molecules.
MFE - 23是一种单链Fv（scFv）抗体分子，因其对肿瘤标志物癌胚抗原（CEA）具有高亲和力而用于靶向结直肠癌。ScFv分子由肽连接的抗体V(H)和V(L)结构域形成，其中许多会形成二聚体。我们最近获得的MFE - 23晶体结构表明，它形成了一种不寻常的两个单体背靠背对称缔合结构，这与结构域交换双抗体结构一致。中子散射和建模拟合表明，在低于1 mg/ml的治疗相关浓度下，MFE - 23以紧密的V(H)-V(L)连接单体形式存在。尺寸排阻凝胶色谱显示，MFE - 23的单体和二聚体形式可以分离，且这两种形式的比例取决于起始MFE - 23浓度。通过分析超速离心在九个MFE - 23浓度下进行的沉降平衡实验表明，存在一个可逆的单体 - 二聚体自缔合平衡，缔合常数为1.9×10³ - 2.2×10³ M⁻¹。使用时间导数g(s(*))方法进行的沉降速度实验表明，MFE - 23 - His的重均沉降系数与浓度有关，从单体的1.8 S增加到二聚体的约3 - 6 S。这两个值与根据MFE - 23晶体结构计算的值一致。关于MFE - 23的热稳定性，通过¹H NMR和FT - IR光谱进行的变性实验表明，该分子在高达47℃时是稳定的，在此温度之后变性是不可逆的。MFE - 23的二聚化是根据双抗体结构的一个新模型进行讨论的，在该模型中，单体中的V(H)和V(L)结构域能够解离并重新缔合形成二聚体或双抗体，但两个单体之间会形成背靠背的对称接触。溶液中的这种二聚化归因于MFE - 23单体C末端表面的互补性质。其他七个scFv分子的晶体结构表明，虽然MFE - 23中形成背靠背对称二聚体的接触残基并不完全保守，但原则上这些分子也可以形成背靠背接触。这为创建其他形式的scFv分子提供了可能性。
