Nikula T K, Bocchia M, Curcio M J, Sgouros G, Ma Y, Finn R D, Scheinberg D A
Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Mol Immunol. 1995 Aug;32(12):865-72. doi: 10.1016/0161-5890(95)00052-g.
Although iodine-131 is the most widely used radionuclide for radioimmunotherapy, direct radiolabeling methods yield decreased immunoreactivity of the antibody as a function of increased iodine incorporation. We have studied the amino acid sequences of a therapeutic IgG (HuM195), and in particular its complementarity determining regions (CDR), in order to correlate the iodination of tyrosine residues in the antigen binding site with changes in immunoreactivity. The CDR contained an overabundance of tyrosines relative to an expected random distribution of amino acids. In contrast, lysine residues that can be used for ligand attachment were evenly distributed throughout the IgG. HuM195 was first trace labeled with 111In and then labeled with stable 127I at various specific activities. The immunoreactivity of each product was determined using the 111In tracer. The immunoreactivity measured after varying levels of iodination fit a theoretical curve that was generated based on the assumption that a single iodine incorporation anywhere on a tyrosine residue in a CDR destroys the immunoreactivity of the antibody. Similar theoretical curves for antibody fragments (Fab, Fv) suggest an even faster decrease in immunoreactivity with increasing iodination. A review of the sequences of other therapeutic IgG shows that a similar overabundance of tyrosine residues is found in the CDRs. Using enzyme digestion, the distribution of iodine on different parts of the antibody was also studied. The iodinated residues were distributed non uniformly throughout the IgG, with the heavy chain variable region tyrosines having a higher propensity for iodine incorporation than tyrosines in the other regions of the IgG. The common abundance of tyrosine in the CDR of IgG and its correlation with loss of function have important implications for therapeutic use of high specific activity radioiodinated monoclonal antibodies or fragments.
尽管碘 - 131是放射免疫治疗中使用最广泛的放射性核素，但直接放射性标记方法会随着碘掺入量的增加而导致抗体免疫反应性降低。我们研究了一种治疗性IgG（HuM195）的氨基酸序列，特别是其互补决定区（CDR），以便将抗原结合位点中酪氨酸残基的碘化与免疫反应性的变化相关联。相对于预期的随机氨基酸分布，CDR中酪氨酸含量过高。相比之下，可用于连接配体的赖氨酸残基在整个IgG中均匀分布。HuM195首先用111In进行微量标记，然后用不同比活度的稳定127I进行标记。使用111In示踪剂测定每种产物的免疫反应性。在不同碘化水平后测量的免疫反应性符合基于以下假设生成的理论曲线：CDR中酪氨酸残基上任何位置的单个碘掺入都会破坏抗体的免疫反应性。抗体片段（Fab、Fv）的类似理论曲线表明，随着碘化增加，免疫反应性下降得更快。对其他治疗性IgG序列的回顾表明，在CDR中发现了类似的酪氨酸残基过量情况。使用酶消化法，还研究了碘在抗体不同部位的分布。碘化残基在整个IgG中分布不均匀，重链可变区酪氨酸比IgG其他区域的酪氨酸更倾向于掺入碘。IgG的CDR中酪氨酸的普遍丰富及其与功能丧失的相关性对高比活度放射性碘化单克隆抗体或片段的治疗应用具有重要意义。
