Colcher D, Pavlinkova G, Beresford G, Booth B J, Choudhury A, Batra S K
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA.
Q J Nucl Med. 1998 Dec;42(4):225-41.
Monoclonal antibodies (MAbs), because of their inherent specificity, are ideal targeting agents. They can be used to deliver radionuclides, toxins or cytotoxic drugs to a specific tissue or malignant cell populations. Intact immunoglobulin (IgG) molecules have several practical limitations of their pharmacology; their relatively large size of approximately 150,000 daltons leads to a slow clearance from the blood pool and the body resulting in significant exposure to normal organs with limited quantities delivered to tumors. The IgG molecule shows a relatively poor diffusion from the vasculature into and through the tumor. Attempts to modify the pharmacology of the Ig molecule have classically involved the use of proteases to generate F(ab')2 and Fab' fragments with molecular weights of approximately 100,000 and 50,000 daltons, respectively. Fv fragments of IgG are one of the smallest size functional modules of antibodies that retain high affinity binding of an antigen. Their smaller size, approximately 25,000 daltons, enables better tumor penetration and makes them potentially more useful than a whole antibody molecule for clinical applications. Molecular cloning and expression of the variable region genes of IgG has greatly facilitated the generation of engineered antibodies. A single-chain Fv (scFv) recombinant protein, prepared by connecting genes encoding for heavy-chain and light-chain variable regions at the DNA level by an appropriate oligonucleotide linker, clears from the blood at much faster rate than intact IgG. The scFv fragment can retain an antigen-binding affinity similar to that of a monovalent Fab' fragment; this however, represents a relative decrease in binding affinity when compared to intact antibodies. The scFv with its faster clearance and lower affinity results in a lower percent-injected dose localizing in tumors when compared to the divalent IgG molecule. This may be adequate for imaging but probably not for therapy. The valency of the MAb fragment is critical for the functional affinity of an antibody to a cell surface or a polymeric antigen. In attempts to generate multivalent forms of scFv molecules, non-covalently linked scFv dimeric and trimeric molecules, disulfide linked dimeric scFvs, as well as covalently linked chimeric scFvs have been studied. These multivalent scFvs generally have a higher functional affinity than the monovalent form resulting in better in vivo targeting. Another way to alter the pharmacology of the scFvs is to modify its net charge. Charge-modified scFvs with desired isoelectric points (pI), have been prepared by inserting negatively charged amino acids on the template of the variable region genes. This can help to overcome undesirable elevations in renal uptake seen with most antibody fragments. In conclusion, genetic manipulations of the immunoglobulin molecules are effective means of altering stability, functional affinity, pharmacokinetics, and biodistribution of the antibodies required for the generation of the "magic bullet".
单克隆抗体（MAbs）因其固有的特异性，是理想的靶向剂。它们可用于将放射性核素、毒素或细胞毒性药物递送至特定组织或恶性细胞群体。完整的免疫球蛋白（IgG）分子在药理学上存在一些实际限制；其相对较大的尺寸约为150,000道尔顿，导致从血池和体内清除缓慢，从而使正常器官受到大量辐射，而输送到肿瘤的量有限。IgG分子从血管系统向肿瘤内和穿过肿瘤的扩散相对较差。经典的改变Ig分子药理学的尝试涉及使用蛋白酶生成分子量分别约为100,000和50,000道尔顿的F(ab')2和Fab'片段。IgG的Fv片段是抗体中最小的具有保留抗原高亲和力结合功能的模块之一。其较小的尺寸约为25,000道尔顿，使其具有更好的肿瘤穿透性，并且在临床应用中可能比完整抗体分子更有用。IgG可变区基因的分子克隆和表达极大地促进了工程抗体的产生。通过用合适的寡核苷酸接头在DNA水平连接编码重链和轻链可变区的基因制备的单链Fv（scFv）重组蛋白，从血液中清除的速度比完整IgG快得多。scFv片段可保留与单价Fab'片段相似的抗原结合亲和力；然而，与完整抗体相比，这代表结合亲和力相对降低。与二价IgG分子相比，scFv具有更快的清除率和更低的亲和力，导致注射剂量在肿瘤中的定位百分比更低。这对于成像可能足够，但可能不适用于治疗。单克隆抗体片段的价态对于抗体与细胞表面或多聚体抗原的功能亲和力至关重要。在尝试生成scFv分子的多价形式时，已经研究了非共价连接的scFv二聚体和三聚体分子、二硫键连接的二聚体scFv以及共价连接的嵌合scFv。这些多价scFv通常比单价形式具有更高的功能亲和力，从而在体内靶向性更好。改变scFv药理学的另一种方法是改变其净电荷。通过在可变区基因模板上插入带负电荷的氨基酸，制备了具有所需等电点（pI）的电荷修饰scFv。这有助于克服大多数抗体片段在肾脏摄取中出现的不良升高。总之，免疫球蛋白分子的基因操作是改变“神奇子弹”所需抗体的稳定性、功能亲和力、药代动力学和生物分布的有效手段。
