Olafsen T, Rasmussen I B, Norderhaug L, Bruland O S, Sandlie I
Department of Biology, University of Oslo, Norway.
Immunotechnology. 1998 Oct;4(2):141-53. doi: 10.1016/s1380-2933(98)00014-1.
The monoclonal antibody (mAb) TP-3 binds selectively to human and canine osteosarcoma (OS) cells and is therefore a potential candidate for use as a targeting agent in radioimmunoimaging and therapy of OS metastases. However, intact murine mAbs have several drawbacks such as large size, delayed blood clearance and high immunogenicity, all of which can be overcome by genetic engineering.
To construct and express bivalent and multivalent TP-3 scFv fragments from the mammalian expression vector, pLNO. This vector has unique restriction sites for simple cassette cloning of any individual variable (V) and constant (C) genes and has previously been used for expression of intact chimeric TP-3 mAbs and Fab fragments. Furthermore, it is also suitable for expression of any modified V region, such as a scFv fragment, fused to any modified C region or to non-immunoglobulin protein sequences.
Six different constructs were made; three scFv-CH3 fragments that differed in the design of linker between the scFv fragment and the IgG CH3 domain. These constructs were also made with the IgM secretory tailpiece (microtp) attached to the C terminus.
All constructs were secreted as bivalent antibody fragments with a molecular weight of about 100 kDa. A band corresponding to a dimer appeared in all the supernatants from TP-3 scFv-CH3 producing cells, whether microtp was present or not, whereas higher orders of multimers were not seen. However, pulse chase analyses of the cells revealed that a small fraction of higher order polymers was formed from genes including the fragment encoding microtp and that microtp conferred retention both to monomers and intermediate polymers. The recombinant TP-3 antibody fragments were shown to bind human OS cells.
Recombinant mAb fragments can be designed and cloned into the mammalian expression vector, pLNO. This vector is flexible in the sense that the genes encoding such fragments can be expressed from either cDNA or from genomic DNA. A microtp attached to the CH3 domain in these fragments was sufficient to drive polymerization, however inefficiently and intracellular retention of both monomers and intermediate polymers was observed.
单克隆抗体（mAb）TP - 3可选择性结合人和犬骨肉瘤（OS）细胞，因此是骨肉瘤转移灶放射免疫成像和治疗中用作靶向剂的潜在候选物。然而，完整的鼠源单克隆抗体有几个缺点，如分子量大、血液清除延迟和免疫原性高，所有这些都可通过基因工程克服。
从哺乳动物表达载体pLNO构建并表达二价和多价TP - 3单链抗体片段（scFv）。该载体具有独特的限制性酶切位点，便于任何单个可变（V）基因和恒定（C）基因的简单盒式克隆，此前已用于完整嵌合TP - 3单克隆抗体和Fab片段的表达。此外，它还适用于表达与任何修饰的C区或非免疫球蛋白蛋白序列融合的任何修饰的V区，如scFv片段。
制备了六种不同的构建体；三种scFv - CH3片段，其scFv片段与IgG CH3结构域之间的接头设计不同。这些构建体的C末端还连接了IgM分泌尾肽（microtp）。
所有构建体均以分子量约100 kDa的二价抗体片段形式分泌。无论是否存在microtp，来自产生TP - 3 scFv - CH3的细胞的所有上清液中均出现了一条对应于二聚体的条带，而未观察到更高阶的多聚体。然而，对细胞的脉冲追踪分析显示，一小部分高阶聚合物由包括编码microtp的片段的基因形成，并且microtp赋予单体和中间聚合物滞留能力。重组TP - 3抗体片段显示可结合人骨肉瘤细胞。
重组单克隆抗体片段可设计并克隆到哺乳动物表达载体pLNO中。该载体具有灵活性，因为编码此类片段的基因可以从cDNA或基因组DNA中表达。连接到这些片段中CH3结构域的microtp足以驱动聚合，然而效率不高，并且观察到单体和中间聚合物在细胞内的滞留。
