Hombach A, Sircar R, Heuser C, Tillmann T, Diehl V, Kruis W, Pohl C, Abken H
Klinik I fur Innere Medizin, Universitat zu Koln, D-50924 Koln, Germany.
Int J Mol Med. 1998 Jul;2(1):99-103. doi: 10.3892/ijmm.2.1.99.
We recently described the generation and expression of a chimeric T cell receptor with specificity for the tumor antigen TAG72 consisting of the single chain antibody (scFv) B72.3-scFv and the gamma chain of the FcepsilonRI receptor. The corresponding chimeric receptor containing the zeta chain of the TCR as signalling unit is not functionally expressed reflecting that the requirements for functional expression of chimeric receptors containing the gamma signalling chain are apparently different compared to those containing the CD3zeta signalling chain of the TCR. We describe a novel set of chimeric anti-TAG72 receptors including in their extracellular moiety the constant immunoglobulin CH2/3 domains that allow stable expression of chimeric gamma as well as zeta receptors. We designed anti-TAG72 receptors that consist of a scFv fragment derived from an anti-TAG72 second generation antibody (CC49) and of the CH2/3 domains of the human IgG and intracellularily either of the zeta or gamma signalling chain. The recombinant CC49-CH2/3-zeta and CC49-CH2/3-gamma DNA, respectively, was transfected into MD45 T cells and expressed under control of the RSV LTR. Both receptors were found on the cell membrane of transfected cells as demonstrated by flow cytometry analysis using an anti-human IgG Fc antibody directed to the CH2/3 immunoglobulin domains of the chimeric receptor. Specific cross-linking of the chimeric zeta as well as the gamma receptor by antigen or anti-human Ig antibodies resulted in specific activation of transfected cells. Our results demonstrate that both the gamma chain and the zeta chain++ containing receptor are stably expressed and convert T cells to specificity for the TAG72 antigen. This receptor design will facilitate efficient generation of genetically modified peripheral T cells and may provide valuable tools for the cellular immunotherapy of TAG72+ tumors.
我们最近描述了一种对肿瘤抗原TAG72具有特异性的嵌合T细胞受体的产生和表达，该受体由单链抗体（scFv）B72.3-scFv和FcepsilonRI受体的γ链组成。包含TCR的ζ链作为信号传导单元的相应嵌合受体没有功能性表达，这反映出与包含TCR的CD3ζ信号传导链的嵌合受体相比，包含γ信号传导链的嵌合受体的功能性表达要求明显不同。我们描述了一组新型的嵌合抗TAG72受体，其细胞外部分包含恒定免疫球蛋白CH2/3结构域，可实现嵌合γ受体和ζ受体的稳定表达。我们设计了抗TAG72受体，其由源自抗TAG72第二代抗体（CC49）的scFv片段、人IgG的CH2/3结构域以及细胞内的ζ或γ信号传导链组成。分别将重组CC49-CH2/3-ζ和CC49-CH2/3-γ DNA转染到MD45 T细胞中，并在劳氏肉瘤病毒长末端重复序列（RSV LTR）的控制下表达。使用针对嵌合受体的CH2/3免疫球蛋白结构域的抗人IgG Fc抗体进行流式细胞术分析表明，两种受体均存在于转染细胞的细胞膜上。抗原或抗人Ig抗体对嵌合ζ受体和γ受体的特异性交联导致转染细胞的特异性激活。我们的结果表明，包含γ链和ζ链的受体均稳定表达，并使T细胞对TAG72抗原具有特异性。这种受体设计将有助于高效产生基因改造的外周T细胞，并可能为TAG72 +肿瘤的细胞免疫治疗提供有价值的工具。
