Chin Li-Te, Chu Chishih, Chen Han-Min, Hsu Shu-Ching, Weng Bor-Chun, Chu Chi-Hong
Graduate Institute of Life Science, Fu-Jen Catholic University, Taipei, Taiwan, RoC.
BMC Biotechnol. 2007 Aug 23;7:51. doi: 10.1186/1472-6750-7-51.
The ability to acquire fully human monoclonal antibodies (mAbs) with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG) molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags) such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4) because proper hosts are lacking.
Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC) by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (gamma4lambdahuCD152), which contains variable (V) regions of the heavy chain and the light chain derived from the VH3 and V lambda human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, gamma4lambdahuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4 lambdas as revealed by the isoelectric focusing (IEF) analysis. Furthermore, gamma4lambdahuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2) of CD152, whereby it can react to activated CD3+ cells.
In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, gamma4lambdahuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4 lambda mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may otherwise prevent the access of whole IgG1 molecules.
除了有前景的免疫疗法外，获得具有预定义特异性的完全人源单克隆抗体（mAb）的能力对于开发用于分析受体功能的分子标签至关重要。然而，大多数已获得的亲和力成熟且完整的人免疫球蛋白G（IgG）分子实际上源自单个人B细胞，由于缺乏合适的宿主，尚未广泛用于研究保守的自身抗原（Ag），如CD152（细胞毒性T淋巴细胞抗原4，CTLA-4）。
在此，我们开发了一种优化方案，通过使用人CD152的选定表位对外周血单核细胞（PBMC）进行定点体外免疫，CD152是参与T细胞活化下调的重要受体。所得稳定的三瘤细胞系持续产生抗CD152 mAb（γ4λhuCD152），其重链和轻链的可变（V）区分别源自VH3和Vλ人胚系基因，但显示出不寻常的IgG4同种型。有趣的是，等电聚焦（IEF）分析显示，γ4λhuCD152具有髓系单克隆IgG4λ中不常见的碱性pI。此外，γ4λhuCD152以纳摩尔亲和力特异性结合包含CD152推定的第二个互补决定区（CDR2）的细胞外成分，从而能够与活化的CD3+细胞反应。
在特定细胞耗竭和条件培养基的背景下，成功获得了针对保守自身抗原的人抗体的体外诱导，并由此获得了对CD152的CDR2具有高亲和力的相对碱性的mAbγ4λhuCD152。这种具有指定CDR2特异性的人IgG4λmAb的应用可能会影响并有利于原位和异位CD152标记，因为它不影响内源性B7配体的结合，并且可以定位到受限的免疫突触中，否则可能会阻止整个IgG1分子的进入。
