Transplantation Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Transplantation. 2011 Apr 27;91(8):833-40. doi: 10.1097/TP.0b013e31820f07ff.
It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function.
The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb.
One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells.
These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.
人们推测，抗体介导的 CD28 选择性共刺激分子阻断优于细胞毒性 T 淋巴细胞抗原 4（CTLA4）-Ig。这是基于这样一个前提，即特异性阻断 CD28 允许通过 CTLA-4 进行抑制信号，从而进一步抑制 T 细胞功能。
从狗外周血单核细胞中克隆犬（ca）CD28 的细胞外结构域。用 caCD28/鼠 IgG2a 融合蛋白免疫小鼠。通过用 splenocytes 与小鼠 NSO 细胞融合产生杂交瘤，并通过 ELISA 筛选 caCD28 结合。在混合白细胞反应中测试单克隆抗体（mAb）的激动和拮抗活性。使用板结合抗 CD3 和抗 CD28 激动剂 mAb 扩增犬调节性 T 细胞。
克隆了一种激动剂和七种拮抗剂犬（ca）CD28 mAb。结合研究表明，一种激动剂（5B8）和一种拮抗剂（1C6）mAb 与 caCD28/caIgG1 融合蛋白和表达于 CD4+和 CD8+外周血 T 细胞上的 CD28 结合良好。拮抗剂抗体以剂量依赖的方式阻断混合淋巴细胞反应（MLR），类似于 CTLA4-Ig，而激动剂抗体则增强 MLR。当与抗 caCD3 联合用于刺激淋巴细胞增殖时，5B8 优于 1C6。此外，激动剂 mAb 5B8 与抗 CD3 mAb 一起诱导犬调节性 T 细胞 100 倍增殖。与未经处理的对照细胞相比，抗 caCD28（1C6）和 CTLA4-Ig 等效地抑制了同种反应性靶细胞的细胞毒性 T 淋巴细胞介导的杀伤。
这些研究表明，可以产生具有激动或拮抗功能的小鼠抗 caCD28 mAb。
