Anti-CD3 Collabody特性分析與糖尿病NOD小鼠治療應用

Abstract

Anti-CD3的專一性療法在治療異體器官移植及第一型糖尿病具有良好前景。藉由減低抗體之Fc 與Fc 受體結合能力減少副作用，可以因而增加anti-CD3抗體用於治療的可能性。本研究利用一段能自我形成三股螺旋結構的類膠原蛋白胜肽序列 (Gly-Pro-Pro)10，生產出不含Fc結構，以三倍體Fab形式的anti-mouse CD3 Collabody， h145CSA。我們將同質三倍體的h145CSA成功地以可溶性蛋白質的形式分泌至CHO 細胞培養上清液中，並且利用親合性及分子篩管柱層析的方式，製備出高純度且適合作為治療用的藥物。與兩價、FcR 結合力降低的免疫球蛋白h145chIgGAA相比，三價的h145CSA具有較佳和TCR/CD3 complex的結合強度、以及使T 細胞表面TCR/CD3 complex表現量下降的能力。與兩價的抗體相比，只有三價的h145CSA可以造成細胞凋亡，且使Th1細胞凋亡比例較Treg細胞高。有趣的是，以h145CSA處理的Th1細胞，其FasL 的表現量明顯高於Treg細胞，且 FasL中和性抗體可以阻斷h145CSA造成的Th1細胞凋亡。h145CSA投與正常活體小鼠，只會造成極低的T細胞活化以及低量細胞激素的產生。投與正常小鼠多次劑量h145CSA，造成脾臟中CD4+ T細胞減少的能力與兩價抗體相同，但它使CD4＋ T細胞回復到正常量的速度比兩價抗體更快速。以h145CSA治療剛發病第一型糖尿病之NOD小鼠的功效也比兩價抗體h145chIgGAA明顯更有效，此與在h145CSA治療小鼠的胰臟及腸繫膜淋巴結中，存有較低比例的CD4+IFNγ+ T 細胞以及Treg/Th1 比值較高有相關性。本研究結果顯示，三價非Fc結構的anti-CD3 Collabody具有治療第一型糖尿病之潛能。

Specific anti-CD3 treatment is deemed to be a promising therapy for allograft rejection and type 1 diabetes (T1D). Fc receptor (FcR) reduced-binding antibodies, by avoiding adverse effects of Fc and FcR interaction, have good therapeutic potential. We generated a trivalent, non-Fc-containing anti-mouse-CD3 Collabody, h145CSA, by using a triplex-forming collage-like peptide (Gly-Pro-Pro)10 to drive the trimerization of the Fab fragments. h145CSA was successfully produced as a soluble protein in the culture supernatant of CHO cells and could be purified as a homotrimer by affinity and size-exclusion chromatography to high purity, suitable for use as therapeutics. Trivalent h145CSA exhibited better binding strength and greater potency in downregulation of the TCR/CD3 complex compared to FcR reduced-binding format IgG, h145chIgGAA. Moreover, while h145CSA induced a significantly higher percentage of Th1 cell apoptosis than Treg cells, h145IgGAA did not. Interestingly, FasL was significantly up-regulated in h145CSA-treated Th1 cells compared to Treg cells. Neutralizing antibody to FasL blocked h145CSA-induced Th1 cell apoptosis. Administration of h145CSA invoked minimal T cell activation and cytokine production in normal mice. Multiple dosing of h145CSA depleted splenic CD4+ T cells, which was comparable to bivalent antibodies, yet it was characterized by more rapid CD4+ T cell recovery kinetics than treatment with other bivalent antibodies. Importantly, h145CSA was more potent than h145chIgGAA in inducing long-lasting remission in recent-onset diabetic NOD mice. Its therapeutic effect was accompanied by a significantly lower percentage of CD4+IFN+ T cells and a higher Treg/Th1 ratio in pancreatic and mesenteric lymph nodes. The results of our study demonstrate that trivalent non-Fc anti-CD3 Collabody has potential to be used in treatment of T1D.