Investigating the therapeutic potential of anti-GITR antibodies

重新激活抗肿瘤免疫反应，避免不必要的自身免疫，是抗癌免疫疗法的主要目标。阻断检查点抑制物以防止免疫抑制的抗体在临床上取得了成功。糖皮质激素诱导的肿瘤坏死因子相关受体(GITR)是一种细胞表面共刺激受体，与OX40和4-1BB属于同一肿瘤坏死因子超家族。GITR在T调节细胞(T-regs)上高表达，在幼稚T细胞和记忆效应T细胞(T-EFs)上低表达。T-regs是一种免疫抑制细胞类型，在多种癌症中被认为与阻止抗肿瘤反应的激活有关(3)。由于T-regs上GITR的高表达，用治疗性抗体去除这些细胞被认为是一种抗肿瘤治疗方法。活化的T-EFs显著增加GITR的表面表达。GITR途径的刺激增加了T-Jeff的增殖和促炎细胞因子的表达，如IL-2和IFNy。此外，GITRL表达水平较高的肿瘤有更多的浸润性CD4+和CD8+T细胞，并延迟生长。考虑到GITR在调节免疫反应中的作用，针对这种表面蛋白的抗体目前正在作为抗肿瘤免疫疗法进行研究。南安普顿大学抗体和疫苗小组(AVG)开发了一组抗人GITR(抗huGITR)抗体。在这个项目中，我们将在体外和体内表征抗huGITR抗体的功能。研究问题、目的和目的利用表达人GITR表面蛋白的人GITR敲入转基因小鼠模型(HuGITRKI)了解抗huGITR抗体对免疫反应的影响。目的1.鉴定人GITR蛋白的表达，并研究不同的抗huGITR抗体在体外如何改变免疫反应。测定抗GITR抗体的结合亲和力和结合表位的动力学。为了确定GITR表达的动力学，在体外用不同的刺激激活人PBMC和来自huGITRKI转基因小鼠的脾细胞。然后用流式细胞仪测量表面蛋白的变化。检测各抗GITR抗体激活和处理后CD4+、CD8+T细胞的细胞增殖情况。确定GITR抗体如何影响细胞信号转导。用表面等离子体共振法测定抗GITR抗体的动力学和结合亲和力。V.确定每个抗GITR抗体的最佳结合表位。

Re-activating anti-tumour immune responses, and avoiding unwanted autoimmunity, is a primary goal of anti-cancer immunotherapies. Antibodies that block checkpoint inhibitors, to prevent immune inhibition, have been clinically successful. Glucocorticoid-Induced TNFR-Related (GITR) is a cell surface co-stimulatory receptor that belongs to the same tumour necrosis factor (TNFR) superfamily as OX40 and 4-1BB. Constitutive expression of GITR is high on T regulatory cells (T-regs), but low on both naïve and memory effector T cells (T-effs). T-regs are an immune-suppressive cell type that, in multiple cancers, have been implicated in preventing the activation of anti-tumour responses (3). Because of high GITR expression on T-regs, depletion of these cells with therapeutic antibodies has been proposed as an anti-tumour therapy. Activated T-effs dramatically increase GITR surface expression. The stimulation of the GITR pathway increased T-eff proliferation and the expression of pro-inflammatory cytokines like IL-2 and IFNy. Furthermore, tumours with higher levels of GITRL expression had increased numbers of infiltrating CD4+ and CD8+ T cells and delayed growth. Considering the role GITR plays in modulating immune responses, antibodies directed against this surface protein are currently being investigated as anti-tumour immuno-therapeutics.The University of Southampton antibody and vaccine group (AVG) have developed a panel of anti-human GITR (anti-huGITR) antibodies. In this project, we will characterise the function of anti-huGITR antibodies in vitro and in vivo. Research question, aims and objectives Understand how anti-huGITR antibodies influence immune responses using a human GITR knock-in transgenic mouse model (huGITRKI) that expresses human GITR surface protein.Aim 1. Characterise human GITR protein expression and investigate how different anti-huGITR antibodies alter immune responses in vitro. Determine the kinetics of binding affinity as well as the binding epitopes of the anti-GITR antibodies. I. To determine the kinetics of GITR expression, human PBMCs and splenocytes from huGITRKI transgenic mice will be activated in vitro with various stimuli. Changes in surface protein are then measured using flow cytometry. II. Measure cellular proliferation of CD4+ and CD8+ T cells post activation and treatment with each anti-GITR antibody. III. Determine how GITR antibodies influence cell signalling. IV. Determine the kinetics and binding affinity of anti-GITR antibodies using surface plasma resonance.V. Determine the optimal binding epitope of each anti-GITR antibody.