ISVHLD2015:双特异性抗体构建调解对特定的重新定位和消除HBV阳

StephenW

Code: O-14

Abstract: 317

Bispecific antibody constructs mediate specific retargeting towards and elimination of HBV-positive hepatocytes and tumor cells

Bohne F.M.1, Hasreiter J.1, Quitt O.1, Bockmann J.-H.1, Momburg F.2, Protzer U.1

1Helmholtz Centre Munich / Institute of Virology, Munich, Germany, 2DKFZ / National Tumor Centre, Heidelberg, Germany

Background & Aim: Chronic hepatitis B is characterized by exhausted effector cells, incapable of eradicating the virus. To circumvent this limitation, HBV-specific retargeting of immune effector cells using bispecific monoclonal antibody (BiMab) constructs is a promising therapeutic approach. In this study, effector cells are supplied with BiMab that redirect T cells towards HBV infected cells and activate their cytotoxic potential. Methods: We constructed tetravalent BiMab harboring two single chain fragment (scFv) binding domains. The first scFv targets the small HBV-envelope protein (HBs) on the plasma membrane of infected hepatocytes. The second binding motif engages T cells via CD3 and co-stimulates them via CD28. The two binding moieties are connected through an IgG1-derived Fc-domain and dimerize through disulfide bonding in the hinge region. This results in tetravalent homodimers comprising two scFv-binders for the target antigen and two for effector cell recruitment. Results: In co-culture experiments, the redirection of and immune effector cells towards HBs-positive hepatoma cells by the BiMab induced specific elimination of target cells and activation of effector cells. Co-administration of HBs/CD3- and HBs/CD28-specific BiMab constructs resulted in synergistic and mediated up to 96% specific elimination of HBs-positive target cells in co-cultures with PBMC. Interestingly, also singly administered BiMab were capable of inducing T-cell activation. Sorted CD4+ T cells displayed superior cytotoxicity in comparison to CD8+ T cells. The isolated subpopulations of naïve, central and effector memory T cells hinted at CD8+ naïve T cells to be incapable of BiMab-mediated killing. Intracellular cytokine staining showed polyfunctional activation mediated by the biMab, resulting in simultaneous secretion of IFNγ, IL-2 and TNFα. Importantly, biMab were also able to redirect T cells to HBV-infected HepaRG cell resulting in specific killing. Furthermore, soluble HBs, mimicking the high viral loads in patient serum failed to induce effector cell activation even when added to non-infected cells. Finally, first in vivo experiments in immunodeficient mice transplanted with HBV-positive hepatoma cells to induce subdermal tumors showed that transferred human PBMC mediated a marked decrease in tumor size upon BiMab injection indicating a functional homing in tumors. Conclusion: Retargeting of T cells towards HBV-positive target cells using bispecific antibody constructs is a promising new immunotherapeutic approach to treat chronic hepatitis B and associated hepatocellular carcinoma.

StephenW

编号：O型14
摘要：317
双特异性抗体构建调解对特定的重新定位和消除HBV阳性肝细胞和肿瘤细胞
Bohne FM1，Hasreiter J.1，Quitt O.1，Bockmann J.-H.1，Momburg F.2，Protzer U.1
1Helmholtz中心慕尼黑/病毒学，慕尼黑，德国，2DKFZ /国家肿瘤中心，德国海德堡研究所
背景与目的：慢性乙肝的特点是耗尽效应细胞，消除无能的病毒。规避使用双特异性单克隆抗体（BiMab）免疫效应细胞的这种限制，HBV特异性重定位构造是一个有希望的治疗方法。在这项研究中，效应细胞被提供有BiMab重定向T细胞对乙肝病毒感染的细胞，并激活它们的细胞毒性潜力。
方法：构建四价BiMab窝藏两个单链片段（scFv）的结合结构域。第一单链抗体靶向于感染的肝细胞的质膜的小的HBV包膜蛋白（HBs）中和。第二结合基序接合的T细胞通过CD3和通过CD28共刺激它们。两个结合部分通过一个IgG1的衍生的Fc结构域连接，并且二聚化，通过在铰链区二硫键。这导致四价二聚体包括两个scFv的结合剂为效应细胞募集的靶抗原和两种。
结果：在共培养实验中，重定向和免疫效应细胞对HBs抗体阳性肝癌细胞由BiMab诱导特异性消除靶细胞和活化效应细胞的。 HBs抗体/ CD3-和HBs抗体/ CD28特异性BiMab构建体共同施用导致协同和介导高达96％的特定消HBs抗体阳性靶细胞共培养用的PBMC。有趣的是，还单独施用BiMab均能够诱导T细胞活化的。选的CD4 + T细胞相比，CD8 + T细胞显示优良细胞毒性。暗示了CD8 +幼稚T细胞幼稚，中央和效应记忆T细胞的分离亚群是没有能力BiMab介导的杀伤。细胞内细胞因子染色显示多官能活化由biMab介导，导致IFNγ，IL-2和TNFα的同时分泌。重要的是，biMab还能够重定向T细胞以产生特异性杀伤HBV感染HepaRG细胞。此外，可溶性HBs抗体，模仿在病人血清中的高病毒载量未能当加入到非感染细胞，诱导效应细胞的活化甚至。最后，先在移植了HBV阳性肝癌细胞诱导皮下肿瘤免疫缺陷小鼠体内实验表明，转移的人PBMC介导时BiMab注射在肿瘤大小的显着降低指示肿瘤的官能归巢。
结论：T细胞对使用的双特异性抗体构建体的HBV阳性靶细胞的重定位是一个有希望的新的免疫治疗的方法来治疗慢性乙型肝炎和相关的肝细胞癌。