Chimeric IgG/A Tumor Immunotherapy

描述（由申请方提供）：抗体依赖性细胞毒性（ADCC）是治疗性单克隆抗体（mAb）杀死肿瘤细胞的重要机制。然而，目前的治疗性IgG mAb的杀伤不是最佳的。无效的ADCC为肿瘤提供了潜在的逃逸机制，并且可以被靶向以改进基于抗体的癌症疗法。临床试验中使用的大多数肿瘤定向mAb是人IgG 1，其可以通过与Fc <$RIIIa（CD 16）结合来激活补体和/或募集NK细胞用于ADCC。几个研究小组已经表明，伊加通过与Fcú RI（CD 89）结合并招募中性粒细胞来触发强效ADCC。我们的目标是创造一类新的有效的抗肿瘤抗体，可以激活各种各样的免疫细胞类型轴承无论是Fc？RI或Fc？RIIIa.这些新型嵌合重链和相关轻链将在植物中表达以产生具有特异性N-聚糖结构和增强的抗体依赖性细胞毒性（ADCC）活性的抗体。 伊加结合Fc的结构研究？RI和IgG 1结合到Fc？RIIIa表明含有IgG 1和伊加结构域并结合肿瘤细胞靶的嵌合抗体将结合并激活免疫效应细胞上的两种受体。作为概念的证明，我们将连接抗HER 2抗体的Fab结构域的Fc融合？1 CH 1-CH 2-CH 3结构域和Fc？2个CH 2-CH 3结构域。将构建仅IgG 1和仅IgA 2版本作为对照。预期IgG 1的Fc赋予三个益处：（a）通过FcRn结合延长血清半衰期; (b)纯化使用蛋白A;和（c）增强细胞募集和激活的自然杀伤（NK）细胞通过Fc？受体结合预期IgA 2 Fc通过Fc？RI结合。 最近的研究表明，IgG Fc糖基化的重要性Fc？R结合和最大化ADCC。尤其是，核心的缺失。1-6)-岩藻糖和平分N-乙酰葡糖胺（GlcNAc）残基的存在下，每个增强Fc结合Fc？RIIIa.通过在转基因本氏烟草（Nicotiana benthamiana）背景中共表达我们的重组抗体沿着特异性糖基转移酶，我们将产生具有对于参与ADCC最佳的N-聚糖结构的抗体。 我们将使用我们的植物表达系统产生六种变体抗体形式（伊加、IgG和嵌合IgG/A，每种具有两种不同的N-糖基化类型），它们都带有相同的抗HER-2/neu/c-Erb-B2 Fab区，并评估它们在体外针对标准乳腺癌细胞系指导ADCC的能力。我们在宾夕法尼亚大学的合作者将评估这些重组抗体在同时表达HER 2/neu和人Fc的转基因小鼠中缩小表达HER 2的同基因肿瘤的能力。RI.

DESCRIPTION (provided by applicant): Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism by which therapeutic monoclonal antibodies (mAbs) kill tumor cells. However, killing by current therapeutic IgG mAbs is not optimal. Inefficient ADCC provides a potential escape mechanism for tumors and can be targeted to improve antibody- based cancer therapies. Most of the tumor-directed mAbs used in clinical trials are human IgG1, which can activate complement and/or recruit NK cells for ADCC by binding to Fc¿RIIIa (CD16). Several groups have shown that IgA triggers potent ADCC by binding to the Fc¿RI (CD89) and recruiting neutrophils. Our goal is to create a new class of potent anti-tumor antibodies that can activate a wide variety of immune cell types bearing either the Fc¿RI or Fc?RIIIa. These novel chimeric heavy chains and associated light chains will be expressed in plants to produce antibodies with specific N-glycan structures and enhanced antibody dependent cellular cytotoxicity (ADCC) activity. Structural studies of IgA binding to the Fc?RI and IgG1 binding to the Fc?RIIIa suggest that a chimeric antibody containing both IgG1 and IgA domains, and bound to a tumor cell target, will bind and activate both receptors on immune effector cells. As proof of concept we will link the Fab domains of an anti-HER2 antibody to a fusion of the Fc ?1 CH1-CH2-CH3 domains and Fc ?2 CH2-CH3 domains. IgG1-only and IgA2-only versions will be constructed as controls. The Fc of IgG1 is expected to confer three benefits: (a) prolongation of serum half-life via FcRn binding; (b) purification using Protein A; and (c) augmented cellular recruitment and activation of natural killer (NK) cells via Fc? receptor binding. The IgA2 Fc is expected to confer augmented cellular recruitment and activation of polymorphonuclear cells (PMN) via Fc?RI binding. Recent studies have demonstrated the importance of IgG Fc glycosylation for Fc?R binding and maximizing ADCC. In particular, the absence of core ?1-6)-Fucose and the presence of a bisecting N- acetylglucosamine (GlcNAc) residue each enhance Fc binding to Fc?RIIIa. By co-expressing our recombinant antibodies along with specific glycosyltransferases in a transgenic Nicotiana benthamiana background we will produce antibodies with N-glycan structures that are optimal for participating in ADCC. We will produce six variant antibody forms (IgA, IgG and chimeric IgG/A, each with two different N- glycosylation types), all bearing the same anti-HER-2/neu/c-Erb-B2 Fab region, using our plant expression system and evaluate their ability to direct ADCC in vitro against standard breast cancer cell lines. Our collaborators at the University of Pennsylvania will evaluate the ability of these recombinant antibodies to shrink HER2-expressing syngeneic tumors in transgenic mice expressing both HER2/neu and human Fc?RI.