Chimeric IgG/A Tumor Immunotherapy

描述(申请人提供)：抗体依赖性细胞毒性(ADCC)是治疗性单抗(MAbbs)杀死肿瘤细胞的重要机制。然而，目前的治疗性免疫球蛋白单抗的杀伤效果并不理想。低效的ADCC为肿瘤提供了一种潜在的逃逸机制，并可被靶向改进基于抗体的癌症治疗。大多数临床试验中使用的肿瘤导向单抗是人IgG1，它可以通过与Fc？RIIIa(CD16)结合来激活补体和/或为ADCC招募NK细胞。几个小组已经证明，IgA通过与Fc？RI(CD89)结合并招募中性粒细胞来触发有效的ADCC。我们的目标是创造一种新的有效的抗肿瘤抗体，可以激活多种免疫细胞类型，携带Fc？RI或Fc？RIIIa。这些新的嵌合重链和相关轻链将在植物中表达，以产生具有特定N-糖链结构的抗体和增强抗体依赖的细胞毒性(ADCC)活性。 结合Fc？RI的IgA和结合Fc？RIIIa的IgG1的结构研究表明，同时包含IgG1和IgA结构域的嵌合抗体与肿瘤细胞靶点结合，将结合并激活免疫效应细胞上的这两种受体。作为概念证明，我们将把抗HER2抗体的Fab结构域与Fc？1CH1-CH2-CH3结构域和Fc？2CH2-CH3结构域的融合联系起来。仅限IgG1和仅限IgA2的版本将被构造为控件。IgG1的Fc有望带来三个好处：(A)通过FcRN结合延长血清半衰期； (B)使用蛋白A进行纯化；以及(C)通过Fc增强自然杀伤(NK)细胞的细胞募集和激活？受体结合。Ig A2 Fc有望通过Fc？RI结合增强中性粒细胞(PMN)的细胞募集和激活。 最近的研究表明，Ig G Fc糖基化对Fc？R结合和ADCC最大化具有重要意义。特别是，核心1-6)-岩藻糖的缺失和N-乙酰氨基葡萄糖(GlcNAc)残基的存在都增强了Fc与Fc？RIIIa的结合。通过将我们的重组抗体与特定的糖基转移酶在转基因烟草背景中共表达，我们将生产出具有最适合参与ADCC的N-糖链结构的抗体。 我们将利用我们的植物表达系统制备六种不同的抗体形式(IgA、Ig G和嵌合的Ig G/A，每种抗体都带有两种不同的N-糖基化类型)，它们都带有相同的抗HER-2/neu/c-Erb-B2 Fab区域，并评价它们在体外引导ADCC对抗标准乳腺癌细胞株的能力。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HER2/neu和人类Fc？RI的转基因小鼠中缩小HER2表达的同基因肿瘤的能力。

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.