Improving the functional affinity of human anti-glycan monoclonal antibodies (mabs) for cancer therapy

在过去的十年中，抗体已成为制药行业的主要增长领域之一。它们结合了对目标（“抗原”）的高度特异性和招募强大的免疫系统介导效应的能力。事实上，抗体是免疫系统通常消除感染因子的主要机制之一。我们在生产具有临床价值的抗癌抗体方面有着良好的记录（我们的两种抗体目前正在临床试验中），我们正在努力生产更多。然而，我们观察到，在允许抗体用于患者所需的分子程序中，它们通常会失去其效力的很大一部分。我们相信，我们现在可以解释为什么会发生这种情况，我们正在寻求资金来进行必要的改变，以避免这种效力的丧失。抗体通常是通过用目标“抗原”免疫小鼠而产生的。从反应小鼠中分离出产生抗体的细胞，并使这些细胞永生。然后对产生的抗体进行反应性和临床适用性筛选，并克隆编码抗体的基因。抗体中不负责目标特异性的区域（“恒定区域”）随后被其人体等效区域取代，这一过程被称为抗体的“人源化”。这对于创建临床有用的试剂至关重要；如果大部分小鼠区域仍然存在，患者体内就会产生免疫反应，使抗体失效。我们观察到，在这种“人源化”过程之后，抗体与靶标的结合强度降低，抗体的许多临床有益作用也丧失了。我们认为，这种降低的效力是由于抗体的原始小鼠“亚类”被替换，称为小鼠IgG3，因为当涉及其他小鼠亚类时，我们没有观察到它。先前的研究人员已经提出，小鼠IgG3抗体可能在与目标结合后结合在一起，这是其他抗体亚类所没有的。这稳定了结合并增强了抗体的临床效力。我们的建议是确认小鼠igg3具有这种作用的区域，并将这些区域转移到人类IgG1中，这是临床上最常用的人类亚类。这是可行的，因为尽管抗体在序列上非常相似，但它们仍然有独特的差异。与其单独研究这些众多的差异，我们建议改变人类IgG1表面的整个斑块，使其与小鼠IgG3相似。通过这种方式，我们将以一种更省时、更节约成本的方式涵盖差异，并且还可以在效果需要多个同时差异的情况下获取任何更改。一旦我们分离出对影响负责的整体区域，我们将逐个调查区域变化，以准确识别哪些变化是重要的。我们有两种候选抗体，我们已经显示，通过从小鼠IgG3到人类IgG1的变化，它们的效力分别降低了100倍和10倍。这些是测试人类IgG1中哪些改变重新引入这种失去的效力的理想候选者。我们相信，这项工作将导致一些非常令人兴奋的前景。它将允许使用针对临床有用靶点的抗体，否则这些靶点在“人源化”阶段就会失败；它将允许使用较低剂量的抗体，副作用的风险较低；它将提高现有抗体的效力，提高它们的临床效果。因此，我们认为拟议的项目是对资源的极好利用，具有患者和经济效益，包括我们的抗体和许多其他治疗性抗体。

Over the last decade, antibodies have become one of the major growth areas in the Pharmaceutical industry. They combine a high level of specificity for their target (the 'antigen') with the ability to recruit powerful immune system-mediated effects. Indeed, antibodies are one of the main mechanisms by which the immune system normally eliminates infectious agents.. We have a proven track record in the production of clinically valuable anti-cancer antibodies (two of our antibodies are currently in clinical trials) and we are striving to produce more. However, we have observed that during the molecular procedures necessary to allow an antibody to be used in patients, they often lose a substantial fraction of their potency. We believe that we now have an explanation for why this might be occurring, and we are seeking the funding to make the changes necessary to avoid this loss of potency.Antibodies are typically produced by immunising mice with a target 'antigen'. Antibody-producing cells are isolated from responding mice and the cells immortalised. The antibodies being produced are then screened for reactivity and clinical applicability, and the genes encoding the antibodies are cloned. The areas of the antibody that are not responsible for the target specificity (the 'constant regions') are then replaced with their human equivalent, in a process referred to as the 'humanisation' of the antibody. This is essential to create a clinically useful reagent; if the majority of the mouse areas remain, an immune response can develop in patients that negates the effectiveness of the antibody. We have observed that after this 'humanisation' process, the antibodies bind less strongly to their target and that many of the clinically beneficial effects of the antibodies are lost. We believe that this reduced potency is due to the replacement of the original mouse 'subclass' of the antibody, called mouse IgG3, as we do not observe it when other mouse subclasses are involved. Previous investigators have suggested that mouse IgG3 antibodies might combine together once bound to their target, in a way that other antibody subclasses do not. This stabilises binding and amplifies the clinical potency of the antibody. Our proposal is to confirm the areas of mouse IgG3s that are having this effect, and to transfer these areas to human IgG1, the most common human subclass used clinically. This is feasible because, although the antibodies are very similar in sequence, they nevertheless have unique differences. Rather than investigate these numerous differences individually, we propose to alter whole patches of the surface of human IgG1 to resemble mouse IgG3. In this way, we will cover the differences in a more time- and cost-efficient way, and also pick up any changes where multiple simultaneous differences are necessary for the effect. Once we have isolated which overall area is responsible for the effect, we will investigate the area change by change to identify exactly which changes are important. We have two candidate antibodies in which we have shown a hundred-fold and ten-fold reduction in potency, respectively, introduced by the change from mouse IgG3 to human IgG1. These make ideal candidates to test which alterations in the human IgG1 re-introduce this lost potency. We believe that this work will result in a number of very exciting prospects. It will permit the use of antibodies against clinically useful targets that would otherwise have failed at the 'humanisation' stage; it will allow the use of lower doses of antibody, with a lower risk of side-effects; and it will increase the potency of currently available antibodies, increasing their clinical effectiveness. We therefore consider that the proposed project is an excellent use of resources with patient and economic benefits encompassing our antibodies and many other therapeutic antibodies.