C-Terminal Selective Ligation to Access Homogeneous Antibody Conjugates

抗体-药物偶联物（adc）是最令人兴奋的新型靶向抗癌治疗药物之一，在过去8年中有4种adc获得临床批准。它们由一种抗体组成，抗体是一种蛋白质大分子，与癌细胞表面过度表达的抗原特异性结合，并附着在细胞毒性化学“弹头”上。他们的目标是克服现有化疗药物的局限性，通过将细胞毒性药物特异性地递送到癌细胞，从而减少与损害健康组织相关的副作用。为了最大限度地提高adc实现其治疗潜力的机会，必须改进其设计和化学结构。关键的是，目前将药物附着到抗体上的方法依赖于非选择性化学反应，这种化学反应产生具有可变药理学特征的高度复杂的偶联物混合物。相反，现在广泛接受的是，下一代优化adc的附着方法必须是位点选择性的。在这个项目中，我们提议开拓一种新的adc生成方法，通过延长蛋白链将药物附着在抗体的特定位置，产生优异的均匀偶联物。至关重要的是，我们的策略不需要对抗体进行基因工程，以纳入反应性手柄，因此直接适用于天然的“现成”抗体。这将使世界各地的研究人员最大限度地获得同质adc，并确保产量尽可能保持高，最终降低这些相对复杂的生物制药的成本。此外，它将形成adc，控制2种药物的装载，这是及时的，因为许多新的超强弹头只能在这种装载下被容忍，因为它们是如此疏水。我们将通过靶向存在于绝大多数临床验证抗体上的c端半胱氨酸来实现这一目标。这是一个具有挑战性的目标，因为虽然已经建立了各种各样的方法来选择性修饰肽/蛋白质上的各种氨基酸，但在c端缺乏可靠的修饰策略。因此，c端修饰策略的发展也将为更广泛的生物偶联领域提供根本性的进步。我们的策略利用半胱氨酸反应试剂，这些试剂将被设计为通过高度有利的6元环中间体从cys转移到c端。这些反应将导致酰胺键的形成，这种键已经被很好地表征，并且已知在体内非常稳定。总的来说，这种c端修饰策略将代表一种生产下一代adc的最佳方法，并促进这些令人兴奋的靶向治疗的更广泛成功。

Antibody-Drug Conjugates (ADCs) represent one of the most exciting classes of new targeted anti-cancer therapeutics, with 4 ADCs achieving clinical approval over the last 8 years. They consist of an antibody, which is a protein macromolecule that specifically binds to an antigen overexpressed on a cancer cell surface, attached to a cytotoxic chemical 'warhead'. They aim to overcome the limitations of existing chemotherapeutics by delivering the cytotoxic drug specifically to the cancer cells, and thus reducing the side-effects associated with damaging healthy tissue. To maximise the chances for ADCs achieving their therapeutic potential, their design and chemical construction must be improved. Critically, current approaches for the attachment of the drug to the antibody have relied on unselective chemistries, which generate highly complex mixtures of conjugates with variable pharmacological profiles. Instead, it is now widely accepted that the attachment method for the next generation of optimised ADCs must be site-selective. In this project we are proposing to pioneer a new approach for the generation of ADCs, which attaches the drugs at specific locations on the antibody by extending the protein chain, generating superior homogeneous conjugates. Our strategy, crucially, will not require genetic engineering of the antibodies to incorporate reactive handles, and is thus applicable directly to native 'off-the-shelf' antibodies. This will maximise the accessibility of homogenous ADCs to researchers across the world and ensure that the production yields are maintained as high as possible, ultimately reducing the cost of these relatively complex biopharmaceuticals. Moreover, it will form ADCs with a controlled loading of 2 drugs, which is timely as many of the new ultra-potent warheads can only be tolerated at such loadings as they are so hydrophobic. We will achieve this goal by targeting the C-terminal cysteines present on the vast majority of clinically validated antibodies. This is a challenging aim as whilst a wide variety of methods have been established for the selective modification of various amino acids on peptides/proteins, there is an absence of a reliable strategy for modification at the C-terminus. As such, the development of a C-terminal modification strategy would also provide a fundamental advance in the broader field of bioconjugation. Our strategy exploits cysteine reactive reagents, which will be designed to transfer from Cys-to-C-terminus via highly favourable 6-membered ring intermediates. The reactions will result in the formation of amide linkages, which are already extremely well characterised and known to be robustly stable in vivo. Overall this C-terminal modification strategy will represent an optimum approach for producing next generation ADCs and facilitate the wider success of these exciting targeted therapies.