C-Terminal Selective Ligation to Access Homogeneous Antibody Conjugates

抗体-药物偶联物（ADC）代表了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获得了临床批准。它们由抗体组成，抗体是一种蛋白质大分子，它特异性结合癌细胞表面过表达的抗原，附着在细胞毒性化学“弹头”上。它们旨在通过将细胞毒性药物特异性地递送到癌细胞来克服现有化疗药物的局限性，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。重要的是，目前用于将药物连接到抗体的方法依赖于非选择性化学，其产生具有可变药理学特征的缀合物的高度复杂的混合物。相反，现在广泛接受的是，下一代优化ADC的连接方法必须是位点选择性的。 在该项目中，我们提出了一种用于生成ADC的新方法，该方法通过延伸蛋白质链将药物附着在抗体上的特定位置，生成上级均质缀合物。我们的策略，至关重要的是，将不需要基因工程的抗体纳入反应手柄，因此直接适用于天然的“现成的”抗体。这将最大限度地提高全世界研究人员对同质ADC的可及性，并确保生产产量尽可能高，最终降低这些相对复杂的生物制药的成本。此外，它将形成具有2种药物的受控载量的ADC，这是及时的，因为许多新的超强弹头只能在这样的载量下耐受，因为它们是如此疏水。 我们将通过靶向绝大多数临床验证抗体上存在的C-末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多种方法来选择性修饰肽/蛋白质上的各种氨基酸，但缺乏可靠的C末端修饰策略。因此，C-末端修饰策略的开发也将在更广泛的生物缀合领域中提供根本性的进步。我们的策略利用半胱氨酸反应试剂，这将被设计为通过高度有利的6元环中间体从Cys转移到C-末端。反应将导致酰胺键的形成，其已经被非常好地表征并且已知在体内是稳健稳定的。总的来说，这种C端修饰策略将代表生产下一代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.