Antibody drug conjugates (ADCs)

Antibody drug conjugates (ADCs) are complex molecules consisting of antibodies linked to bioactive cytotoxic payloads. ADCs combine the targeting ability of antibodies with the tumor-killing ability of cytotoxic drugs. Antibodies attach themselves to antigens on the surface of tumor cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cells, which then take up or internalize the antibody along with the attached cytotoxin. After internalization, the cytotoxin kills the tumor. This targeting capability limits side effects and provides a wider therapeutic window than other chemotherapeutic drugs.

The ADC consists of 3 parts: an antibody that provides the ADC to target and potentially elicit a therapeutic response, a payload that elicits the desired therapeutic response, and a linker that attaches the payload to the antibody. The payloads of many antitumor ADCs are natural product-based molecules, some of which covalently interact with their targets. Common payloads include the tubulin inhibitors monomethylauritin A MMAE and monomethylauritin F MMAF, the DNA-binding agent calicheamicin and the topoisomerase 1 inhibitor SN-38, and glucocorticoid receptor modulators (GRMs). In addition to small molecule payloads, other molecules such as siRNA have also been studied.

For linkers, it is necessary to ensure that the cytotoxic payload is shed as little as possible before reaching the target cell, thereby improving safety and limiting doses, there are cleavable and non-cleavable types, both of which have been tested in preclinical and clinical trials and proven to be safe. The non-cleavable linker will keep the drug inside the cell, and when the entire ADC enters the target cell, the antibody is degraded to amino acids, and the resulting amino acid, linker, and cytotoxic drug complex is considered the active drug. A cleavable linker is dissociated by enzyme in the target cell. The cytotoxic payload can then escape from the target cell and attack neighboring cells in a process known as "bystander killing." For the latter, another type currently under development adds an additional molecule between the cytotoxin and the cleavage site, which would enable the creation of ADCs with greater flexibility without changing the kinetics of cleavage.

An important parameter in ADC design is DAR, the drug-to-antibody ratio, which represents the level of payload loading on the ADC, which has a significant impact on the potency and safety of the product. With the emergence of various new antibody-derived alternative products, a new ADC concept, namely Anything-Drug Conjugates, has emerged, which can include various small antibody fragments, such as diabodies, Fab, scFV and bicyclic peptides.

In addition to the considerations for preparing the components of the ADC, an important step in its production is the conjugation of the linker-payload to the antibody. A variety of methods are available for linker-payload conjugate to the antibody, including complete cysteine coupling, cysteine coupling after reduction of interchain disulfide bonds, through the introduction of unnatural amino acids for site-specific coupling, site-specific enzymatic coupling to the introduced substrate sequence.

Compared to antibody production, the structural complexity of ADCs makes their development and production more challenging, necessitating careful selection of purification techniques to remove free linker-payload and impurities such as aggregates. TFF is a common technique to remove small molecule impurities, such as stabilizers, from antibody samples. TFF can also be used to exchange the starting buffer with a solution suitable for the coupling reaction. Antibody modification followed by a coupling step yields a primary crude ADC solution that can be purified by TFF or its combination with an appropriate chromatography step. Usually, in order to simplify the operation and improve the yield, a single TFF step may be selected, but due to the existence of non-ideal DAR substances and to achieve the target purity requirements, the industrial purification of ADC usually requires the use of chromatography steps, and proper chromatography mode selection is one of the major challenges in ADC purification. For example, ADCs would have different physicochemical properties compared to the original antibody due to the covalently coupled hydrophobic linker-drug, so hydrophobic interaction chromatography was explored for the separation of such mixtures, and size exclusion chromatography was also tested. For removal of non-protein impurities such as payloads and aggregates. In addition, mixed-mode chromatography such as ion exchange and hydroxyapatite has also been reported for the purification of ADCs.