Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required?

Abstract

Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in 'bystander killing'. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation.

Figure 1

Schematic diagram of antibody drug conjugate. An antibody drug conjugate consists of a cancer-targeting vehicle, usually the whole or fragment of a monoclonal antibody, connected by a cleavable or non-cleavable linker to a potent cytotoxic warhead drug. mAb, monoclonal antibody; Fab, fragment antigen binding; SIP, small immune protein.

Figure 2

Possible mechanism(s) of action are shown for an ADC with a diffusible drug (MMAE) attached with a cathepsin B cleavable linker. The canonical processing pathway for an ADC involves (1) binding of the ADC to the target antigen and (2) internalisation. (3) The ADC is transported to the lysosome where the linker is cleaved, producing free diffusible drug (4). This free drug can then bind to microtubules (5) or DNA (depending on drug type) within the target cells to induce cell cycle arrest and ultimately result in cell death. The free drug can also diffuse out of the target cell (6) and penetrate surrounding ‘bystander’ cells to cause cell death. After ADC binding to the target antigen (7) but before internalisation, an alternative route for ADC processing is cleavage by extracellular enzymes (such as cathepsin B), which are released by surrounding tumour cells and tumour-associated macrophages (TAMs) and which generate diffusible drug from the ADC (8). This free drug can then penetrate surrounding ‘bystander’ cells resulting in cell death. Also, the ADC-bound target tumour cell may be internalised through Fc-mediated phagocytosis (9), which upon degradation of the target tumour cell would also result in release of free, diffusible drug (10).