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. 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. Keywords: antibody drug conjugate, bystander killing, non-internalising, cathepsin B Ideally, anti-cancer treatments should specifically target and kill Bax inhibitor peptide V5 tumour cells while leaving normal, healthy tissues relatively unscathed. The therapeutic ratio of the maximum tolerated to the minimally efficacious dose of many of the drugs used in conventional cytotoxic chemotherapy is relatively low, resulting in unwanted side-effects. Nevertheless, these drugs are relatively effective against cancer cells because normal cells have more efficient mechanisms for effluxing cytotoxins, repairing DNA, and clearing dead and damaged cells. Together, these mechanisms limit toxicity even among the rapidly dividing cells of the bone EMR1 marrow and gastrointestinal epithelium before the next cycle of chemotherapy is given. Immunotherapy has now joined cytotoxic chemotherapy as an effective systemic treatment for cancer, and antibody-based therapy has contributed most significantly to this burgeoning area. Monoclonal antibodies (mAbs) may directly stall or kill tumour cells by binding tumour cell-specific antigens, or indirectly kill tumour cells by inhibiting the immune checkpoint molecules that restrain tumouricidal lymphocytes. Building on these advances, antibody drug conjugate (ADC) therapy has emerged with worldwide marketing approvals for the indications of breast cancer and lymphoma. An ADC is divided into three distinct parts C the tumour-targeting mAb, the linker, and the cyotoxic drug, also known as the payload or warhead (Figure 1). Modern ADC technology is successful because the exquisite specificity of mAbs is stably coupled with the extreme potency of new cytotoxins. Therapeutic mAbs are commonly directed at antigens on the surface of tumour cells themselves or the cells or other components of the supporting tumour stroma. Ideally, these antigens are expressed at minimal levels on normal healthy tissues but are highly expressed by tumour cells or within the tumour microenvironment. The payload drugs used with ADCs are orders of magnitude more potent than conventional cytotoxic drugs, such as chemotherapeutic drugs, and as such are too potent to use safely as free drugs for cancer therapy. Moreover, advances in drug/linker chemistries, which enhance plasma stability and control cytotoxin release at the tumour site, have critically enabled modern ADC technology. Consequently, ADCs are expected to have a higher therapeutic ratio than conventional systemic chemotherapy. Open in a separate window 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. Two ADCs are currently FDA-approved for cancer treatment; ado-trastuzumab emtansine (KADCYLA, Bax inhibitor peptide V5 Roche/Genentech, San Francisco, CA, USA) for the treatment of patients with human epidermal growth receptor 2 (HER2)-expressing, metastatic breast cancer (Verma three out of five complete responders) when given at equivalent molar concentrations (Gebleux (Sutherland tumour cell killing (Li anti-tumour activity of the ADC. Given that these results were not reproduced with a MMAF-containing ADC, the membrane permeability of the released warhead and its ability to diffuse through the tumour seemed to be required for bystander killing. Although internalisation of vc-MMAE-based ADCs provides one way in which they exert anti-tumour activity, studies indicate that ADC internalisation may not be required for anti-tumour action of these ADCs. For example, targeting the poorly internalised antigen CD21 with an ADC containing vc-MMAF was not effective, whereas conjugation of the same antibody with vc-MMAE showed potent anti-tumour activity in a preclinical lymphoma model (Polson stability resulting in differences in activity of the ADC, and in some cases different cleaved drug products, whereas substitution with a non-cleavable linker abrogated any anti-cancer activity, confirming that a cleavable linker was essential for any anti-cancer activity of this non-internalising ADC (Dal Corso (Oflazoglu treatment for human tumour xenografts, which had a significant content of tumour Bax inhibitor peptide V5 associated macrophages (TAMs). This anti-tumour effect was mediated by the Fc-mediated uptake and processing of the ADC by the TAMs, which then resulted in extracellular release of MMAE and bystander tumour cell killing. It is therefore clear that immune cells, in particular macrophages, are required for immunotherapeutic efficacy, and these effects may be dependent on Fc-mediated phagocytosis. However, ADCs with site-directed mutation to reduce Fc-binding still show efficacy, meaning that in some cases Fc-dependent effector functions are not solely required for tumour responses to ADCs (McDonagh et al, 2008). Conclusion ADCs are revolutionising the field of cancer treatment by harnessing the specificity of tumour-targeting mAbs with the stability and high.