Release date:2022/5/23 15:00:36

    Antibody-drug conjugates (ADCs) are a large class of drugs for the treatment of cancer, and have been increasingly favored in recent years. ADCs is mainly composed of three parts, including a monoclonal antibody, a cytotoxin, and a linker. The antibody is used to specifically target tumor cells, cytotoxin plays the role of cell killing, and linker plays the role of linking and releasing toxin. In 2013, the launch of T-DM1 marked the beginning of ADC drugs entering the field of solid tumor treatment. The most difficult aspect of ADC treatment in solid tumors is the heterogeneous expression of target antigens (Ag) in metastatic tumor tissue, which can make drugs acting on the primary tumor ineffective against metastatic tumor tissue. The bystander effect of ADC is expected to solve this problem. 

Basic structure of ADC

Figure 1 Basic structure of ADC

The Concept of ADC Bystander Effect

So what is the bystander effect? The so-called bystander cells are actually the cells surrounding the tumor cells (Fig. 2), including heterogeneous tumor cells and the above-mentioned metastatic tumor cells. These nearby cancer cells can be Ag+ or Ag-. The bystander effect is that the drug can also kill these cells. Not all ADCs have this effect, and it is often related to the biochemical properties of the linker and cytotoxin.

Schematic diagram of bystander effect of ADC

Figure 2 Schematic diagram of bystander effect of ADC
(doi: 10.1038/bjc.2017.367)

The Mechanism of ADC Bystander Effect

The bystander effect, which kills cells surrounding the target tumor cells, is quite different from ADCs without this effect (Figure 3). There are many factors that influence the bystander effect.

Firstly, the stability of the chemical bond between the antibody and the linker is one of the influencing factors. ADCs without bystander effect will not release cytotoxins before reaching tumor cells, so the chemical bond is very stable and will not be easily broken. ADCs with relatively weak stability will lead to the early release of cytotoxins (Fig. 2, ⑧). Instead, they have the opportunity to reach cells near tumor cells, resulting in a bystander effect.

Secondly, the stability of the linker is the same, and it is also one of the factors that lead to the bystander effect (Fig. 2, ⑧). The linkers are usually divided into cleavable linkers and non-cleavable linkers. The former includes chemically labile linkers (eg, pH-sensitive hydrazone bonds, disulfide bonds) and enzyme-labile linkers (eg, dipeptides, which can be cleaved by histone B), while the latter are mainly covalent linkers (eg, thioether bonds) , must be internalized into cells and broken down by lysosomes to release toxins.

The nature of cytotoxins is also an important factor affecting the bystander effect. Toxins with low polarity easily penetrate the cell membrane, so after being released in tumor cells, it is still possible to diffuse through the cell membrane to the outside of the tumor, thereby acting on surrounding cells (Fig. 2)., ⑥), resulting in a bystander effect, and toxins with high polarity are difficult to penetrate the cell membrane. There are also special cases here. For example, although the polarity of the cytotoxin of T-DM1 is not very high, due to its incomplete cleavage, a positively charged lysine remains on the toxin, which makes it impossible to penetrate the cell membrane, so T -DM1 won't produce a bystander effect.

In addition, when the target tumor cells finally disintegrate, their internal cytotoxins are released and diffuse to surrounding cells, exerting a bystander effect (Fig. 2, ⑩).

The mechanism of action of ADC

Figure 3 The mechanism of action of ADC without bystander effect (top) and with bystander effect (bottom)
(doi: 10.1007/s11912-022-01266-4)

T-DM1 and T-DXd

There have been more and more studies on the bystander effect, and T-DM1 and T-DXd are the most typical examples. T-DXd (aka DS-8201a) consists of trastuzumab (targeting HER2), toxin DXd (topoisomerase I inhibitor), and a tetrapeptide linker, which is linked to the cysteine of the antibody amino acid residues (Figure 4). T-DM1 consists of trastuzumab, toxin DM1 (tubulin polymerization inhibitor), thioether linker (non-cleavable) (Figure 4). Both need to be internalized into tumor cells and cleaved by lysosomes to release toxins, but T-DXd has better membrane permeability and produces a bystander effect, while T-DM1 does not have this function because it is positively charged.

T-DXd and T-DM1

Figure 4 T-DXd and T-DM1 and their cleaved toxin structures
(Anti-HER2-DXd (2) differs from T-DXd in that the cleaved toxin DXd (2) has a positive charge in its amino group in body fluids)
(doi: 10.1111/cas.12966)

(HER2+) human breast cancer cell KPL-4 and (HER2-) human breast cancer cell MDA-MB-468 were mixed and cultured and tested for cell viability. It was found that both cells could be killed by T-DXd, while T-DM1 only killed KPL-4 (Figure 5). In vivo, in xenograft experiments with (HER2+) human gastric cancer cells NCI-N87 cells and luciferase-expressing (HER2-) MDA-MB-468 cells, T-DXd was able to reduce luciferase signal in mice, whereas T-DM1 does not (Figure 6). In addition, the bystander effect of T-DXd can only act locally, which means that there is little systemic toxicity.

Cell viability in mixed culture

Figure 5 Cell viability in mixed culture of (HER2+)KPL-4 and (HER2-)MDA-MB-468  
(doi: 10.1111/cas.12966)

Bystander effect of T-DXD

Figure 6 Bystander effect of T-DXd in (HER2+)NCI-N87 and (HER2-)MDA-MB-468 xenografts
(doi: 10.1111/cas.12966)


Preclinical studies have shown that in addition to T-DXd, many other ADCs also have bystander effects, such as sacituzumab govitecan (SG), tisotumab vedotin (TV), enfortumab vedotin (EV), anetumab ravtansine (BAY 94–9343).

In addition, a large number of clinical trials have also begun to investigate bystander effects, which have expanded the clinical indications of ADCs. There are currently five ADC drugs for the treatment of solid tumors, including T-DM1, T-DXd, SG, TV and EV. Although the bystander effect seems to be very meaningful for efficacy, whether it will bring toxicity also needs to be considered. More and more preclinical/clinical trials are beginning to focus on these issues to guide the design of next-generation ADCs.

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[1]  Giugliano F, Corti C, Tarantino P, Michelini F, Curigliano G. Bystander effect of antibody-drug conjugates: fact or fiction? Curr Oncol Rep. 2022 Mar 19. doi: 10.1007/s11912-022-01266-4.
[2]  Ogitani Y, Hagihara K, Oitate M, Naito H, Agatsuma T. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity. Cancer Sci. 2016 Jul;107(7):1039-46. doi: 10.1111/cas.12966.
[3]  Staudacher AH, Brown MP. Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required? Br J Cancer. 2017 Dec 5;117(12):1736-1742. doi: 10.1038/bjc.2017.367.

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