In recent years, antibody-drug conjugates (ADCs) have achieved substantial success in cancer therapy. Typically, an antibody or peptide in a conjugated drug acts as a precisely located “navigator” while a small-molecule drug acts as a “bullet”, and a linker acts as a “bond” between the first two. After drug administration, the navigator takes the bullet to the tumor site and releases it to kill tumor cells, thus playing an anti-tumor effect.
Factors to consider when selecting a linker
Among the ADC drugs on the market, linkers can be divided into four types, namely, non-cleavable and three cleavable linkers (enzyme-cleavable, acid-cleavable, and GSH-cleavable linkers). Linker design plays a key role in modulating conjugate drug stability in systemic circulation. It also regulates the efficiency of payload release in the tumor. Several other key linker parameters, such as linker branching, charge, polarity, and hydrophilicity, will also impact the pharmacokinetics (PK), efficacy, and toxicity of the conjugate drug.
Influence of linkers on drug properties
Pharmacokinetics and toxicity: Hydrophilicity is crucial to ADC development. Low hydrophilicity of linkers will cause many problems, such as low coupling efficiency, ADC deposition in plasma, and non-specific ADC uptake in body tissues. Burke et al. have verified a strong correlation between ADC hydrophobicity, non-specific uptake rate, payload release, and resulting toxicity.
By adjusting PEG length, the non-specific tissue distribution of ADCs in vivo can be regulated, reducing non-target-dependent toxicity. Methods to improve the hydrophilicity of the linker include attaching structures like PEG, sulfonate, or phosphate to the linker.
Efficacy: The design of linkers also has a significant effect on the efficacy of ADCs since it directly influences the efficiency of payload delivery. To improve ADC efficacy, the development direction is to avoid or reduce payload resistance. Most coupled drugs employ cytotoxic drugs as payloads. However, many cytotoxic drugs are substrates of the xenobiotic transporter MDR1, which is a major cause of cytotoxic drug resistance. Most MDR1 substrates are hydrophobic. Kovtun verified that if maytansine-like compounds are attached to antibodies via a hydrophilic linker, the resulting metabolites will be hydrophilic and therefore may not be MDR1 substrates, thus avoiding MDR1-mediated resistance.
ADC efficacy may also be affected by the “bystander effect”. It involves two action mechanisms. When the conjugated drug reaches the vicinity of the tumor cell, it releases cytotoxic payloads inside the cell through endocytosis by binding to the tumor cell antigen. In some cases, the payload may be membrane permeable enough to spread out of the cell and kill surrounding tumor cells. Or, instead of internalizing, the conjugated drug reaches the tumor cell microenvironment, where the linker breaks and releases its payload, killing not only target cells but also nearby non-targeted cells. The “bystander effect” generally requires cleavable linkers. Non-cleavable linkers generally do not exert the bystander effect since the degraded charged amino acid drug lacks cellular permeability.
Extracellular cleavage experiments on linkers
BOC Sciences can establish in vitro tests to assess the stability of the linker and to identify its metabolites according to whether or not the drug linker is broken and the way it is broken. If it is an acid-cleavable linker, different pH buffers can be used to simulate the acidic environment in the body. If it is a VC-PABC linker, cathepsin B can simulate intracellular enzyme cleavage. For the reduced disulfide bond, GSH stability can be tested in the buffer solution. For non-cleavable linkers, their stability can be evaluated in human lysosomes. In vitro, stability tests or metabolite identification tests can also be performed on other substrates such as liver S9 or liver homogenate, kidney S9 or kidney homogenate, plasma, and tumor cells. BOC Sciences hopes that the establishment of these extracellular cleavage experiments will facilitate conjugated drug R&D in the industry.
Discussion
With the development of conjugated drugs and experience, linkers have been regarded as vital elements. The four traditional types of linkers have their own characteristics. In the design of conjugated drugs, linker advantages should be considered while disadvantages should be avoided. Some linkers with new mechanisms are also being developed, such as the release of payloads by foreign objects, which is a new direction in the development of cleavable linkers. As these technologies are gradually refined, more precisely delivered conjugate drugs are expected to come to market.