After more than 20 years of development, ADC drug is becoming the main force in cancer treatment, which, compared with traditional chemotherapy, has a wider application scope. However, minimizing ADCs’ non-target toxicity meanwhile maximizing efficacy remains a challenge.
In order to achieve a balance between safety and effectiveness, it is necessary to adjust and optimize the antibody, linker, payload, and reasonable combination among them.
Antibody: the Starting Point of ADC Design
The mechanism of action of ADC begins with the binding to the target cells, so the antibody should be highly specific to the target antigen. Ideally, this antigen should be overexpressed on the surface of tumor cells but not or very low in normal tissues. A lack of high specificity or cross-reaction with other antigens will cause unpredictable side effects and reduce the treatment effect.
When an ADC binds to the antigen on the target cell surface, it is then internalized and transported to the lysosomal compartment, where it is degraded, resulting in the release of the payload. The degree of internalization depends on the affinity of the antibody to the target and the binding antigen density. High-level internalization is not necessarily beneficial to the treatment of solid tumors, because the rapid degradation of ADC limits its ability to penetrate tumor masses. Therefore, the optimal affinity of the antibody depends on the nature of the target. Uninternalized ADCs can also play a therapeutic role through the so-called “bystander effect”, in which payloads can penetrate the cell membranes of neighboring cells. This mechanism is very important for the treatment of solid tumors.
The specificity of ADC to target cells can be improved by using bispecific antibodies. For example, bispecific ADCs targeting HER2 and prolactin receptor (PRL-R) have been shown to kill target cells more effectively than ADCs targeting HER2 (expressing both HER2 and PRL-R), suggesting that the coupling of ADC target (HER2) with rapid internalization protein (PRL-R) promotes rapid internalization and lysosome degradation, which helps to reduce accidental toxicity in patients with low HER2 expression.
The mAbs of most anti-tumor ADCs are IgG1 antibodies, which can trigger Fc-mediated immune functions such as ADCC, CDC, and ADCP. However, most ADCs for solid tumors did not pass phase II clinical trials. An important reason is that the effectiveness of ADC is affected by its size that limits the tumor penetration rate. In addition to the size of IgG, excessive binding of FcRn will cause ADCs to return to extracellular circulation, increasing the exposure to healthy tissue, which not only limits the release of intracellular payload, but also may lead to extracellular toxicity. Fc γ R cross-reacts with endothelial cells and the immune system, which may also lead to off-target toxicity. Smaller binding forms have been explored to make up for these shortcomings, for example, antigen-binding fragment (Fab), single-domain antibody fragment (VHH), and single-chain antibody (scFv).
Linkers: a Key Factor in the Formation of Medicinal Properties of ADC
The linker plays a key role in ensuring that the payload remains attached to the antibody during systemic circulation, which is then released after ADC internalization. Most ADCs are characterized by cleavable linkers and release cytotoxins through reduction, acidity, or proteolysis of linkers according to the cellular physiological environment. The non-cleavable linkers will be degraded as with the antibodies released after ADC internalization.
The milestone in ADC design is the approaches to attaching payloads to antibodies. The first generation of ADC is based on lysine conjugation. The large amount of lysine in the structure of the antibody leads to the wide heterogeneity of ADC. In 2000, Mylotarg became the first ADC approved by the FDA for early successful treatment of acute myeloid leukemia, which couples Calicheamicin with anti-CD33 monoclonal antibody through lysine side chain. However, about half of the drug substances are made up of unbound antibodies. These “naked” mAbs bind to ADC targets competitively, limiting the drug effectiveness. Mylotarg voluntarily withdrew from the market in 2010 because in subsequent clinical trials, the ADC combined chemotherapy had no benefit but increased lethal toxicity compared with chemotherapy alone. The toxicity is mainly related to the non-specific release of its acid cleavable linker and payload. Mylotarg was re-approved in 2017 after a change in the dose. Subsequent generations of ADCs contain lower levels of naked mAbs, including other lysine-based conjugates, such as Kadcyla.
Most approved ADC, including Adcetris in 2011 and the ADCs approved in 2019/2020, are based on cysteine binding that, compared with lysine binding, has less heterogeneity, and easier-to-predict attachment site, contributing to lower risk of blocking the antigen-binding region.
Further improvements have been achieved using the binding technique of specific sites. Although all approved ADCs use traditional lysine or cysteine binding chemical methods, the use of site-specific binding strategies for ADCs in clinical development has increased significantly in recent years. There are a variety of ways to achieve this, including the use of engineering cysteine, unnatural amino acids, enzyme-assisted ligation, and sugar chain remodeling. The homogeneity of these ADCs is greatly increased, making the treatment safer, more effective, and more precise.
A common challenge in ADC design is the hydrophobicity of payloads, which can lead to problems associated with water solubility, aggregation, and rapid removal. In addition, cancer cells up-regulate the expression of MDR1 to transport hydrophobic compounds and produce drug resistance. One way to overcome this problem is to use polyethylene glycol (PEG) linker, which significantly improves solubility in water. Sacituzumab-govitecan (IMMU-132) is coupled with SN-38 through a cleavable Maleimide connector with a short polyethylene glycol unit. The use of hydrophilic connectors may also help to combat ADC drug resistance.
In addition, conjugation chemistry can be used to change the degree of bystander effect. When non-polar drug derivatives are released from ADC, they cross the biofilm into neighboring cells, while charged compounds do not. Cleavable linkers might produce neutral drugs that can more easily cross the cell membrane and kill surrounding cells. On the contrary, the bystander effect was not observed using the uncleavable thioether connector, because the amino acid-connector-cytotoxin complex formed by the decomposition of the uncleavable linker has been positively charged and could not pass through the hydrophobic lipid bilayer of the target cell.
Cytotoxic Payload: ADC Tumor Killing Warhead
Cytotoxic drugs are usually chosen to conjugate with antibodies because of their high potency. Most payloads are small hydrophobic molecules that, once released from ADC, can cross the biofilm and then disrupt critical cellular processes, leading to cell death. The most common are tubulin inhibitors, such as maitansine, which account for more than half of ADCs in clinical development, and many are already on the market. Other cytotoxic payloads with different mechanisms include DNA alkylating agents and cross-linking agents, topoisomerase I inhibitors, and RNA polymerase inhibitors.
The drug antibody ratio (DAR) of ADC depends on the effectiveness of the payload. For ADCs containing maytansine, the DAR of about 4 is the standard, and lower DAR may lead to the loss of activity, while higher DAR usually lead to toxicity. Although ADC has a higher therapeutic index than traditional chemotherapy, it is still necessary to distract efficacy from toxicity. In order to achieve this, different methods have been adopted. One is to use very strong toxins such as the pyrrolobenzodiazepine (PBD) with a DAR of about 2 to reduce the minimum effective dose.
Bystander effect may also be affected by cytotoxic drugs in addition to the linker. For example, MMAE is neutral and can pass through the biofilm, while MMAF produces metabolites with charged carboxyl terminal phenylalanine residues that cannot pass through the biofilm, so MMAF is less toxic to neighboring cells than MMAE.
In addition to traditional cytotoxins, more and more payloads with new mechanisms are incorporated into ADC design. These drugs include Toll-like receptor (TLR) and interferon gene stimulator (STING) agonist, which are used to activate anti-tumor immune response.
The immune checkpoint ADC is also coming to the fore. Recent studies cover the design of ADC targeting programmed cell death 1 ligand 1 (PD-L1), which is designed to have dual chemical activity. It not only has the characteristics of immune regulation, but also can exert the effect of cytotoxin through endocytosis.
In the past decade, ADC has developed into a mature biopharmaceutical product through improvements in its design. Of course, there are still space for improvement and strong market driving force for the design and further optimization of ADC drugs. It is believed that the quantity and quality of ADC will be greatly improved in the next decade.