Antibody Drug Conjugates (ADCs)

Overview

Antibody drug conjugates (ADCs) is formed by the coupling of monoclonal antibody (mAb) and small molecule drugs through the linker. The targeting of mAb to tumor cells enables small molecule drugs to play a role in specific tumor tissues, which can effectively reduce the high toxic side effects of traditional small molecule drugs and improve the overall treatment efficiency. Currently, there are 13 ADC drugs on the market worldwide. According to Grandview data, as ADCs come on the market one after another and indications expand to more disease areas, the industry scale of ADCs will maintain rapid growth in the future, and it is estimated that the market scale will reach 21 billion US dollars in 2025, and the compound growth rate will exceed 50% in 2020-2025.

The mechanism of action of ADCs

The injected ADC binds to the target cell antigen, and the ADC-antigen complex enters the cell through clathrin-mediated endocytosis and is encapsulated in the endosome. In endosomes, when linker is cleavable in ADC, the drug can be released by hydrolysis, protease cleavage, disulfide reductive cleavage, etc. The non-cutting linker goes to the next stage. Endosomes fuse with lysosomes. The proton pump on the lysosome creates an acidic environment to assist the lysosome in the complete degradation of the ADC, where the ADC containing non-cleavable Linker is cleaved. The drug is released into the cytoplasm and causes apoptosis of target cells by inserting it into DNA or inhibiting microtubule polymerization. When the target cell dies, the active cytotoxic load may also kill the surrounding tumor cells¹.

Component design of an ADC

• Antibody

The antigen targeted by an antibody in an ADC must be highly expressed in tumor tissue and low or no expression in normal tissue. Generally, these antigens can be divided into two categories, namely, tumor-specific Antigen (TSA) expressed only in Tumor tissues and tumor-associated Antigen (TAA) expressed highly in Tumor tissues but low in normal tissues. In addition, these antigens need to be able to be expressed in tumor cell membranes and mediate the entry of ADCs into cells to further enable small molecule drugs to function in cells. Some commonly used targeted antigens include HER2, BCMA, CD20, etc^2-3.

Antibodies should meet the requirements of high specificity, strong target binding ability, low immunogenicity and low cross-reactivity, so as to achieve more efficient uptake of ADCs by tumor cells and longer half-life of ADCs in serum. Current clinical and preclinical ADCs often select IgG as the antibody to target the antigen. Among them, IgG1 is the most studied and adopted antibody due to its good balance between long blood half-life and strong immune activation and its high natural abundance. In addition to IgG1, IgG4 is also frequently used in the design of some ADCs requiring high immunogenicity due to its low Immune activating effect⁴.

• Linker

The selection of linkers is also one of the biggest challenges in the development of ADCs. Linkers are selected to ensure that the final ADCs is stable while circulating in the body for a long time, and that the small molecule drug can be released in a specific way to further cytotoxicity after entering the cell. In short, Linkers can be classified as chemically unstable, enzymatically unstable and non-cracking.

• Chemically unstable Linkers are cleaved by ph-dependent mechanisms, which means that they are sensitive to the acidic pH of lysosomes. (eg. Mylotarg® and Besponsa®)
• Enzymatically unstable linkers are relatively stable and cleaved by lysosomal proteases. (eg. Adcetris®)
• Non-cracking linkers can ensure the high stability of ADCs in vivo circulation. It mainly includes maleyl type and mercaptan hexanamide type linker, which can couple small molecule drugs with antibodies by forming amide bond and thioether bond. (eg. Kadcyla®)

Table 1. Summary of commonly used linkers for the development of ADCs

• Small molecule drug

Small molecule drugs are the key factors determining the lethality of ADC. In addition to being highly toxic, they also need to be water-soluble and stable enough. At present, the toxin drugs in clinical use can be divided into two categories according to the mechanism of action:

• Microtubule inhibitors, representative Auristatins (such as MMAE, MMAF, MMAD), maytansine and its derivatives (such as DM1, DM4), which block the cell cycle by combining with microtubules to prevent the aggregation of microtubules⁵.
• DNA damage agents, such as Calichemicin, Duocarmycins, Doxorubicin and Calicheamicin, bind to DNA trenches and promote alkylation, breaking or cross-linking of DNA chains. Other small molecules, such as alpha-amatoxin, a selective RNA polymerase II inhibitor, are also being investigated⁶.

Clinical application

ADCs have made great progress in recent 10 years, and the treatment window has been expanding.  Currently, a total of 13 ADCs have been approved worldwide (Table 1), among which 5 targets of CD22, CD30, CD33, CD79b and BCMA are indicated for hematoma. The indications for HER2, Nectin-4 and TROP-2 are solid tumors⁷. In addition to the 12 ADCs already on the market, there are 155 ongoing clinical trials of ADCs worldwide as of March 2021, according to the Clinical Trials database.

Table 1. The overview of the 12 ADCs on the market

An important factor affecting ADCs efficacy – DAR

Drug loading is the key design parameter of ADCs⁸. Drug-to-antibody Ratio (DAR) can be obtained using HPLC-MS and other testing methods. DAR is essential for the later stages of ADCs development. ADCs are taken up by tumor cells in limited quantities during in-vivo circulation, so a higher DAR is generally beneficial for increased potency. However, the small molecule drugs used in ADCs have strong hydrophobicity, so when the DAR value is too high, it will cause drug aggregation, leading to the reduction of circulating half-life in vivo and the improvement of toxic side effects, which leads to the infeasibility of excessively high DAR. The DAR value of pre-clinical and clinical ADCs generally ranges from 2 to 8.

References:

1. Dan, N.; et al. Antibody-Drug Conjugates for Cancer Therapy: Chemistry to Clinical Implications[J]. Pharmaceuticals. 2018, 11(2):32-.
2. Joubert, N.; et al. Antibody-Drug Conjugates: The Last Decade. Pharmaceuticals (Basel). 2020, Sep 14;13(9):245.
3. Yu, B.; et al. BCMA-targeted immunotherapy for multiple myeloma. J Hematol Oncol. 2020, Sep 17;13(1):125
4. Thomas, A.; et al. Antibody–drug conjugates for cancer therapy[J]. Lancet Oncology, 2016.
5. Polakis, P.; Antibody drug conjugates for cancer therapy. Pharmacol. Rev. 2016, 68, 3–19.
6. Watanabe, C.M.; et al. Transcriptional effects of the potent enediyne anti-cancer agent calicheamicin gamma(i)(1). Chem. Biol. 2002, 9, 245–251.
7. Khongorzul, P.; et al. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020, Jan;18(1):3-19.
8. Hamblett, K. J. Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate[J]. Clinical Cancer Research. 2004, 10(20):7063-7070.

ADC Targets

Bioanalysis of ADCs

ADCs present unique bioanalytical challenges as they tend to be complex heterogeneous mixtures of multiple species with a range of drug-to-antibody ratios. ADCs typically incorporate both large and small molecule characteristics, and so there are diverse bioanalytical methods, including ligand-binding assays and LC-MS methods, to quantify these species in serum/plasma for PK studies and strategies for assessing immunogenicity. As part of the clinical phase of drug development, the immunogenicity of the ADC will need to be determined. Our experts can define the strategy for assessing immunogenicity and apply sensitive immunoassays. View More

Creative Diagnostics provides key antibodies and assays for ADCs bioanalysis:

• Quantitative bioanalytical assays for preclinical and clinical studies
• Total antibody assays
• Total conjugated ADC assays
• Immunogenicity assessment – anti-drug antibody (ADA) assays

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