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Interpretation of ADC drugs (3) | An article to analyze the difficulties of ADC drug development

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The complex and diverse structure of ADC drugs, as well as the complexities of in vivo actions and metabolic processes, bring multiple challenges to CMC research and preclinical research. Different tumor microenvironments and target proteins are different. For ADC drugs, specific consideration should be given to the selection of antibodies, small-molecule toxins, linkers and connection modes. In the era of pan-conjugation, each conjugated drug should find its position based on its own characteristics, form a differentiated competitive strategy, and take the road of innovative research and development to meet the unmet clinical needs.

 ADC drug design elements

ADC is a complex structure composed of three parts: antibody + linker + small molecule toxin, which determines that its preparation process will be more complicated. In the preparation and production process, it needs to go through multiple synthetic steps and dissolve in various solvents, and small molecule toxins need to maintain chemical structure and properties during these processes. The success of an ADC drug typically depends on the design of the following five elements:

Target Antigen

The selection of targeted antigens is a critical part of ADC drug design. Need to meet:

1. Specificity, high expression in tumor cells, low expression or no expression in normal cells;

2. The target antigen must be a tumor cell surface antigen;

3. Internalization and so on.

Theoretically, ADC drugs can release toxins outside tumor cells, without cellular internalization, to kill tumor cells through the "bystander effect". But in fact, most of the current realization of ADC drug efficacy is based on the drug release after internalization. Therefore, after the antibody in the ADC drug is combined with the tumor cell surface antigen, the ADC-antigen complex must be able to effectively induce the internalization process, enter the tumor cell, and achieve the effective small molecule drug through the appropriate intracellular transport and degradation process.

ADC internalization was detected by Confocal immunofluorescence.png

ADC internalization was detected by Confocal immunofluorescence


Antibodies in ADC drugs require:

1. It is highly specific to the selected antigen. Lack of specificity may result in off-target toxicity or premature clearance;

2. High affinity with the target antigen;

3. Low immunogenicity, thus ensuring that ADC drugs have a longer circulation time in the blood and smoothly enter tumor cells.


There are two types of linkers: non-cleavable linkers and cleavable linkers. Linkers need to consider a balance of stability and release efficiency. Non-cleavable linkers have more stability advantages, and the release efficiency of the cleavable linker is higher.

Conjugation technology

Coupling technology connects antibodies and small molecule toxins together through linkers, involving chemical reactions, antibody modification and transformation and other related technologies. The conjugation technology used for ADC drugs is closely related to their final drug-to-antibody ratio (DAR), and the value and distribution of DAR can significantly affect the properties of ADC drugs. Too large a DAR may cause the ADC drug to accumulate and then be eliminated in the circulatory system; if a DAR is too small, the ADC drug may not achieve the best therapeutic effect. DAR between 2 and 4 is the most preferred for ADC drugs. At present, the commonly used coupling techniques can be divided into two categories: random coupling and site-specific coupling. DS-8201 adopts site-specific conjugation technology, and the drug-to-antibody ratio is as high as 8, which has better curative effect.

Small molecule toxin

Small-molecule toxin is the main component of the killing activity of ADC drugs. When selecting small molecule toxins, it is necessary to comprehensively consider multiple factors such as toxicity and modifiability: 1. Compared with general chemical drugs, it has higher toxicity; 2. Modifiability; 3. Appropriate hydrophilic and hydrophobic balance; 4. High stability, etc.

The role of ADCC&CDC

After the ADC drug enters the blood, its antibody part recognizes and binds to the surface antigen of the target cell; then the ADC-antigen complex is mediated by the endocytosis pathway into the cell. In the cell, the cleavable linker is sensitive to the microenvironment in the tumor cell and will be affected by pH value, or cleaved by proteases and certain chemicals; ADC drugs carrying non-cleavable linkers are digested by lysosomes, thereby releasing the drug . Small molecules of certain ADC drugs can penetrate cell membranes to further kill surrounding tumor cells, a bystander-killing effect. In addition, ADC also has the immune effect of antibodies such as ADCC and CDC.

ADCC (antibody-dependent cell-mediated cytotoxicity) is an antibody-dependent cytotoxicity and an important immune defense mechanism of the immune system against viral infections and tumor diseases. Generally, it is mediated by natural killer cells (NK) (sometimes neutrophils and eosinophils can also mediate ADCC), the Fab end of the antibody binds to the target cell surface antigen, and the Fc end binds to the Fc receptor CD16 on the surface of NK cells. The target cells are brought closer to the NK cells, and the NK cells are activated to release granzymes and perforin, etc., which eventually lead to the lysis of the target cells.

CDC (complement dependent cytotoxicity) is a complement-dependent cytotoxicity mediated by a series of complement proteins (C1-C9) abundantly present in serum. C1q binds to the Fc domain of cell surface antibody molecules to trigger the CDC response. Complement binds to the corresponding antigen on the cell membrane surface through specific antibodies, activates the classical pathway of complement, and forms a membrane attack complex to lyse the target cell. Many anti-tumor antibodies, such as those raised against CD20, CD52, human leukocyte antigen (HLA)-class II, carcinoembryonic antigen (CEA), glycolipid antigen, etc., can induce CDC responses.

The mechanism of action of ADCC and CDC.png

The mechanism of action of ADCC and CDC[1]

Dual-target ADC

The study found that dual-target ADCs have better therapeutic effects than the combined administration of two single-target ADCs. Dual-target ADC formats hold great potential for the treatment of refractory breast cancer and other tumors. For example, M1231, co-developed by Sutro Biopharma and EMD Serono, is a bispecific antibody ADC targeting EGFR and MUC1. M1231 is linked to the Hemiasterlin cytotoxin via a cleavable Val-Cit SUTRO linker. Hemiasterlin is a tripeptide that exerts its cytotoxicity by binding to tubulin, disrupting normal microtubule dynamics.

For another example, in the following, researchers designed new branched linkers with azide and methyltetrazine groups as orthogonal click reaction sites. Plus payload modules (including DBCO or TCO, PEGn, GluValCit cleavable adaptors, PABC groups, and toxin molecules MMAE or MMAF as click pairs). A homogeneous ADC with dual payloads is synthesized. The GluValCit linker subsystem ensures ADC efficacy in vivo while minimizing premature degradation of the linker in animal plasma. The selection of dual conjugates of MMAE and MMAF enables ADCs to target a variety of breast cancer cells.

Molecular design and conjugation strategy of dual-target ADC.jpg

Molecular design and conjugation strategy of dual-target ADC[2]

ADC bystander effect

ADC drugs induce tumor cell death by inhibiting tumor DNA replication or arresting the cell cycle. After the ADC drug enters the blood circulation, it binds to the targeted antigen receptors on the surface of tumor cells to form ADC antigen complexes, which are endocytosed by tumor cells, and then degraded by lysosomes. Cytotoxins are released intracellularly and bind to the DNA minor groove or Tubulin, inhibits tumor DNA replication or arrests the cell cycle, induces tumor cell death. Hydrophobic small-molecule toxins can also diffuse through cell membranes and produce killing activity on adjacent tumor cells, known as the bystander effect.

Schematic diagram of the bystander effect of ADC drugs.jpg

Schematic diagram of the bystander effect of ADC drugs[3]

Both SYD985 and T-DM1 are ADCs targeting HER2, with SYD985 being more potent in cytotoxicity assays due to its stronger bystander effect. KRCH31 cells were HER2 positive and ARK-4 cells were HER2 negative, and the two cell lines were co-cultured (labeled with dye) and then further analyzed for cell death by FACS. The data show that SYD985 causes the killing of bystander cells (ARK-4). As shown in the figure below, when KRCH31 cells co-cultured with ARK-4 were treated with SYD985, the lethality of KRCH31 was not increased, but the co-culture of KRCH31/ARK-4 made HER2 low/non-expressing ARK-4 cells a bystander effect significantly enhanced. Minimal bystander cytotoxicity was detected when KRCH31/ARK-4 co-cultures were treated with T-DM1.

In vitro bystander effect.png

In vitro bystander effect[4]

The Medicilon team has more than 200 tumor cancer cell lines, and a variety of selectable ADC target protein expression positive and negative tumor cells. In addition, the Medicilon team has extensive experience in cell labeling and FACS-based cell viability analysis capabilities.

 ADC drug CMC research

Includes 4 parts:

1. Antibodies;

2. Drug-loaded-linker intermediate;

3. ADC APIs;

4. Preparation part.

In addition, some special quality control indicators need to be introduced. Such as the ratio of drug to antibody, the junction site of drug loading and antibody, and the distribution of drug loading in ADC drugs. In addition, the free drug load and antibody in the ADC need to be quantitatively controlled, and the effect of the drug load on the binding efficacy of the antibody and the target and the stability of the ADC in human plasma also need to be studied.

Selection of relevant species for ADC preclinical research

In general, toxicity studies for bulk ADCs can be performed in one animal species. At the same time, the dual properties of small molecule compounds and antibody drugs need to be considered. If the small molecule compound is a new compound or its toxicity profile is unclear, its toxicity needs to be investigated separately in at least one relevant animal species. A separate test can be carried out, or a separate dosing group can be set up in the toxicity study of ADC. If the small molecule compound is a marketed drug, there is no need for a separate study of its toxicity. If the antibody partially targets a new target or has special safety concerns, the targeted pharmacological effects, Fc effects of the antibody, and the potential toxicity caused by the targeted release of small molecule compounds should be considered by using transgenic animals or alternative molecules. research. Medicilon can help customers to complete a full set of studies such as plasma stability and in vitro hemolysis tests, pharmacodynamics studies, pharmacokinetics, toxicity studies, and safety evaluation of ADC drugs.

    Medicilon ADC preclinical R&D solution

Medicilon has in-depth exchanges with customers in the formulation of the preclinical integrated research plan of ADC. The backbone of scientific research combines the characteristics of each case with years of practical experience and technical accumulation, and carefully submits high-quality experimental plans and results to customers. . Up to now, Medicilon has undertaken more than 100 major IND application biopharmaceutical projects, including monoclonal antibodies, double antibodies, polyclonal antibodies, ADCs, viral vaccines and fusion proteins. As of May 2022, Medicilon has successfully helped 10 ADC drugs to be approved for clinical use, and has multiple ADC projects under development. Medicilon has completed toxin small molecules: DM1, MMAE, Exatecan, Dxd, SN38, etc. Medicilon has completed targets: Her2, Her3, Trop2, Claudin 18.2, CD33, Muc1, FR, etc.

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