What are the most important things to pay attention to during the preparation of antibody-conjugated drugs?

Antibody conjugated drugs are formed by linking monoclonal antibodies targeting specific antigens and small molecule cytotoxic drugs through a linker. It has both the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting properties of antibody drugs. Since the first ADC (Mylotarg) was approved for the treatment of CD33-positive acute myeloid leukemia, several ADCs for the treatment of cancer have been developed.

From selecting the right antibody to the final product, the entire development process of ADC is an arduous and challenging task. Clinical pharmacology is one of the most important tools for drug development. Using this tool helps to find the best dose of the product, so as to maintain the safety and effectiveness of the product in the patient population. Unlike other small or large molecules that usually only measure one part/metabolite for pharmacokinetic analysis, ADCs need to measure multiple parts to characterize their PK characteristics. Therefore, an in-depth understanding of the clinical pharmacology of ADCs is essential for selecting safe and effective doses in the patient population.

Pharmacokinetic Overview Of ADC

Pharmacokinetics is an indispensable part of clinical pharmacology and modern drug development. The main purpose of pharmacokinetic research is to obtain the absorption, volume of distribution, clearance rate, half-life, accumulation after multiple administrations, various disease states, and the effects of age, weight and gender on the pharmacokinetics of the drug. Information. These pharmacokinetic parameters can be used to design the optimal dosing regimen for patients.

It should be recognized that, unlike small molecules and therapeutic proteins (antibody/fusion proteins), the PK of ADCs is very complex because ADCs are composed of several components. Not only must the PK of the monoclonal antibody be considered, but also the PK of the cytotoxic molecules and the physical and chemical properties of the binding. Because the molecular weight of the monoclonal antibody accounts for more than 90%, the PK of the different components of the ADC is greatly affected by its PK. The PK characteristics of total antibody (ADC+mAb) provide the best assessment of ADC stability and integrity. Conjugates and coupling sites also play an important role in maintaining the stability and PK of ADCs. The following table lists the characteristics of FDA-approved ADCs and PK.

Pharmacokinetic Characteristics Of ADC

Generally speaking, four processes are involved in the body after administration. These processes are absorption, distribution, catabolism and clearance.


Most antibodies are usually given by intravenous injection or infusion, and antibodies can also be given by SC route. However, for ADC, the current route of administration is intravenous injection or infusion. Due to the response to cytotoxic payloads and local deposition of cytotoxic substances, SC administration may not be suitable for ADC.


The distribution of drugs in the body can be described by the volume of distribution. Due to their size and polarity, the distribution of antibodies and ADCs is usually limited to blood vessels and interstitial spaces.

The initial distribution of ADCs is generally limited to blood vessels, and the volume of distribution is generally equal to blood volume. Subsequently, ADCs can be distributed to the interstitial space. In addition, ADC distribution will also be affected by target antigen expression and endocytosis.

The distribution and accumulation of ADC in the same tissue can produce undesirable (toxic) pharmacological effects, which are due to the release of cytotoxic drugs or metabolites after the ingestion of ADC.


ADC decomposing or metabolic processes in vivo include antibody catabolism and small molecule drug metabolism in vivo. ADCs release effector molecules in the cell (non-cleavable linker) or circulatory system (cleavable linker) before reaching the tumor cells. Unbound antibodies and antibody fragments follow the metabolic pathways of antibodies to produce amino acids through enzymatic hydrolysis, which are reused by the body.

Free small-molecule drugs/small-molecule drugs with amino acid residues/linker’s small-molecule drug metabolites that may be formed after ADC cleavage or catabolism will further undergo liver CYP450 enzyme metabolism, and potential drug drugs may also occur interaction.

In addition to the nature of ADC itself, antigen expression, receptor/cell density, FcRn-mediated circulation, Fcγ interaction, receptor-mediated endocytosis, immunogenicity, etc., all affect the catabolism of ADC.


ADC is also eliminated through catabolism and excretion. ADC can be degraded after entering the lysosome through a specific pathway that binds to the target, and is cleared from the body after releasing small molecule drugs; it can also be cleared through non-specific pinocytosis, which involves the recycling process involving FcRn .

ADCs, antibodies, peptides with larger molecular weights and amino acid fragments cannot be filtered and excreted through the glomerulus, but are re-absorbed and utilized in the form of amino acids. Free small molecule drugs, peptides with smaller molecular weight and small molecule drugs linked by amino acids, and antibody fragments with smaller molecular weight can be excreted through glomerular filtration. At the same time, small molecule drugs and metabolites can also be eliminated through enzymatic metabolism or excreted into the feces through transporters.

ADC Biological Analysis

ADC has several components to characterize the PK characteristics of these components, several analysis methods are required, as described below:

1.ELISA immunoassay determines the kinetic curve of the conjugate and total antibody.
2.TFC-MS/MS, quantify free drugs/metabolites.
3.High resolution mass spectrometry is used for in vivo drug-to-antibody ratio analysis.

In addition, two types of ELISA immunoassays are used to quantitatively measure ADC analytes: the first type of analysis measures total antibodies, ADCs with DAR greater than or equal to zero. The second analytical method measures drug-binding antibodies, defined as ADCs with a DAR greater than or equal to 1.

Other analytical methods are size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC). SEC is the most commonly used liquid chromatography (LC) technique to determine the number of aggregated antibodies. This technique can also be used for ADCs. Although HIC is a traditional technique for protein separation, purification, and characterization, this technique is now being used for ADC characterization and analysis.

Cytotoxic Payload

ADC cytotoxic payload should have the following characteristics:

1.Payloads of molecules should be small in size, lack immunogenicity, and soluble in water buffers so that they can be easily coupled.
2.The cytotoxic payload should have appropriate fat solubility.
3.The target of the payload should be located inside the cell.
4.The payload should be stable in the blood.

Currently, commonly used cytotoxic drug effector molecules are microtubule inhibitors (auristatins/maytansinoids), DNA damaging agents (calicheamicin/duocarmycins/anthracyclines/pyrrolobenzodiazepine dimers) and DNA transcription inhibitors (Amatoxin/Quinolinealkaloid (SN-38)). Several ADC drugs that have been approved for marketing use a total of 6 different small molecule drugs, of which 3 ADC drugs use MMAE as a conjugate drug, 2 drugs use Calicheamicin as a conjugate drug. MMAF , DM1, SN-38, Dxd are also successfully used.

Drug To Antibody Ratio(DAR)

DAR refers to the average number of payload molecules attached to a single monoclonal antibody, usually between 2 to 4 molecules. In rare cases, DARs of up to 8 can be safely achieved by using hydrophilic linker payloads, such as Enhertus and Trodelvys. DAR is very important for the determination of the efficacy of ADCs, and DAR may affect the stability of drugs in the circulation, PK and the toxicity of ADCs.

Studies have shown that ADCs with high DAR values ​​(7-14) have a faster clearance rate and lower in vivo efficacy compared with ADCs with DAR values ​​<6. The DAR value and its effect on stability and PK also depend on the coupling position and the size of the linker.

Lysine or cysteine ​​is usually modified to produce ADC. Lysine is one of the most commonly used amino acid residues to connect substrates and antibodies. Lysine usually exists on the surface of antibodies, so it is easy to couple.

Other amino acids such as cysteine and tyrosine can also be modified, and maleimide was used to modify cysteine to synthesise ADCs such as Adcetriss, Polivys, Padcevs, Enhertus, Trodelvys and Blenreps.


Linker is an indispensable part of ADC, which determines the drug release mechanism, PK, therapeutic index and safety of ADC. Early ADC linkers were chemically unstable, such as disulfide and hydrazone. These linkers are unstable in the circulation and have a short half-life, usually one to two days. The latest generation of linkers are more stable in the systemic circulation, such as peptide and glucuronic acid linkers. The two most common connectors are as follows:

Cleavable Linker

The cleavage linker is sensitive to the intracellular environment, and releases free effector molecules and antibodies through the combined action of catabolism and dissociation in the cell, such as acid cleavage linkers and protease cleavage linkers. They are usually stable in the blood, but will rapidly cleave in a low pH and protease-rich lysosome environment, releasing effector molecules. In addition, if effector molecules can transmembrane, tumors can be eliminated by exerting a potential bystander effect.

Non-cleavable Linker

The non-cleavable linker is a new generation of linker. Compared with the cleavable linker, it has better plasma stability. Since non-cleavable linkers can provide greater stability and tolerance than cleavable linkers, these linkers reduce off-target toxicity and also provide a larger therapeutic window.


In 11 clinical trials targeting 8 ADCs, the baseline incidence of ADAs was between 1.4% and 8.1%, and the incidence of ADAs after baseline was between 0-35.8%. These values ​​are in the range of therapeutic monoclonal antibodies. In general, the incidence of ADA for ADCs is less in patients with hematological tumors than in patients with solid tumors; most ADAs are directed against the monoclonal antibody domain of ADCs. In addition, in most patients, the hapten-like structure of these ADCs does not pose a greater risk of immune response than therapeutic monoclonal antibodies.

ADC Pharmacokinetic Model

The application model method can integrate PK, efficacy and safety data to meet the needs of ADC drug research and development at different stages, such as: target selection, antibody affinity, linker stability, animal to human external Push, dose selection and adjustment, ER correlation research (exposure-response relationships), DDI research, etc. Because ADC has multiple clearance pathways (dissociation and catabolism) and the complex PK characteristics of multiple analytes, its kinetic model is also more complicated.

Different models have different applications. For example, two-compartment model and PBPK model can be used to describe the stability characteristics of ADC with parameters such as clearance rate, dissociation, and metabolic rate. At present, non-compartmental models, population pharmacokinetic models, mechanism-based models, and physiological-based models are all used in ADC pharmacokinetic research.


In the development process of ADC drugs, clinical pharmacology plays a very important role. Through the continuous development of bioanalytical technology, the PK/PD characteristics of ADC drugs are clarified thoroughly and comprehensively, which is important for promoting the development of more low-toxic and efficient ADC drugs. Important. ADC drugs will also show more powerful advantages in the field of tumor treatment.


1. ClinicalPharmacology of Antibody-Drug Conjugates. Antibodies (Basel). 2021 May21;10(2):20.