July 24, 2021

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The main points and challenges of ADC drug PK/PD research

The main points and challenges of ADC drug PK/PD research

The main points and challenges of ADC drug PK/PD research



The main points and challenges of ADC drug PK/PD research.  Antibody-Drug Conjugate (ADC) is composed of antibodies, linkers and small molecule toxins.

It has the advantages of high targeting and high cytotoxicity. At the same time, due to its structural diversity and complexity, as well as circulation The low content of small molecule toxins released in the system has brought many challenges to its pharmacokinetic research…



ADC molecular design


In the ADC molecule:

①The choice of target and antibody is the starting point of ADC drug design and is the decisive factor for drug indications. The selected target antigen should usually be tumor or disease-related and high-level expression;

②The linker should be stable enough during the ADC’s internal circulation, and the ADC drug should be able to quickly release the small molecule toxins in a highly active form after entering the target cell;

③Small molecular toxins should have a highly effective killing effect on tumor cells. Targets, antibodies, linkers and other factors can affect the effective safety of ADC drugs. The following will discuss them one by one.


1.   Target antigen and antibody


After determining the target indications, the first thing to consider is which antigens have specific and high-level expression on the surface of such tumor cells. Ideally:

The selected antigen should be highly uniformly expressed on the surface of the target cell, and not expressed or less expressed on the surface of normal tissues or cells;


Antigens should be non-secretory. Secreted antigens can bind to ADC drugs or naked antibodies in the circulatory system of the body, resulting in a decrease in ADC drugs that bind to tumor cells, affecting the efficacy and safety of the drugs;


After ADC drug is combined with antigen, it needs to have a proper endocytic pathway and a certain endocytosis rate, and release small molecule toxins through enzymatic degradation in the cell.


Currently, lack of efficacy and off-target toxicity are the main challenges facing ADC drugs. One of the important reasons is the low-level expression of the target antigen and the limited internalization rate.

Researchers are currently developing methods to solve low antigen expression and low internalization speed, such as the use of anti-tumor angiogenesis antibodies or bispecific antibodies to design non-internalized ADC drugs:

① Anti-angiogenic antibodies are used to avoid the internalization process, but off-target effects may occur and affect normal angiogenesis. Careful selection of target antigens and corresponding antibodies is required;

② Use bispecific antibodies to target two non-overlapping epitopes of an antigen to enhance the affinity between the antibody and the antigen.

In ADC drugs designed by ROSSIN and others, diabodies lacking the Fc region are used to target the antigen, and additional chemical activators are used to cleave the linker outside the tumor cells to release free small molecule drugs and penetrate into the tumor cells. This method avoids insufficient internalization caused by the interstitial pressure and epithelial barrier of tumor cells, thereby improving anti-tumor activity.

Bispecific antibodies can also selectively bind to two different antigens on tumor cells, thereby reducing off-target toxicity.



Studies have shown that some ADC drugs can use the physical and chemical properties of the linker and the tumor microenvironment to release free small molecule toxins, thereby killing adjacent tumor cells that are negative for antigen expression. This process is the bystander killing effect.

After internalization, some ADC drugs can be metabolized to release uncharged, cytotoxic metabolites that can penetrate the cell membrane and kill adjacent cancer cells that are negative for antigen expression. The bystander killing effect is of great significance to tumor cells with uneven antigen expression.

It should be noted that even for the same target, different tumor types will affect the therapeutic effect of ADC, that is, the same ADC drug may show different PK characteristics and effective safety in patients with different indications. For example, Besponsa is an antibody conjugate of Inotuzumab that targets CD22 and Ozogamicin. It was approved by the FDA in 2017 for adult relapsed and refractory B-cell acute lymphoblastic leukemia, but Besponsa is used in relapsed and refractory non-Hodgkins. The phase III clinical trial of lymphoma was terminated due to poor efficacy.


Among immunoglobulin G (IgG) antibodies, IgG1, IgG2, and IgG4 are usually used to develop therapeutic biological products (with a half-life of about 18 to 21 d). IgG3 has a low binding rate to the FcRn receptor, leading to its clearance rate It is faster (half-life is about 7 d) and therefore less used.

At present, many ADC drugs use IgG1 subtype. IgG1 subtype can exert antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to further improve ADC activity. However, there are also certain drawbacks. The combination of ADC drugs and effector cells may affect their targeting of tumor cells, reduce the aggregation of drugs in target cells, and prevent drug molecules from entering target cells.

At the same time, it is also necessary to consider the molecular weight of the antibody selected. When the molecular weight of the antibody is too large, it is difficult to penetrate the capillary endothelial layer and the extracellular space. If the molecular weight of the antibody is too small, it may affect its half-life in vivo.

In general, the ideal antibody should have good targeting function, effectively deliver small molecule drugs to target cells, and at the same time have low immunogenicity, and the antibody should have a suitable linking site and linker couple. After the antibody is combined with the antigen, it can have a certain endocytosis speed and a suitable endocytic pathway, and the selected antibody can maintain all or part of the function of the naked antibody.

For example, the first approved single-agent ADC drug enmetrastuzumab (Kadcyla, T-DM1) for the treatment of solid tumors is composed of trastuzumab and the small molecule microtubule inhibitor DM1 (derived from maytansine) The antibody part is trastuzumab, which maintains the ADCC activity of the naked antibody.



2.  Small molecule drugs

Factors such as the limitation of antibody’s tumor penetration ability, low antigen expression and limitation of endocytosis efficiency can cause low concentration of small molecule toxin drugs in cells, so small molecule toxins need to have high cytotoxicity. Under normal circumstances, the small molecule toxin target of ADC drugs is located in the cell. If the ADC drug cannot be transported into the cell, it will affect the effectiveness and safety of the drug, and it may affect the normal next to it outside the cell or after dissociation. Cells are toxic.

In addition, it is necessary to consider the impact of small molecules on the overall properties of ADC drugs, for example, it may affect the endocytosis efficiency of ADC drugs, the polarity of ADC drugs, and immunogenicity. At the same time, the small molecule toxin usually needs to have proper solubility in the aqueous buffer solution to facilitate coupling with the antibody, and the coupled small molecule toxin should have a certain degree of stability. The small molecule toxins currently used mainly include maytansine, alistatin, anthracyclines and camptothecin analogs.


3.  Linker

The selected linker needs to be able to exist stably in plasma to avoid the premature release of small molecule toxins and damage to normal tissues or cells.

When the ADC drug is endocytosed into the target cell, the selected linker needs to be able to quickly release effective active ingredients. In addition, the influence of the molecular weight and polarity of the selected linker on the overall properties of ADC drugs should also be considered.

Linkers can be divided into cleavage type and non-cleavage type. The cleavage type linker can use the difference between the tumor microenvironment and the normal physiological environment to release small molecule toxins that may penetrate the membrane and produce bystander effects. Non-cleavable linkers usually disconnect the antibody from the linker after the antigen-antibody complex enters the intracellular lysosome.

The two types of linkers have their own advantages and disadvantages. The non-cleavable linker is more stable than the cleavable linker, which can reduce off-target toxicity and improve the phenomenon of multi-drug resistance (MDR); The passive diffusion of metabolites produced by linkers is easier to enter the cell to produce bystander killing effects, which is of great significance to tumors with heterogeneous target antigen expression, but is more prone to off-target than the non-lysed type. Compared with cleavable linkers, non-cleavable linkers are more stringent in antigen selection.

Enmetrastuzumab (Kadcyla) uses a non-cleavable thioether linker to connect with maytansine derivatives. Due to the ADC’s intracellular metabolism, ionized metabolites are produced and the permeability is poor. The impact of ® is small, and Kadcyla has shown acceptable safety. The nature of the linker will have a greater impact on the metabolic pathway of the drug in the body, and has an important impact on the design of ADC drugs.

4. Glycotype affects half-life


The efficacy of ADC drugs mainly depends on the concentration of small molecule toxins in tumor cells. Therefore, the drug-antibody ratio (DAR) is an important factor influencing the efficacy of ADC drugs. At present, many studies are devoted to improving the DAR of ADC drugs in order to increase the concentration of drugs in tumor cells.


However, studies have found that it is not that the higher the DAR, the better the efficacy. This may be related to factors such as the polarity of small molecule toxins. From a safety perspective, the higher the DAR, the toxicity to normal tissues may also increase. In the study of Zhang et al., when DAR was increased to a certain level, the activity of ADC drugs did not increase further. Choosing an appropriate DAR is of great significance for the effective concentration in tumor cells.



The connection site is related to the homogeneity of ADC drugs and is also one of the important considerations in the design of DAR molecules.

Cysteine ​​(8) and lysine residues (80) on antibodies are more likely to be modified by chemical reactions, so they are often used as binding sites for effector molecules. In the early development of ADC, the lysine on the antibody is usually selected as the binding site, because there are up to 80 lysine residues on each antibody, resulting in great heterogeneity. There are only 8 free cysteines on each antibody that can be connected to the linker through disulfide bonds. Using cysteine ​​as the connection site helps reduce the heterogeneity of ADC. JUNUTULA et al. reported a new type of THIOMAB-drug conjugate (TDC), which uses engineered site-specific cysteine, has a clearer DAR, and has less heterogeneity.

In the “Expert Consensus on Antibody Conjugation Drug Quality Control and Preclinical Evaluation” issued by China Food and Drug Control Institute on July 20, 2018, the main drug coupling sites, DAR and drug loading distribution are important for ADC drug quality control. component.




PK characteristics of ADC drugs


The absorption, distribution, metabolism and elimination of ADC drugs are critical to the understanding of the relationship between PK and PK/PD, and will affect the selection of candidate molecules during the drug development process.

Since the structure of ADC drugs includes both macromolecular antibodies and small molecular toxins, a mixed method may be required to characterize their ADME properties. Because ADC drugs are mostly administered intravenously in clinical practice, their absorption characteristics are not discussed here.

In terms of molecular weight and space volume, the main body of ADC drug structure is antibody, so it shows many pharmacokinetic characteristics similar to naked antibody, and has the main pharmacokinetic characteristics and mechanism of action of antibody drugs, such as target Spot-mediated drug clearance, FcRn receptor circulation and non-specific protease degradation. 

In general, ADC drugs are usually administered intravenously, and their distribution is similar to antibody drugs. They also have metabolic and clearance pathways for antibodies and small molecules. They are non-linear at low doses and linear at high doses.


One of the most important characteristics of ADC drugs is their diversity. Due to the difference in the number and/or binding sites of the small molecule toxins coupled to the antibody, the ADC is a mixture of multiple different molecules.

When the ADC enters the body, the small molecule toxin is removed from the ADC through enzymatic hydrolysis or chemical reaction. The gradual dissociation of drugs further increases the diversity of ADC drugs in the body. This ever-changing diversity is one of the important challenges for ADC drug PK research.


1. Distributed

The spatial structure of ADC drugs is mainly composed of antibodies, so the distribution in the body is usually similar to that of unbound antibodies.

The initial distribution of ADC drug is mainly confined to the blood vessel. The volume of distribution of the central compartment is similar to the volume of plasma (-50 mL·kg -1), and then expands into the interstitial space, and the steady-state distribution volume is about 150-200 mL ·Kg -1. Similar to naked anti-antibody, ADC drugs are difficult to pass through vascular epithelial cells, with low tissue distribution and slow spreading.

They are more distributed in tissues with high blood flow, such as liver, kidney, lung, spleen, and heart. Similar to naked antibodies, the distribution of ADC drugs will also be affected by the target antigen expression and internalization rate. Drugs that distribute naked antibodies to non-target tissues through non-specific or specific binding of antigens usually do not have pharmacological effects.

However, in ADC drugs, small molecule toxins or their analogs will be released later, so they are distributed in the same tissues. And accumulation may produce clinically significant pharmacological/toxic effects. Understanding the distribution of ADC drugs is important for understanding pharmacological/toxic effects.

Tumor cells or normal tissues may release antigens into the circulatory system, combine with ADC drugs to eliminate ADC drugs and affect their distribution. The complex formed by the combination of ADC drug and soluble antigen can be taken up and cleared by the liver, and a large amount of small molecule toxins are released in the liver during this process, causing potential hepatotoxicity. Studies in rodents have shown that the binding of antibodies to monomethylamantadine E (MMAE) will affect its tissue distribution, and will increase liver uptake compared with unbound antibodies; similar phenomena have been seen in other studies. , The combination of small molecule toxins has a significant impact on the distribution of ADC drug CMD-193 in normal human tissues and tumors: the uptake of tumors is reduced and more distributed in the liver.


In the above case, the antibody distribution study of ADC drugs used the method of labeling antibodies. But at the same time, understanding the tissue distribution of free and bound small molecule toxins is also very important. Some researchers have carried out dual radioisotope labeling studies on antibodies and small molecule toxins. The results show that small molecule toxins MMAE and antibodies are in most tissues. The distribution is similar, but the concentration of small molecule toxins in the liver is higher than that of antibodies.

2.  Metabolism and excretion


Antibodies enter cells mainly through target-mediated and non-specific uptake, and are eliminated from the body through proteolysis.

Unlike naked antibodies, ADC metabolism has its own unique characteristics, which can release cytotoxic metabolites through two different pathways (uncoupling and catabolism). ①Uncoupling: The linker is cleaved to release free small molecule toxins and retain the antibody skeleton; ②Catabolism: The antibody part in ADC drugs is proteolyzed into peptides/amino acids, and free small molecule toxins are produced at the same time, or with connection These metabolites can still have high cytotoxicity.

Generally, two metabolic pathways occur simultaneously in the body, and which pathway is the main one depends on factors such as linker stability, binding site, and total drug loading.


For ADC drugs with linkers (such as disulfide bonds) that are susceptible to enzymatic or chemical cleavage, the release of cytotoxic drugs through the uncoupling process may be the main way. If it is a non-cleavable linker, the metabolic pathway in the body may be mainly catabolism to release free small molecules and their structural analogs.


For example, enmetrastuzumab (Kadcyla) with a non-cleavable conjugate is metabolized in vivo to form effector molecules with amino acid residues and/or linkers. Among them, the concentration of MCC-DM1 in plasma Cmax is Much higher than free DM1.




The free small molecule toxins and their structural analogs produced by ADC drug metabolism will continue to undergo metabolism and biotransformation in the body (such as through cytochrome P450 enzyme metabolism). In theory, there are also drug interactions with other small molecule therapeutic drugs. -Drug Interaction, DDI) may affect the blood concentration of ADC drug catabolites or other combined drugs. However, given the low concentration of small molecule toxins released by ADC drugs in the circulatory system of the body, the risk of DDI is usually low.




PK research considerations for ADC drugs

1.  Target analyte

The main content of ADC drug PK research includes the stability of ADC drug, blood concentration-time curve, distribution, metabolism and excretion process, etc.;

If the small molecule drug is a new compound, it is recommended to comprehensively apply in vivo and in vitro research methods, qualitative and/or quantitative detection methods, system exposure to small molecule drugs, plasma protein binding and excretion characteristics, tumor and normal tissue uptake/distribution characteristics, etc. Carry out detailed research, and when necessary, study the system exposure, metabolite spectrum, distribution, shedding mode, and breaking point of small molecule drug metabolites.

The analytes commonly used to characterize the PK characteristics of ADC drugs include bound antibodies (antibodies conjugated to at least one small molecule toxin), total antibodies (antibodies conjugated and unconjugated to small molecule toxins), bound effector molecules, and free small molecules Toxins and their analogs. The content and meaning of the PK of different analytes are different, which constitutes the overall picture of ADC drug metabolism in the body as a whole.

There are two ways to eliminate ADC drug concentration in the body:

①The antibody part is disintegrated by enzymatic degradation;

②The small molecule toxin is completely dissociated from the antibody (that is, DAR becomes 0).

The only way to influence the concentration of total antibody is ①. Therefore, it is usually observed that the ADC drug has a faster clearance rate. The difference between the clearance rate of the total antibody concentration is the rate at which the effector molecule is completely dissociated from the ADC drug. The side reflects the stability of the ADC drug in the blood, that is, compared to the ADC given The clearance rate of total antibody and bound antibody after drug treatment, the observed difference reflects the rate at which the effector molecule is completely dissociated from the ADC drug.

The effect of conjugated drugs on antibody metabolism can be compared by the total antibody PK measured by the naked antibody and ADC drugs, so as to evaluate the effect of small molecule drugs on the antibody clearance rate after being connected to the antibody. In some studies of ADC drugs, it has been found that after combining with small molecule toxins, the clearance of antibodies may be accelerated, and ADC drugs with high DAR have a faster clearance.


2.  Immunogenicity

Similar to other macromolecular biological therapies, ADC drugs can also induce immune response in the human body to produce anti-drug antibodies (Anti-therapeutic Antibody, ATA). Both internal factors (product-related) and external factors (patient-related) may affect the incidence of ATA.

For example, ADC drug-related variants (such as tertiary structure deformation) may increase the risk of immunogenicity. The ATA produced in the body can neutralize ADC drugs, which is a way to clear ADC drugs, and increase the clearance rate of ADC drugs themselves and naked antibodies. Like monoclonal antibodies, the immunogenicity of ADC drugs needs to be strictly monitored and evaluated during clinical trials.


3.  PK/PD analysis

PK/PD modeling can quantitatively reflect the relationship between drug dosage and pharmacological effects (response), and is an important part of new drug development. A comprehensive assessment of the exposure-response (ER) relationship can provide recommendations for the patient’s dosage, frequency of medication, and dosage adjustments. Compared with unconjugated naked antibodies, ADC drugs usually have a narrower therapeutic index, so it is more necessary to improve ER analysis to guide clinical research and actual medication.

PK/PD analysis in the development of ADC drugs has its own characteristics and challenges. For example, ADC drugs may have multiple pharmacological mechanisms of action (such as target-specific toxicity and non-specific toxicity), and metabolism in the body will produce multiple activities. For analytes (such as ADC, total antibodies, free small molecules and their structural analogs), different analytes have different pharmacological/toxic effects. The analytes should be fully investigated when conducting PK/PD studies.

The existence of multiple active substances in the body makes the establishment of ER relationship more complicated. The selected key analytes that drive the action of the drug are different, and the results of ER relationship modeling may also be different. For example, in the development and application of T-DM1, in the ERPK/PD model established by the applicant, the driving analytes used are the AUC and Cmax of T-DM1 predicted by NCA, the AUC of total antibody, and the Cmax of DM1. The ER model shows that there is no significant correlation between exposure and drug efficacy.

The E R model established by the FDA during the review process uses the driving analytes as the AUC and Cmin of T-DM1 predicted by the model. The model results show that for subjects with low exposure, it is possible to consider increasing the dose of the drug to improve the efficacy.


4.  Combining multiple studies

The research of ADC drug metabolism mechanism and metabolites requires a combination of in vitro research and in vivo research, animal research and human research, collaborative and multi-pronged. Reasonable in vitro studies and animal studies (including catabolism studies in cell lines expressing targets and cross-species plasma stability studies) can help clarify the metabolic mechanisms and pathways of ADC, and can be used to identify ADC catabolites and establish The relevance of preclinical species, etc., provide references for clinical trials in humans.

For example, during the development of T-DM1, two material balance studies were carried out in rats to explore the metabolic pathways and recovery rates of ADC and DM1 in rats respectively, laying a foundation for the clinical research of T-DM1 in humans. At the same time, the study found that DM1 is mainly metabolized by CYP3A4/5. Therefore, it is recommended not to use strong CYP3A4 inhibitors in combination with strong CYP3A4 inhibitors in the T-DM1 instructions, and continue to complete the PK study of T-DM1 in patients with liver injury after the market.




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