The absorption, distribution, metabolism and elimination (ADME) of antibody-drug conjugate (ADC) drugs are crucial to the understanding of their pharmacokinetic (PK) and PK/PD relationships, which will influence the selection of candidate molecules during drug development. Since the structural composition of ADC drugs includes both macromolecular antibodies and small molecule toxins, a mixed approach may be required to characterize their ADME properties.

PK Characteristics of ADC Drugs

In terms of molecular weight and spatial 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-mediated drug clearance, FcRn receptor cycle and non-specific protease degradation. Comparison of the main PK characteristics of ADC drugs with small molecule drugs and antibody drugs is shown in Table 1. In general, ADC drugs are usually administered intravenously, and their distribution is similar to that of antibody drugs. They have the metabolism and clearance pathways of both antibodies and small molecules, which are nonlinear at low doses and linear at high doses.

Table 1. Comparison of small molecule drugs, monoclonal antibodies and ADCs.

One of the most important features of ADC drugs is their diversity. Due to the difference in the number and/or binding sites of small molecule toxins coupled to antibodies, ADC is a mixture of different molecules. When ADC enters the body, small molecule toxins are gradually dissociated from ADC drugs through enzymatic hydrolysis or chemical reaction, which further increases the diversity of ADC drugs in the body. This ever-changing diversity is one of the important challenges in the PK research of ADC drugs.

Distributed

The spatial structure of ADC drugs is mainly composed of antibodies, so the distribution in vivo is usually similar to that of unbound antibodies. The distribution of ADC was mainly confined to the blood vessels in the early stage after drug administration. The distribution volume of the central chamber was similar to that of the plasma (~ 50 mL·kg^-1), and then extended to the tissue space. The steady-state distribution volume was about 150~200 mL·kg^-1. Similar to naked antibody, ADC drugs are difficult to pass through vascular epithelial cells, have low tissue distribution and slow diffusion, and are more distributed in tissues with large blood flow, such as liver, kidney, lung, spleen and heart.

Similar to naked antibody, the distribution of ADC drugs is also affected by the expression and internalization rate of target antigen. Drugs usually do not have pharmacological effects on the distribution of naked antibodies to non-target tissues through non-specific or specific binding of antigens. However, in ADC drugs, the distribution and accumulation in the same tissues may produce clinically significant pharmacological/toxic effects due to the subsequent release of small molecule toxins or their analogues. Understanding the distribution of ADC drugs is of great significance for understanding pharmacological/toxic effects.

Tumor cells or normal tissues may release antigens into the circulatory system, combine with ADC drugs to remove ADC drugs and affect their distribution. The complex formed by the combination of ADC drugs and soluble antigens can be taken up and removed by the liver, and in the process, a large number of small molecule toxins are released in the liver to cause potential hepatotoxicity. Studies in rodents have shown that the binding of antibodies to monomethyladamantanamine E (MMAE) affects its tissue distribution. Compared with unbound antibodies, it increases liver uptake. In the above cases, the antibody distribution of ADC drugs was studied by the method of labeling antibodies. However, it is also important to understand the tissue distribution of free and bound small molecule toxins. Some researchers have carried out dual radioisotope labeling studies on antibodies and small molecule toxins. The results show that the distribution of small molecule toxin MMAE and antibodies in most tissues is similar, but the concentration of small molecule toxins in the liver is higher than that of antibodies.

Metabolism and Excretion

Antibodies enter cells mainly through target-mediated and non-specific uptake, and are cleared from the body through proteolysis. Different from naked antibody, the metabolism of ADC has its unique characteristics, which can release cytotoxic metabolites through two different pathways (uncoupling and catabolism).

• Uncoupling: The linker is cleaved to release free small molecule toxins and retain the antibody skeleton;
• Degradation: Some of the antibody proteins in ADC drugs are hydrolyzed into peptides/amino acids, and produce free small molecule toxins, or small molecule toxins with linkers, or small molecule toxin analogues with amino acid-linkers. These metabolites can still have high cytotoxicity.

Usually, the two metabolic pathways occur simultaneously in the body, and which pathway is dominant depends on factors such as linker stability, binding site, and total drug loading. For ADC drugs with linkers (such as disulfide bonds) that are easily cleaved by enzymes or chemical cleavage, the release of cytotoxic drugs through the uncoupling process may be the main pathway. If it is a non-cleavage linker, the metabolic pathway in vivo may be dominated by the release of free small molecules and their structural analogues by catabolism. For example, in vivo metabolism of Kadcyla, a non-cleavage conjugate, results in the formation of effector molecules with amino acid residues and/or linkers, in which the C_max value of MCC-DM1 in plasma is much higher than that of free DM1.

ADC Analysis and Characterization

BOC Sciences provides comprehensive ADC analysis and characterization services. Our analytical services provide comprehensive support for the development and quality control of ADCs and are designed to help researchers and developers ensure the safety and efficacy of these complex therapeutic molecules.

• ADC In Vitro Analysis
• ADC In Vivo Analysis
• ADC ADME Characterization
• ADC DMPK Analysis
• Bioanalytics
• Bioassays
• DAR and Drug Distribution Analysis
• Stability Analysis Services

PK Considerations for ADC Drugs

Target Analyte

The main contents of the PK study of ADC drugs include the stability of ADC drugs, blood concentration-time curve, distribution, metabolism and excretion process. If small molecule drugs are new compounds, it is recommended to comprehensively apply in vitro and in vivo research methods, qualitative and/or quantitative detection methods, to systematically study the systemic exposure of small molecule drugs, plasma protein binding and excretion characteristics, and uptake/distribution characteristics of tumors and normal tissues. If necessary, the system exposure, metabolite profile, distribution, shedding mode, and break point of small molecule drug metabolites should be studied.

The analytes commonly used to characterize the PK characteristics of ADC drugs include binding antibodies (antibodies coupled to at least one small molecule toxin), total antibodies (antibodies coupled to and uncoupled with small molecule toxins), binding effector molecules, free small molecule toxins and their analogues. The PK of different analytes reflects different contents and meanings, which constitutes the whole picture of ADC drug metabolism in vivo. There are two ways to eliminate the decrease of ADC concentration in vivo:

1) The antibody was partially disintegrated by enzymatic degradation;

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

The pathway that affects the concentration of total antibodies is only pathway 1). Therefore, it is usually observed that the ADC drug has a faster clearance rate. The difference in the clearance rate from the total antibody concentration is the rate at which the effector molecules are completely dissociated from the ADC drug, which reflects the stability of the ADC drug in the blood. In other words, the difference in the clearance rate of the total antibody and the binding antibody after ADC administration reflects the rate at which the effector molecules are completely dissociated from the ADC drug.

The effect of coupling drugs on antibody metabolism can be compared by giving naked antibodies and total antibody PK measured by ADC drugs, so as to evaluate the effect of small molecule drugs on antibody clearance rate after connecting to antibodies. In some studies of ADC drugs, it was found that binding to small molecule toxins may accelerate the clearance of antibodies, and ADC drugs with high DAR clearance faster.

Fig. 1. Pharmacokinetics and pharmacodynamics of antibody-drug conjugates (Pharmaceutics. 2023, 15(4): 1132).

Immunogenicity

Similar to other macromolecular biotherapy, ADC drugs can also induce an immune response in the human body to produce anti-therapeutic antibody (ATA). Both internal factors (product-related) and external factors (patient-related) may affect the incidence of ATA. For example, related variants of ADC drugs (such as tertiary structure deformation) may increase the risk of immunogenicity. The ATA produced in the body neutralizes the ADC drug, which is a clearance pathway for ADC drugs, increasing 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.

PK/PD Analysis

PK/PD modeling can quantitatively reflect the relationship between drug dose and pharmacological action (response), which is an important part of new drug research and development. A comprehensive assessment of the Exposure-Response (ER) relationship can provide recommendations for patient dosage, medication frequency, and dose adjustment. For unbound naked antibodies, ADC drugs usually have a narrow therapeutic index, so it is more necessary to improve ER analysis to guide clinical research and practical medication.

The PK/PD analysis in the development of ADC drugs has its own characteristics and challenges. For example, ADC drugs may have multiple pharmacological mechanisms (such as target-specific toxicity and non-specific toxicity) at the same time, and in vivo metabolism produces a variety of active analytes (such as ADC, total antibodies, free small molecules and their structural analogues). Different analytes have different pharmacological/toxic effects, and 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 results of ER relationship modeling may also be different due to the different key analytes selected to drive the drug action. For example, in the development and application of T-DM1, the AUC and C_max of T-DM1 predicted by NCA, the AUC of total antibody and the C_max of DM1 were used in the ER PK/PD model established by the applicant. The established ER model showed that there was no significant correlation between exposure and drug efficacy. The driving analytes used in the ER model established by FDA during the review process were the AUC and C_min of T-DM1 predicted by the model. The model results showed that for subjects with low exposure, increasing the dose could be considered to improve the efficacy.

Combination of Multiple Studies

The study of ADC drug metabolism mechanism and metabolites requires a combination of in vitro and in vivo studies, animal studies and human studies, working together and multi-pronged. Reasonable in vitro and animal studies (including catabolic studies in cell lines expressing the target and cross-species plasma stability studies) can help to elucidate the metabolic mechanisms and pathways of ADC, and can be used to identify ADC catabolic products and establish the correlation of preclinical species, providing a reference for clinical trials in humans.

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

ADC Products from BOC Sciences