Pharmacokinetic/pharmacodynamic (PK/PD) studies are an integral part of the drug development process, using analytical methods to detect the distribution of test substances and biomarkers in normal or disease model animals to describe drugs Time-related effects and pharmacodynamics after administration. PK/PD studies contribute to a better understanding of the relationship between drug exposure, efficacy, and toxicity, and are important tools to guide the design of further preclinical and clinical evaluations. We can obtain PK parameters such as area under the curve, maximum plasma concentration or other parameters to determine drug exposure. We can detect biomarkers such as cytokine levels, protein expression levels or other necessary parameters related to drug efficacy/toxicity.
Unlike small molecules and antibodies, the PK of ADCs is very complex because ADCs consist of several components. Not only the PK of the mAb, but also the PK of the cytotoxic molecule and the physicochemical properties of the binding should be considered. The PK of the different components of the ADC is greatly influenced by the PK of the mAb because it accounts for more than 90% of the molecular weight. The PK profile of total antibody (ADC + mAb) provides the best assessment of ADC stability and integrity. The binding site also plays an important role in maintaining ADC stability and PK.
PK characteristics of ADCs
PK is the study and characterization of the time course of drug absorption, distribution, metabolism and excretion (ADME). These processes of the ADC are briefly described below.
Following administration, the ADC drug enters the systemic circulation. Typically, the drug is administered orally, intravenously, intramuscularly, and subcutaneously. Each of these methods has its own characteristics, advantages and disadvantages of ADME.
Most antibodies are usually administered by the intravenous bolus or infusion route, but can also be administered by the subcutaneous route. However, for ADCs, the current route of administration is intravenous. Subcutaneous administration may not be feasible for ADCs due to the response to the cytotoxic payload and local deposition of cytotoxic substances.
The structure of ADCs is primarily governed by the antibody backbone, and therefore, ADCs generally behave like unbound antibodies in their distribution. The initial distribution is usually confined to the vascular space, and the distribution of the central ventricular volume is similar to that of the plasma volume (~50 mL/kg). Over time, the distribution expanded into the interstitial space, with a steady-state distribution volume of approximately 150-200 mL/kg. Similar to unconjugated antibodies, ADCs diffuse very slowly in vascular endothelial cells, and convection is thought to be the primary mechanism for antibody transfer from plasma to interstitial fluid.
Of course, similar to non-conjugated antibodies, the distribution of ADCs is also affected by target antigen expression and internalization.
Metabolism and elimination
There are two main pathways in the current understanding of ADC clearance mechanisms: protein degradation and deconjugation.
Similar to mAbs, clearance of ADCs by protein degradation is primarily driven by catabolism mediated by target cell-specific or non-specific cellular uptake, followed by lysosomal degradation. Degradation of ADCs occurs in various tissues, including skin, muscle, and liver, due to uptake by macrophages. For cells with an antibody target, target-mediated clearance occurs when the antibody binds to the target cell and is internalized and degraded. This clearance pathway is saturated and may lead to non-linear PK, especially at low doses or with highly expressed targets. Antibodies also bind to Fc[gamma] receptors expressed on cells of the monocyte-macrophage system (MPS). Interaction with Fcγ receptors also leads to the internalization and catabolism of ADCs.
Deconjugation clearance is typically mediated by enzymatic or chemical cleavage of the linker (eg, maleimide exchange), resulting in the release of cytotoxic drugs from the ADC. Once released from ADCs, cytotoxic drugs may be further metabolized, transported, and eliminated by conventional mechanisms applicable to small molecules.
ADCs have several analytes, and in order to characterize the PK profile of these analytes, several analytical methods are required:
Enzyme-linked immunoassay to detect pharmacokinetic changes of antibody conjugates and total antibody; TFC-MS/MS, to quantify free drug/metabolite; and high-resolution mass spectrometry for in vivo DAR analysis.
Two types of ELISA methods are used to quantitatively measure the analyte of the ADC: the first method measures total antibody, which includes both bound and unbound forms of the ADC. Total antibody PK describes the antibody-related PK behavior of ADCs and provides the best assessment of the in vivo stability and integrity of antibodies over time. The total antibody PK profile of an ADC plays a key role in ADC optimization, especially in assessing the effect of binding and selecting drug loading. A second assay is used to measure the concentration of bound antibody in the systemic circulation, which is defined as an ADC with a DAR greater than or equal to 1. Since detection requires the presence of intact antibodies and cytotoxic drug components of the ADC, the conjugated antibody concentration is often used as the ADC active concentration and is the basis for most ADC PK assays.
Other analytical methods are size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC). SEC is the most commonly used liquid chromatography (LC) technique for the determination of mAb aggregation, and this technique is also applicable to ADCs. While HIC is a traditional technique used to isolate, purify and characterize proteins, this technique is now being used for the identification and analysis of ADCs, which can be screened, analyzed and characterized by using ammonium sulfate buffers and high pressure liquid chromatography systems .
Interpretation of ADC analytes and PK
Although there are several methods available to characterize the PK of ADCs. However, no single analytical approach captures all aspects of the PK behavior of these complex molecules, such as the rate of drug loss from ADCs (i.e., linker stability), the effect of binding on ADC clearance, and ultimately the exposure-response relationship. Therefore, integrating information from multiple assays is critical for deciphering the pharmacological effects of ADCs and optimizing ADCs.
Effects of drug conjugation on antibody exposure and clearance
The conjugation of cytotoxic drugs to antibodies may affect the pharmacokinetic behavior of antibodies. Comparison of the total antibody PK of the ADC with that of the unbound antibody provided information on the effect of binding on antibody clearance (Figure 4). Binding can cause an increase in antibody clearance, which has been observed in several ADCs. Furthermore, conjugates with higher drug-to-antibody ratios (DARs) tended to clear faster than conjugates with lower DARs. Although the effect of increased clearance is not fully understood, it may lead to faster delivery of cytotoxic drug-carrying ADCs to normal organs or tissues, with potential toxicity.
Drug Loss Rate of ADC
Loss of ADC drug results in reduced efficacy and altered toxicity associated with ADC administration. To understand this phenomenon, evaluating the total antibody PK in comparison to the conjugated antibody PK or conjugated drug PK can provide qualitative guidance on the drug loss rate of ADCs. A theoretical plot of this comparison is shown in Figure 5. When comparing total antibody PK and bound antibody PK, it is generally observed that the concentration of bound antibody decreases more rapidly than the concentration of total antibody. The degree of deviation of the curve represents the loss rate of the cytotoxic drug of the ADC. The greater the difference between the PK of the bound antibody and the PK of the total antibody, indicating a faster rate of loss of the cytotoxic drug from the ADC.
Accumulation of unbound antibody
Unbound antibody concentrations (ADC antibodies without cytotoxic drugs) are rarely measured directly, but can be inferred from the concentrations of total and bound antibodies. Since many antibodies used in ADCs have little or no biological activity, the concentration of unbound antibody is rarely calculated.
ADCs are an emerging class of anticancer therapeutics that combine the antigen targeting and favorable pharmacokinetic properties of monoclonal antibodies with the cytotoxic activity of small-molecule chemotherapeutics. The complex structure of ADCs presents unique challenges to characterize the pharmacokinetics (PK) of drugs, as it requires a quantitative understanding of the PK properties of multiple different molecular forms such as ADC conjugates, total antibodies, and unconjugated cytotoxic drugs . Knowing the PK of an ADC, and thus its pharmacokinetic and pharmacodynamic (PK/PD) properties, is critical to the successful development of an ADC drug.