Antibody-drug conjugates (ADC) are formed by linking monoclonal antibodies that target specific antigens with small molecule cytotoxic drugs through linkers, combining the powerful killing effect of traditional small molecule chemotherapy with the tumor targeting ability of antibody drugs. Since the first ADC (Gemtuzumab-ozogamicin, trade name: Mylotarg) was approved for the treatment of CD33-positive acute myeloid leukemia, dozens of ADCs have been developed and approved for cancer treatment.
The entire development process of ADCs, from selecting the appropriate antibody to the final product, is a challenging task. Clinical pharmacology is one of the most important tools in drug development, helping to find the optimal dosage of products to maintain safety and efficacy in patient populations. Unlike other small or large molecules that typically measure one part and/or metabolite for pharmacokinetic analysis, ADCs require the measurement of multiple components to characterize their PK properties. Therefore, a deep understanding of the clinical pharmacology of ADCs is crucial for selecting safe and effective doses in patient populations.
Overview of ADC Pharmacokinetics
Pharmacokinetics is an indispensable part of clinical pharmacology and modern drug development. The main purpose of pharmacokinetics studies is to obtain information about the drug’s absorption, volume of distribution, clearance rate, half-life, accumulation after multiple doses, and the influence of various disease states as well as age, weight, and gender on drug pharmacokinetics. 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 (antibodies or fusion proteins), the PK of ADCs is very complex because ADCs consist of several components. Not only must the PK of the monoclonal antibody be considered, but also the PK of the cytotoxic molecule and the physicochemical properties of the conjugate. Since the molecular weight of the monoclonal antibody accounts for more than 90%, the PK of the different components of ADCs is greatly influenced by its PK. The overall PK characteristics (ADC + mAb) provide the best assessment of the stability and integrity of ADCs. The conjugate and conjugation sites also play important roles in maintaining the stability and PK of ADCs. The table below lists the FDA-approved ADCs and their PK characteristics.
Pharmacokinetic Characteristics of ADCs
Generally, four processes are involved in drug administration: absorption, distribution, metabolism, and elimination.
Most antibodies are usually administered intravenously or by infusion, and antibodies can also be administered subcutaneously (SC). However, for ADCs, the current administration route is intravenously. Due to the response to the cytotoxic payload and local deposition of cytotoxic substances, SC administration may not be suitable for ADCs.
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 typically limited to vascular and interstitial spaces.
The initial distribution of ADCs is generally confined to the vasculature, with a volume of distribution typically equal to blood volume. Subsequently, ADCs can distribute into interstitial spaces. Furthermore, the distribution of ADCs can also be affected by target antigen expression and endocytosis.
The distribution and accumulation of ADCs in the same tissue can lead to adverse (toxic) pharmacological effects due to the release of cytotoxic drugs or metabolites after the uptake of ADCs.
The breakdown/metabolism process of ADCs in the body includes the metabolic processes of the antibody and the small molecule drug. Before reaching tumor cells, ADCs release active molecules either intracellularly (non-cleavable linker) or in the circulation (cleavable linker). Unbound antibodies and antibody fragments follow the metabolic pathways of antibodies, producing amino acids through enzymatic hydrolysis that can be reused by the body.
Free small molecule drugs and/or small molecule drug metabolites linked to amino acid residues and/or linker may further undergo hepatic CYP450 enzyme metabolism and may also undergo potential drug-drug interactions.
In addition to the properties of the ADC itself, the expression of antigens, receptor/cell density, FcRn-mediated circulation, Fcγ interactions, receptor-mediated endocytosis, and immunogenicity can also affect the breakdown and metabolism of ADCs.
ADCs are also eliminated through breakdown/metabolism and excretion. ADCs can enter lysosomes through specific pathways that bind to targets, undergo degradation, and release small molecule drugs for elimination from the body; they can also be cleared through non-specific pinocytosis, a process involving the recycling process mediated by the neonatal receptor (FcRn).
ADCs, antibodies, larger peptides, and amino acid fragments cannot be excreted through glomerular filtration but are reabsorbed in the form of amino acids. Free small molecule drugs, smaller peptides, and small molecule drugs linked to amino acids, as well as smaller antibody fragments can be excreted through glomerular filtration. Meanwhile, small molecule drugs and metabolites can also be eliminated through enzymatic metabolism or excreted into feces via transporters.
ADCs have several components, and to characterize the PK characteristics of these components, several analytical methods are required, as follows:
• ELISA immunoassays to determine the kinetic curves of bound and total antibodies;
• TFC-MS/MS for quantifying free drugs/metabolites;
• High-resolution mass spectrometry for in vivo drug-antibody ratio (DAR) analysis.
In addition, two types of ELISA immunoassays are used to quantitatively measure the analytes of ADCs: the first type measures total antibodies, i.e., ADCs with DAR greater than or equal to zero. The second type measures drug-bound antibodies, defined as ADCs with DAR greater than or equal to 1.
Other analytical methods include size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC). SEC is the most commonly used liquid chromatography (LC) technique for determining the aggregation of antibodies, and this technique can also be used for ADCs. Although HIC is a traditional technique for protein separation, purification, and characterization, it is now being used for ADC characterization and analysis.
ADC cytotoxic payloads should have the following characteristics:
• The cytotoxic payload should have appropriate lipophilicity.
• The target of the payload should be located inside the cell.
• The molecules of the payload should be of small size, lack immunogenicity, and be soluble in buffered aqueous solutions for easy conjugation.
• The payload should be stable in the blood.
Currently, commonly used cytotoxic drug effect molecules include microtubule inhibitors (e.g., auristatins, maytansinoids), DNA damaging agents (e.g., calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers), and DNA transcription inhibitors (Amatoxin and Quinoline alkaloid (SN-38)). Several approved ADC drugs use a total of six different small molecule drugs, three of which use MMAE as the conjugate, two use Calicheamicin as the conjugate, and others successfully applied include MMAF, DM1, SN-38, and Dxd.
Drug-Antibody Ratio (DAR)
The drug-antibody ratio (DAR) refers to the average number of payload molecules attached to a single monoclonal antibody, typically ranging from 2 to 4 molecules. In rare cases, using hydrophilic linkers, payloads can safely achieve DARs of up to 8, as seen in Enhertus and Trodelvys. DAR is crucial for determining the efficacy of ADCs; furthermore, DAR may affect drug stability in circulation, PK, and the toxicity of ADCs.
Studies have shown that ADCs with DAR values < 6 clear more slowly and have reduced in vivo efficacy compared to those with higher DAR values (7 to 14). The impact of DAR on stability and PK also depends on the conjugation site and the size of the linker.
Lysine or cysteine is typically modified to produce ADCs. Lysine is one of the most commonly used amino acid residues for linking substrates and antibodies, as it is often present on the surface of antibodies, making it easy to conjugate. Mylotargs, Kadcylas, and Besponsas all utilize lysine bioconjugation techniques.
Other amino acids such as cysteine and tyrosine can also be modified, with maleimide-modified cysteine used to synthesize ADCs like Adcetriss, Polivys, Padcevs, Enhertus, Trodelvys, and Blenreps.
Linkers are an indispensable part of ADCs, determining the drug release mechanism, PK, therapeutic index, and safety of ADCs. Early ADC linkers were chemically unstable, such as disulfides and hydrazones. These linkers were unstable in circulation, with short half-lives typically ranging from one to two days. The latest generation of linkers is more stable in systemic circulation, such as peptide and glucuronic acid linkers. The two most common linkers are as follows:
Cleavable linkers are sensitive to the intracellular environment, releasing free effect molecules and antibodies through breakdown and dissociation in the cell, such as acid-cleavable linkers and protease-cleavable linkers. They are usually stable in blood but rapidly cleave in low pH and protease-rich lysosomal environments, releasing effect molecules. Additionally, if effect molecules can cross membranes, they may eliminate tumors through potential bystander effects.
Non-cleavable linkers are a new generation of linkers that provide better plasma stability than cleavable linkers. Because non-cleavable linkers can provide greater stability and tolerability than cleavable linkers, these linkers reduce off-target toxicity and provide a larger therapeutic window.
In 11 clinical trials targeting 8 ADCs, the baseline incidence of ADAs ranged from 1.4% to 8.1%, and the incidence of post-baseline ADAs ranged from 0 to 35.8%, values that fall within the range of therapeutic monoclonal antibodies. Overall, the incidence of ADAs for ADCs is lower in patients targeting hematological malignancies than in those targeting solid tumors; most ADAs are directed against the monoclonal antibody domain of ADCs. Furthermore, in most patients, the half-antigen-like structure of these ADCs does not pose a higher risk of immune response compared to therapeutic monoclonal antibodies.
ADC Pharmacokinetic Models
The application of modeling methods can integrate PK, efficacy, and safety data to meet the needs of different stages of ADC drug development, such as target selection, antibody affinity, linker stability, animal-to-human extrapolation, dose selection and adjustment, exposure-response relationships studies, DDI studies, etc. Due to the multiple clearance pathways of ADCs (dissociation and breakdown) and the complex PK characteristics of various analytes, their kinetic models are also complex.
Different models have different applications; for example, a two-compartment model and a PBPK model can describe the stability characteristics of ADCs using parameters such as clearance rate, dissociation, and metabolism rate. Currently, non-compartmental models, population pharmacokinetic models, mechanistic models, and physiological models have all been applied in ADC pharmacokinetic studies.
In the development process of ADC drugs, clinical pharmacology plays a very important role. Through continuously evolving bioanalytical techniques, a comprehensive understanding of the PK/PD characteristics of ADC drugs is crucial for promoting the development of low-toxicity and high-efficacy ADC drugs. ADC drugs are expected to demonstrate even greater advantages in the field of cancer treatment.
References:
1. Clinical Pharmacology of Antibody-Drug Conjugates. Antibodies (Basel). 2021 May 21;10(2):20.
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