Antibody-drug conjugates and how their design shapes the drug
An antibody-drug conjugate joins a monoclonal antibody to a cytotoxic drug through a chemical linker, so that the antibody carries the drug to a tumour cell that bears its target antigen. How the conjugate reaches and kills that cell, and how one conjugate comes to behave differently from another, follow from five design choices: the target antigen, the antibody, the method of conjugation, the linker and the payload.
What an antibody-drug conjugate is and why it exists
Antibody-drug conjugates combine the specificity and targeting of monoclonal antibodies with the potent cytotoxicity of small-molecule drugs, and in 2023 they were among the fastest-growing drug classes in oncology, considered the "biological missiles" of cancer therapy. They are tripartite drugs with three key elements: a tumour-specific monoclonal antibody framework that binds selectively to an antigen on the tumour cell surface, a potent cytotoxic payload, and a chemical linker that attaches the two. Each component can vary widely, so the design is relatively complex, and subtle differences produce immense diversity in the overall structure and in the pharmacological and clinical properties that follow from it. A conjugate's clinical success depends on five factors: the target antigen, the antibody framework, the method of conjugation, the chemical linker and the cytotoxic payload. It is essential for medical communicators to have a basic understanding of each component of the design and of its potential effect on efficacy, on safety and on the ability to target particular degrees of antigen expression and tumour types.
Paul Ehrlich proposed the "magic bullet", the selective transport of cytotoxic drugs to a target tissue, in the early 1900s. Chemotherapy, developed in the 1940s, was the first major step from concept to reality. Its cytotoxic agents lacked high-level specificity and targeting, which caused a high degree of systemic toxicity and has remained a challenge. In the 1970s hybridoma technology and monoclonal antibodies gave an effective way of targeting antigens on the tumour cell surface. They led to targeted therapies, an attractive way of improving tumour selectivity and reducing the systemic toxicity of traditional chemotherapy. Combining chemotherapy with monoclonal antibodies produced the first antibody-drug conjugate. In recent decades the class has expanded rapidly, designed specifically to overcome the shortfalls of chemotherapy. Antibody-drug conjugates combine the precision of targeted therapy with chemotherapy-based strategies that use more potent cytotoxic agents.
How the conjugate reaches and kills the cell
The core concept is to use the specific recognition between antibody and antigen to deliver the cytotoxic drug selectively to the tumour site, where a release mechanism frees the payload, and the details vary with the design. In the general mechanism, the conjugate is given intravenously, recognises its target antigen on the tumour cell surface and binds it to form an antigen–conjugate complex, which the cell internalises by receptor-mediated endocytosis. With a cleavable linker, some payload may be released before internalisation, and inside the cytosol, endosome or lysosome the chemical conditions, such as low pH, high glutathione or thiol levels and proteolytic enzymes, release more. With a noncleavable linker, release typically requires complete degradation of the conjugate. The free payload then destroys the cell through a pathway-specific mechanism, such as microtubule disruption or DNA intercalation.
A hydrophobic or nonpolar payload can also cross cell membranes and diffuse from the target cell into neighbouring tumour cells. This bystander effect extends the antitumour activity to cells with low or no target antigen expression and to cells less accessible from the circulation, such as those of solid tumours. Solid tumours are a challenge both for drug access and because the target antigen varies within them, so designs that release or distribute drug to neighbouring cells with little or no antigen, such as cleavable linkers or nonpolarised payloads, may be an advantage against them.
Target antigen
The target antigen should be tumour-specific or tumour-associated, highly expressed in tumour cells and minimally or not at all in healthy tissue, which gives a favourable therapeutic index and lowers the risk of off-target toxicity. After the conjugate binds, the antigen should internalise efficiently by endocytosis, so that the conjugate enters the cell and the payload is transported and released. It should also be recycled or replenished efficiently on the cell surface without shedding into the circulation, which improves targeting and reduces the risk of toxicity. Ideally it is also expressed homogeneously in tumour cells and is minimally present in the circulation.
Antibody framework
The antibody framework provides the selective binding and can significantly influence efficacy, therapeutic index, pharmacokinetics and pharmacodynamics. Ideally it binds strongly and specifically with minimal cross-reactivity with healthy tissue, internalises efficiently, has low immunogenicity and has a long half-life. Of the five major antibody subtypes, IgA, IgD, IgE, IgG and IgM, IgG is used most often because of its plasma stability and its strong binding affinity for the Fc receptor. Humanised or fully human antibodies are typically favoured over murine or chimeric ones because they are less likely to provoke an immune response.
Method of conjugation
The method of conjugation is how the linker and payload are attached to the antibody, and it is either conventional, also called stochastic, or site-specific. Conventional conjugation, historically the most widely used, alkylates or acylates lysine side chains or reduces disulfide bonds to free cysteines as attachment sites. An antibody such as IgG has plenty of both, with 80 to 100 lysines and 32 to 40 cysteines. It is stochastic, so it yields heterogeneous mixtures with varying conjugation sites and drug-to-antibody ratios, the number of payload molecules per antibody, which often range from 0 to 8 or more. That variability can potentially affect hydrophobicity, charge, polarity, pharmacokinetics and thermostability, and in some cases leaves the conjugate unstable enough to release its payload prematurely, raising the potential for off-target toxicity. High ratios can also cause aggregation, faster metabolism or interference with the antigen-binding region. Lysines are spread across the heavy and light chains, so payload attached near the recognition sites could interfere with binding. Conjugates carrying payload on the heavy chains have lower efficacy in vivo than light-chain conjugates.
Site-specific conjugation was developed to make more homogeneous products with favourable pharmacokinetic and antigen-binding properties. Its methods generally fall into six kinds: engineered cysteines, disulfide rebridging, genetically encoded unnatural amino acids, enzyme-assisted ligation, glycan remodelling or glycoconjugation, and the pClick crosslinker. Engineered cysteines are commonly made with THIOMAB technology, which positions conjugation sites on both heavy and light chains and produces THIOMAB-drug conjugates shown to have improved safety and therapeutic indexes. In disulfide rebridging, the four interchain disulfide bonds of an IgG are reduced and treated with a cysteine-selective cross-linking reagent, which reattaches the chains while installing drug molecules or functional groups that may be further modified. Unnatural amino acids such as p-acetylphenylalanine and p-azidophenylalanine have shown advantages in optimising the conjugate's physical properties and in its efficacy, pharmacokinetic and safety profiles. They give superior in vitro selectivity and efficacy compared with conventional cysteine conjugates, particularly in tumour cells with low antigen expression. Enzyme-assisted ligation uses enzymes such as transglutaminase on genetically engineered sequences or tags. Glycan remodelling exploits the natural glycosylation site at residue N297 of the IgG CH2 domain, but it controls placement less well than the other site-specific methods. The newer pClick method uses a proximity-activated crosslinker that attaches to a specific site without further antibody engineering or post-synthesis treatment.
Linker
The linker bridges antibody and payload, and its chemistry greatly influences specificity, potency, safety and overall activity. It is designed to stay stable in the circulation and to release the payload once the conjugate reaches the tumour. Linkers fall into two main categories, cleavable and noncleavable.
Cleavable linkers exploit conditions specific to cancer cells and come in three kinds: acid-sensitive, glutathione-sensitive and enzymatically cleavable. Acid-sensitive linkers such as hydrazone stay stable at the blood's neutral pH and release the drug in acidic lysosomes or endosomes. Glutathione-sensitive or disulfide linkers are cleaved by the high levels of glutathione and other thiols in the cytosol of cancer cells. The enzymatically cleavable kinds include protease-sensitive peptide linkers, most often dipeptides such as valine-citrulline, which need enzymatic cleavage. Compared with the chemically labile kinds, such as hydrazone and disulfide linkers, peptide linkers give greater control of delivery, better systemic stability and rapid release inside the target cell. Besides peptide-based linkers, the enzymatically cleavable kinds include β-glucuronide-based and phosphate-based linkers.
Noncleavable linkers need internalisation through antigen-mediated endocytosis and lysosomal proteolytic degradation to free the payload. They generally bring greater plasma stability, a longer half-life and less risk of off-target effects. Their action is typically confined to the target cell. The amino acid–drug metabolites they release are also more hydrophilic and polar, which reduces the bystander effect. Common noncleavable linkers are the thioether linker known as SMCC and the maleimide linkers maleimidopropionyl and maleimidocaproyl.
Payload
The payloads are typically 100 to 1,000 times more potent than conventional chemotherapy and fall into two main groups: DNA-damaging agents, which include calicheamicins, duocarmycins, pyrrolobenzodiazepines and camptothecin analogues, and anti-tubulin agents, which include auristatins, maytansinoids and tubulysins.
Calicheamicins such as ozogamicin, isolated in the mid-1980s from the actinomycete Micromonospora echinospora, bind the minor groove of DNA and cause site-specific double-strand breaks, and their high hydrophobicity limits how many can be attached to each antibody. Duocarmycins are potent alkylating compounds from Streptomyces zelensis that, with derivatives such as duocarmazine, alkylate adenine in the minor groove, which leads to strand cleavage and apoptosis. Pyrrolobenzodiazepine dimers such as tesirine derive from anthramycin, an antitumour antibiotic isolated from Streptomyces. They bind the minor groove and form covalent interstrand crosslinks that lead to cell death, and the crosslinks form rapidly and with minimal distortion of the DNA, which is thought to help them persist and evade repair. Camptothecin is a pentacyclic alkaloid from the stem wood of Camptotheca acuminata, a tree native to China, and its derivatives such as govitecan and deruxtecan inhibit topoisomerase I, causing double-strand breaks and cell death.
The auristatins monomethyl auristatin E and F are synthetic analogues of dolastatin 10 from the sea hare Dolabella auricularia, and they inhibit tubulin polymerisation at the same binding site as the vinca alkaloids, such as vincristine and vinblastine, used in conventional chemotherapy. Maytansinoids are benzoansamacrolides from the bark of the African shrub Maytenus ovatus that bind at or near the vinca site to inhibit microtubule assembly and arrest mitosis, and those commonly used in conjugates include two thiomethyl derivatives of maytansine, mertansine and ravtansine. Tubulysins are more recently discovered cytostatic peptides from the myxobacteria Archangium gephyra and Angiococcus disciformis that inhibit microtubule polymerisation during mitosis and induce apoptosis. Tubulysin A is one such payload. Immunomodulators, such as toll-like receptor and STING agonists, are a further class of payload.
Approved conjugates and clinical development
The first antibody-drug conjugate was approved in 2000, and technological advances since have led to an explosion in the number under clinical development. As of 2022, 12 conjugates with 9 associated biomarker targets had been approved for solid tumours and haematological malignancies. In 2023 more than 80 were in clinical trials, which suggests their use will keep increasing. In 2023, 13 had US Food and Drug Administration approval for haematological and solid tumours. In the five years to 2023, 39 clinical trials investigated more than 19 conjugates.
Trastuzumab deruxtecan, a conjugate directed at human epidermal growth factor receptor 2 (HER2), was investigated across solid tumours in the DESTINY trials, which led to approvals in HER2-positive and HER2-low metastatic breast cancer and in gastric or gastroesophageal cancer, and to an accelerated approval in non-small cell lung cancer. Its data in metastatic breast cancer were unprecedented and transformative, and they have played a major part in reshaping how HER2 expression in tumours is assessed and managed. Immunohistochemistry had classified tumours as HER2-positive or HER2-negative. Early HER2-targeted therapies worked only in HER2-positive metastatic breast cancer. This conjugate's design and the bystander effect that follows from it have extended responses to patients with lower levels of HER2 expression.
As cancer incidence rises worldwide, demand for safer, more personalised cancer therapies with fewer side effects will drive further advances in the technology. Because these treatments are being introduced quickly, the need for clear and accurate communication about them will keep growing, and medical communicators must educate themselves and their readers so that readers can approach the literature critically and make informed decisions about the drugs' use.