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Safe and effective drug targeting with ADCs requires careful selection of drug, antibody, and linker.
Antibody-drug conjugates (ADCs) are targeted therapies that combine a specific antibody or antibody fragment linked to a drug. This drug targeting approach has enabled better tumor penetration with less side effects in the treatment of cancer. Developing ADCs, however, requires careful selection of drug, antibody, and linker. David Simpson, CEO of Glythera, and Ian Evetts, commercial director of Glythera, spoke to Pharmaceutical Technology about the challenges involved in optimal ADC design.
Key considerations in ADC development
PharmTech: In ADC development, what are the key considerations when selecting the drug, antibody, and linker?
Glythera: ADCs are generally designed to incorporate highly potent, normally intolerable, anticancer cytotoxics; assuming, of course, that the resulting ADC construct is a stable entity with no de-drugging potential. De-conjugation of the drug–linker component from the antibody outside of the cancer cell is the main driver of an ADCs toxicity and adverse event profile. ADC stability is solely controlled by the linker attachment chemistry. With stable attachment chemistry, highly potent payloads can be selected with confidence, enhancing cell kill potential. Although there is also premise to combine antibodies to mid-potency agents, especially where the antibody itself has anti-tumor effects, the use of mid-potency payloads is generally uncommon amongst ADC innovators.
Drug selection. Preclinical studies aligned with commercial considerations suggest that criteria for the ideal cytotoxic should include:
The cytotoxic also should be chemically tractable and stable while bound to the monoclonal antibody (mAb). In addition, drugs must contain a suitable functional group for conjugation and need to be stable under physiological conditions. It is important that therapeutically appropriate amounts of the cytotoxic are linked to the mAb to optimize the potential effectiveness of the ADC once it reaches the target cancer cell.
In general, we should avoid drugs that are associated with neurotoxicity (e.g., platinum-based therapies or vinca alkaloids and first-generation taxanes).
The drugs currently being used to construct ADCs generally fall into two categories: microtubule inhibitors and DNA-damaging agents, although other drugs such as polymerase II inhibitors are also under investigation. Microtubule inhibitors bind tubulin, destabilize microtubules, and cause G2/M phase cell cycle arrest. DNA-damaging agents include anthracyclines, calicheamicins, duocarmycins, and pyrrolobenzodiazepines (PBDs). These drugs function by binding the minor groove of DNA and causing DNA stand scission, alkylation, or cross-linking. The cytotoxins are highly potent, with free drug IC50 of <10-9 M.
Choice of target antigen. Tolerability of an ADC is also affected by the specificity of antigen expression in cancerous tissue vs. normal tissue; non-specific expression results in toxicity and reduced efficacy due to a reduction in the dose of conjugate available to the tumor. Therefore, the ideal tumor antigen must be specifically localized to the tumor cell-surface to allow ADC binding and, preferably, display differential expression between tumor and normal tissue, with increased expression in cancer cells.
Antigen choice is also dictated by its ability to internalize upon ADC binding. Internalization of an ADC occurs through receptor-mediated endocytosis followed by ADC degradation in the lysosome, and this leads to free drug release for effective cell killing. However, endocytosis is not guaranteed for all cell-surface antigens, and the rate of internalization can vary considerably. Optimal drug release into the cell requires minimal recycling of the ADC to the cell surface as well as enhanced delivery of an internalized antigen/ADC to the lysosome. The ideal tumor antigen, then, should be cell-surface expressed, highly upregulated in cancer tissue, internalized upon ADC binding, and able to release the cytotoxic agent inside the cell.
Antibody selection. Of course, antibody selection also plays a critical part in the success of an ADC. Of a number of key attributes, high specificity for the tumor antigen is essential because an antibody that binds non-specifically or cross-reacts to other antigens can be taken up in normal tissues, inducing toxicity as well as elimination of the ADC before it can reach the tumor. Efficiency of uptake requires high affinity binding to the target antigen (KD < 10 nM), and antibodies should also be minimally immunogenic. Ideally, antibodies should also possess optimal pharmacokinetic properties including relatively long half-lives and slower clearance in plasma.
PharmTech: Why is linker chemistry important?
Glythera: We should clarify the role of chemistry of the linker and the chemistry used for attachment of the drug–linker adduct to the antibody. Linkers are generally selected based on the preferred method for drug release from the antibody (i.e., depending on whether cleavable or non-cleavable linkers are used). However, the attachment chemistry used to conjugate the drug–linker adduct to the antibody determines the overall stability of the ADC and is, therefore, crucial in maximizing efficacy and minimizing intolerability. So, although the selection of linker to attach to a drug is an important factor in the manufacture of an ADC, the ADC stability and, hence, clinical performance is wholly governed by selection of appropriate attachment chemistry to attach the drug–linker adduct to the antibody. Selecting the right linker and attachment chemistry can ensure maximum cell-killing activity by avoiding de-drugging and off-target tolerability.
ADC stability. Developed linkers have all suffered from stability issues largely due to use of maleimide chemistry for attachment of the drug–linker conjugate to the antibody, and reductions in drug: antibody ratios (DAR) over time have been shown for both cleavable and non-cleavable linker formats. In this situation, the drug–linker adduct is cleaved before entering the tumor cell, reducing the potency of the treatment and releasing the cytotoxic drug into systemic circulation.
Several groups have focused on enhancing the stability of ADCs through stabilizing conjugation, although most have concentrated on next-generation maleimide chemistry approaches, primarily via catalysis of ring hydrolysis. Through controlling the microenvironment around the succinimide thioether, stability of maleimide conjugation can be improved, because solvent accessible sites slow down retro-Michael reactions. If cysteine is close to positively charged entities, rapid succinimide hydrolysis occurs, preventing further linker-maleimide exchange. Also, modifying maleimide with the insertion of an amine group can shift the reaction through hydrolysis. However, these approaches don’t necessarily lead to the desired reduction in adverse events or to an increase in in-vivo efficacy, at least for cleavable linker systems, for which less obvious increases in exposure have had no real impact on ADC efficacy. Although ring-opening stabilizes the conjugate, the long-term stability of the ring-opened product is not precisely known. High pH conditions required for mild hydrolysis methods of stabilizing conjugation induce uncontrollable de-drugging and asparagine deamidation.
PharmTech: What recent advances have you seen in linking technologies?
Glythera: A recent development of linker systems has actually been focused on the conjugation platform with a need to improve stability due to significant advances in toxin potency and the potential for off-target toxicity. In addition, our improved understanding of the stoichiometry associated with maximal ADC efficacy has re-focused innovation to alternative conjugation solutions and, in particular, site-directed technologies.
A focus area for many major pharma companies has been to improve the tolerability of ADCs by reducing the de-drugging potential of highly potent toxins from the antibody, which results in free and active drug capable of circulating to alternative and off-target sites. Recent developments have included modification of existing maleimide-based technologies to reduce the potential for the retro-Michael reaction in vivo, to the development of fundamentally changed, novel chemistry solutions that no longer employ the maleimide component, resulting in significantly improved stability profiles and tolerability.
The ongoing innovation in toxin development has improved both potency and broad tissue type efficacy resulting in the increased use of engineered cysteines to overcome the instability issues associated with many of the approved platforms. In addition, we see the development of entirely novel approaches through alternative conjugation targets, many of which focus on the use of sugar-based conjugation to the antibody which, by definition, reduces the potential drug loading and, therefore, reduces the risks associated with high levels of free drug and tolerability.
PharmTech: Can you elaborate on the manufacturing challenges for ADCs?
Glythera: Bringing together biologics and toxic payloads has presented major challenges through the history of drug conjugates and more so now with the rapid expansion of target products entering human trials, which require cGMP manufacturing. Drug substance manufacturing to date has generally been achieved through the combined efforts of multiple partners for the production of both the biologics and toxin chemistry components as drug substance and then their combination safely in a single facility. As the clinical pipeline has grown, with more lead candidates requiring cGMP manufacturing, many contract manufacturing organizations (CMOs) have been quick to adapt to the market needs through expansion of their own biologics or chemistry capabilities with additional conjugation suites. However, as with all contract manufacturing models, the ability to undertake multiple manufacturing campaigns using different toxins and antibodies presents its own challenges in terms of control and segregation.
With the advent of increasingly potent cytotoxins used in the development of ADCs, facility design and the containment systems have required significant redesign to be able to cope with scale, demand, and operator safety. Working procedures have been a keen focus to reduce operator risk of exposure to these toxins through either inhalation or transdermal contamination. .