https://orcid.org/0000-0002-9896-4654
Abstract 摘要
Antibody drug conjugates are an exciting therapeutic modality that combines the targeting specificity of antibodies with potent cytotoxins to selectively kill cancer cells. The targeting component improves efficacy and protects non-target cells from the harmful effects of the payload. To date 15 ADCs have been approved by regulatory agencies for commercial use and shown to be valuable tools in the treatment of cancer.
抗体药物偶联物是一种令人兴奋的治疗方式，它将抗体的靶向特异性与强效细胞毒素相结合，以选择性地杀死癌细胞。靶向成分提高了疗效，并保护非靶细胞免受药物负荷的有害影响。迄今为止，已有 15 种抗体药物偶联物获得监管机构的批准用于商业用途，并被证明是治疗癌症的宝贵工具。The assembly of an ADC requires the chemical ligation of a linker-payload to an antibody. Conventional conjugation methods targeting accessible lysines and cysteines have produced all the ADCs currently on the market. While successful, technologies aiming to improve the homogeneity and stability of ADCs are being developed and tested.
ADC 的组装需要将连接子-药物通过化学连接与抗体结合。传统的结合方法针对可接触的赖氨酸和半胱氨酸，已生产出目前市场上所有的 ADC。尽管成功，但旨在提高 ADC 的均一性和稳定性的技术正在开发和测试中。Here we provide a review of developing methods for ADC construction. These include enzymatic methods, oligosaccharide remodelling, and technologies using genetic code expansion techniques. The virtues and limitations of each technology are discussed.
在这里，我们提供了关于抗体-药物偶联物（ADC）构建方法发展的综述。这些方法包括酶法、寡糖重塑以及使用基因编码扩展技术的技术。讨论了每种技术的优点和局限性。Emerging conjugation technologies are being applied to produce new formats of ADCs with enhanced functionality including bispecific ADCs, dual-payload ADCs, and nanoparticles for targeted drug delivery. The benefits of these novel formats are highlighted.
新兴的结合技术正在被应用于生产具有增强功能的新型抗体药物偶联物（ADCs），包括双特异性 ADCs、双载药 ADCs 和用于靶向药物递送的纳米颗粒。这些新型格式的优势得到了强调。
Introduction 引言
Historically, the more we treat a medical condition the better we become at curing it; but there is a learning process, trial and error, and experimentation that leads to better understanding of disease fundamentals and felicitous treatment. As we become more successful, we also become motivated to try new techniques, optimise dosing, and invent new technologies. However, development of cancer drugs has been impeded by the diverse and heterogeneous nature of this conglomeration of diseases. Current treatments address the one commonality amongst them, their accelerated growth. Thus, chemotherapy drugs that kill rapidly growing cells were developed, but due to systemic administration these drugs have side-effects that impact non-cancerous fast-growing cells like skin, hair follicles and digestive tract, and have a debilitating impact on patients. Chemotherapy remains the first line treatment for most patients due to its long historical use and track record. But in 2000, the U.S. Food and Drug Administration (FDA) approved a new cancer therapy, an antibody drug conjugate (ADC) (Mylotarg, Pfizer/Wyeth) that combined the targeting specificity of an antibody (anti-CD33) with a potent cytotoxin (calicheamicin) (Naito et al. Citation2000; Yu and Liu Citation2019). By targeting specific cells, this drug aimed to improve efficacy, as well as protect non-target cells from the harmful effects of the cytotoxin. These properties would enable 1) the use of highly toxic payloads that were not tolerable as free chemotherapies, but effectively kill tumour cells, and 2) lower dosing regiments to improve patient tolerability while retaining anti-tumour activity. But, in its post-approval study, Mylotarg showed no survival benefits over conventional treatment along with high liver toxicity, and the drug was voluntarily withdrawn in the U.S. in 2010. Nevertheless, the concept of an ADC is compelling, and hundreds of ADCs have been built, studied, and tested. Since Mylotarg’s first approval, 15 ADCs have been approved by the FDA for the treatment of both solid and hematological cancers (, Sasso et al. Citation2023)—including Mylotarg which returned to the market in 2017 with a lower suggested dose and a revised dosing schedule—and over 100 ADCs are currently in clinical trials, with more than 200 ADCs in preclinical development.
历史上，我们对医疗状况的治疗越多，治愈的能力就越强；但这需要一个学习过程，包括试验和错误，以及实验，这些都能更好地理解疾病的基本原理和有效的治疗方法。随着我们变得越来越成功，我们也会受到激励去尝试新技术、优化剂量和发明新技术。然而，癌症药物的开发受到这种疾病群体多样性和异质性的阻碍。目前的治疗方法针对它们之间的一个共同点，即加速生长。因此，开发了能够杀死快速生长细胞的化疗药物，但由于系统性给药，这些药物对非癌性快速生长细胞（如皮肤、毛囊和消化道）产生了副作用，并对患者造成了严重影响。由于其悠久的历史和良好的记录，化疗仍然是大多数患者的首选治疗方法。但在 2000 年，美国。 食品和药物管理局（FDA）批准了一种新的癌症治疗方法，即抗体药物偶联物（ADC）（Mylotarg，辉瑞/惠氏），该药物结合了抗体（抗 CD33）的靶向特异性和强效细胞毒素（卡利克霉素）（Naito 等，2000；Yu 和 Liu，2019）。通过靶向特定细胞，该药物旨在提高疗效，同时保护非靶向细胞免受细胞毒素的有害影响。这些特性使得 1）可以使用作为自由化疗无法耐受的高毒性药物载荷，但有效杀死肿瘤细胞，以及 2）降低剂量方案以提高患者耐受性，同时保持抗肿瘤活性。然而，在其批准后的研究中，Mylotarg 未显示出比常规治疗更好的生存益处，并且伴随有高肝毒性，该药物于 2010 年在美国自愿撤回。尽管如此，ADC 的概念仍然引人注目，已有数百种 ADC 被构建、研究和测试。自 Mylotarg 首次获批以来，FDA 已批准 15 种 ADC 用于治疗实体瘤和血液肿瘤（表 1，Sasso 等）。 2023 年）—包括在 2017 年以较低建议剂量和修订给药方案重新上市的 Mylotarg—目前有超过 100 种抗体药物偶联物（ADCs）正在进行临床试验，超过 200 种 ADCs 处于临床前开发阶段。
Table 1. FDA approved ADCs.
What we have learned over two decades of research, is that each component of an ADC (the antibody, linker, and payload) confers pharmacokinetics and biodistribution properties that provide more on-target activity, but also a concomitant exposure to the rest of the body that results in off-target toxicities (Colombo and Rich Citation2022; Sasso et al. Citation2023). To expand the therapeutic window, much research has been devoted to the development of cleavable and non-cleavable linkers that improve the stability, hydrophobicity, and specific release of the cytotoxic payloads. Ideally, linkers are stable in circulation and release payload in cancer cells. To achieve this dual functionality, linkers with diverse release triggers have been developed including pH sensitive linkers, enzyme cleavable linkers (i.e. specifically cleaved by cathepsins, glycosidases, or sulfatases), and photo-sensitive linkers. The unique properties of these linkers convey functionality to ADCs, but an in-depth analysis of these is beyond the scope of this review (Su et al. Citation2021). In addition, a collection of different cytotoxins with various mechanisms of action and potencies have been used to reduce off-target effects (Beck et al. Citation2017). A critical consideration when designing an ADC is how protein engineering and the chemistry of conjugation can determine the individual components of the conjugate, and consequently, affect its overall stability, PK, efficacy, and safety. Here we discuss advances in protein engineering strategies and emerging technologies that are being developed to improve the functional properties of ADCs. This includes the maturation of conjugation technologies that enable: (1) the construction of more stable, site-selective, and homogenous ADCs, (2) the development of bispecific ADCs to improve specificity, and (3) the re-emergence of radio-immunoconjugates. Additionally, we discuss (4) how functionalization of nanoparticles with targeting agents improves delivery of therapeutics to tumour cells. Targeted nanomedicines display unique biodistribution and retention properties and have high carrying capacities for payloads that are a departure from classical antibody-based delivery methods. All new methods have their advantages and challenges, these will be discussed.
Conventional conjugations and the rise of site-specific ADCs
An obligatory step in ADC design is choosing a strategy for covalently attaching a small molecule drug to the targeting antibody. The most common methods for ADC generation are the stochastic conjugations to native cysteines or lysines. Both methods have been used successfully but generate heterogenous drug loading distributions with respect to both site and number of toxins. Amine coupling is used for the generation of Trastuzumab emtanzine, Inotuzumab ozogamicin, Gemtuzumab ozogamicin and Mirvetuximab soravtansine (see references in ). But, with 40 accessible lysine residues a wide range of drug to antibody ratios can be generated. In contrast, four interchain disulphide bonds of an IgG1 can be reduced to expose the thiol groups of cysteines that can be covalently linked to a variety of electrophilic handles, most commonly to maleimide-bearing synthetic payloads via Michael addition. Depending on the reduction conditions, and payload stoichiometries, ADCs with drug-to-antibody ratios (DAR) of 2,4,6 and 8 are produced. Using the same chemistry, engineered cysteines can be encoded at desired positions and selectively targeted to enable more controlled DAR and orientation of the payload. This technology, used for Loncastuximab tesirine (DAR2) enables a more controlled conjugation product and homogeneous ADC. Optimal drug loading depends in part on the potency of the cytotoxin and the safety profile observed. Low or reduced DARs are preferred for more potent cytotoxins like Pyrrolobenzodiazepine (PBD) in Loncastuximab tesirine (DAR2), and monomethyl auristatin E (MMAE) used in Brentuximab vedotin and Enfortumab vedotin (DAR4). Less potent warheads like the exatecan derivatives tend to require higher drug loads to achieve efficacy. For example, Trastuzumab deruxtecan and Sacituzumab govitecan, both contain eight payloads. However, many factors influence the final drug load choice including receptor density and numbers, antibody properties, drug hydrophobicity, linker chemistry, ADC stability and clearance. Optimal DARs can vary within the same drug class, for example Datopotamab deruxtecan, a Trop-2 targeted ADC for use in locally advanced or metastatic nonsquamous non-small cell lung cancer (biologics licence application pending regulatory decision December 2024), contains the same payload used for Trastuzumab deruxtecan, but is produced with four cytotoxins (Okajima et al. Citation2021). In each case the goal is to maximise both on-target efficacy as well as tolerability. This window, known as the therapeutic index, provides physicians the boundaries for treatment options.
A variable in stochastic ADCs is that any two conjugates with the same DAR are likely regioisomers due to attachment at different sites. The heterogeneity of the population hinders the safety and therapeutic index of ADCs, as each conjugate differs in important properties such as pharmacokinetics, toxicity, propensity to aggregate, and drug release (Hamblett et al. Citation2004; Pillow et al. Citation2014; Lyon et al. Citation2015; Hafeez et al. Citation2020). This results in antibodies bearing suboptimal toxin loads that compete for target engagement and the generation of free toxins that distribute indiscriminately into healthy tissues exacerbating off-target toxicities. In addition, thiol-maleimide Michael addition can undergo the reverse reaction (retro-Michael addition) that is position dependent (Alley et al. Citation2008; Shen et al. Citation2012; Ohri et al. Citation2018), causing premature release of payload and transfer to free thiols (like that found in Albumin and glutathione) and can lead to off-target toxicity (Wei et al. Citation2016). At some sites this can be prevented with a procedure (or by using self-stabilising linkers) that results in the hydrolysis of the succinimide generating more stable adducts that are not susceptible to retro-Micheal addition (Lyon et al. Citation2014; Tumey et al. Citation2014; Christie et al. Citation2015; Dovgan et al. Citation2016). Homogeneous ADCs can be made through stochastic conjugations by targeting all the interchain cysteines and developing ADCs with a DAR of 8. This method disrupts structurally important interchain disulphides but produces ADCs with higher drug loads that show improved in vitro efficacy, but surprisingly less in vivo activity. This loss of activity is attributed to their faster clearance due to increased hydrophobicity (Hamblett et al. Citation2004). Interestingly, a thiol-bridge method using bifunctional drug-linkers to crosslink interchain cysteines was developed to generate homogenous ADCs with a DAR4 (Behrens et al. Citation2015; Bryant et al. Citation2015). This method restores the interchain connections increasing antibody stability and resulting in higher efficacy, improved pharmacokinetics, and tolerability in vivo compared to conventional conjugates. But, despite significant drawbacks most of the approved ADCs make use of stochastic conjugation strategies. The advantages of this conventional conjugation method are the ease of antibody expression, the use of unaltered antibody sequences, and the fast conjugation kinetics (thiol maleimide chemistry has a reaction rate constant of 500 M−1s−1) (Christie et al. Citation2015) which greatly facilitate manufacturing.
To produce more consistent and homogeneous ADCs investigators developed site-specific conjugation methods, like ThiomAb (Junutula et al. Citation2008; Shen et al. Citation2012), that target engineered cysteines at user defined positions. The conjugation process requires three steps: a reducing step to disrupt disulphide bonds and uncap free cysteines, an oxidation step to allow the reformation of interchain disulphides, and finally the conjugation to the engineered cysteines. This approach retains the interchain disulphide bridges of the native antibody improving stability. More importantly, it offers a method to strategically position the conjugation site to improve the hydrophobicity and stability of the conjugate. One site, S239C, was identified as a preferred position for thiol-maleimide conjugation as it enables efficient adduct formation and produces potent ADCs with enhanced conjugate stability (Sutherland et al. Citation2013; Thompson et al. Citation2016; Sussman et al. Citation2018). This site, however, produces ADCs with relatively high hydrophobicity and enhanced clearance, suggesting that the site of conjugation may be important to controlling the exposure of the payload. A systematic analysis of positional cysteine variants highlighted that efficacy of conjugation and stability are positionally dependent, and relatively few sites provide desirable ADCs (Ohri et al. Citation2018). Interestingly, engineered cysteines have been identified that allow direct conjugation without reduction-oxidation potentially simplifying the manufacture of site-specific ADCs (Shinmi et al. Citation2016). The substitution of residue Q124 with a cysteine in the antibody light chain exhibited high thiol reactivity and conjugate formation. Although the conjugate was subject to retro- Michael addition, hydrolysis of the succinimide produced stable ADCs. In the remainder of this article we will describe novel conjugation methods that are being used to construct site-specific ADCs with improved functional properties. These emerging technologies provide alternative conjugation chemistries, that overcome some of the limitations observed with thiol-maleimide conjugates and produce more precise, stable ADCs.
Recent advances in enzymatic conjugations towards ADC generation
Many site-specific approaches have been proposed to generate ADCs of uniform DAR with payloads attached to defined locations. Apart from commonly used means for producing homogenous ADCs, such as cysteine engineering, chemoenzymatic approaches towards homogeneous ADC production have also recently emerged. Indeed, the high specificity and activity of enzymes make them attractive tools for this purpose as evidenced by the growing number of ADCs entering the clinic which involve the use of enzymes in their production (vide infra). Furthermore, the inherent capability of enzymes to catalyse reactions in aqueous solutions and eco-friendly conditions make them ideal for large-scale production of drug substances. Despite these advantages, some challenges exist when considering chemoenzymatic approaches. First, producing large amounts of high-quality and active enzymes for manufacturing purposes can be costly and labour-intensive. Furthermore, the introduction of a recognition sequence in the target antibody that is enzyme-accessible requires engineering of the antibody sequence and could require extensive screening effort to identify an optimal site. Each conjugation method also comes with its own unique set of challenges. Nevertheless, the capabilities of enzymes have made them powerful tools for producing ADCs that enter the clinic. Here, we will provide an overview of chemoenzymatic approaches towards clinically relevant homogenous ADCs and the advantages and drawbacks of each strategy.
Transglutaminase
Transglutaminases (TGs) represent a class of isopeptide bond-forming enzymes that are endogenous to both prokaryotic and eukaryotic species. In nature, transglutaminases form cross-linkages between a wide variety of protein substrates to form biological structures such as hair, skin, and blood clots (Griffin et al. Citation2002). The crosslinking activity of transglutaminases is derived from the active site, consisting of a catalytic triad of Cys, His, and Asp amino acid residues. The net reaction involves a transglutaminase-mediated linkage formed between an acyl donor (typically the side chain amide from a Gln residue) and an acyl acceptor consisting of a primary amine resulting in an isopeptide linkage (). This reaction is facilitated via formation of a thioester intermediate formed between the Cys within the TG active site and the acyl donor, which is susceptible to nucleophilic attack by the amine substrate giving the final product (Oteng-Pabi et al. Citation2014). Transglutaminases originating from eukaryotic species require Ca2+ ions and GTP as cofactors for this reaction, while microbial transglutaminases do not have this requirement. For this reason, transglutaminases from the microbial organism Streptomyces mobaransis represent the majority of industrially relevant TGs used today (Strop Citation2014).
Figure 1. Overall scheme depicting the isopeptide-bond forming reaction between an acyl donor (blue) with a primary amine donor (orange) catalysed by transglutaminase.
The substrate promiscuity of transglutaminases extends the versatility of these enzymes to a wide range of applications. Numerous primary amine donors are suitable for transglutaminase-mediated coupling, showcasing the substrate scope accessible with this technology (Ohtsuka et al. Citation2000a). On the other hand, transglutaminases show somewhat more stringent requirements for the environment surrounding the acyl donor compared to the amine substrate. More specifically, the acyl donor must consist of a Gln surrounded by specific residues, particularly hydrophobic amino acids, and must reside within a loop region with local chain flexibility (Ohtsuka et al. Citation2000b). Owing to this substrate promiscuity, transglutaminases have most frequently been applied to the food industry where they are used to catalyse the crosslinking of meats to enhance flavour, extend shelf life, and improve food texture quality; hence these enzymes have been aptly referred to as ‘meat glue.’ Transglutaminases are also heavily involved in the textile industry, as they perform useful functions such as restoring the strength of fabrics after chemical processing and filling the voids of leather fibres to increase smoothness (Duarte et al. Citation2019).
Figure 2. Schematic overview for the generation of R927C (PF-0664178). A Trop2-targeting mAb with an engineered Q-tag (LLQGA) located at the heavy-chain C-terminus is conjugated to the amine-containing synthetic payload, PF-06380101 (orange) via transglutaminase-mediated ligation to afford a DAR 2 ADC.
The versatility of TG in coupling a wide range of substrates sparked interest in its use towards generating site-specific ADCs by ligating amine-bearing payloads to glutamine side chains of an antibody, a convenient strategy considering the ease in which primary amines can be incorporated into synthetic payloads and the ability of TG to only recognise specific acyl donors. Early works demonstrated that a single glutamine residue (Q295) was suitable for this purpose, however, conjugation could only occur when the glycan moiety residing at N297 was absent, limiting this application to deglycosylated antibodies only (Jeger et al. Citation2010). This limitation can be considered unfavourable, as antibodies lacking the glycan moiety show less stability compared to their glycosylated counterparts (Zheng et al. Citation2011). To expand the number of Gln sites accessible by TG, a team from Pfizer consisting of Strop and others screened a variety of engineered antibodies containing glutamine tags (Q-tags) consisting of a four-residue peptide, LLQG, in their primary sequences to determine whether TG-catalysed conjugation could be directed to other locations (Strop et al. Citation2013). These efforts revealed that the Q-tag could be selectively conjugated with lysine-derived payloads at or near completion when placed at 12 of the 90 total screened sites spanning all constant Fab and Fc domains. Further characterisation revealed that the site of conjugation significantly impacted pharmacokinetics, particularly clearance rates in rodents, attributed to varying degrees of proteolytic degradation of Val-Cit linkers.
The Q-tag approach led to the development of R927C, an ADC featuring anti-Trop2 monoclonal antibody (mAb) featuring the Q-tag, LLQGA, at the C-terminus of the heavy-chain (). The mAb is conjugated to a linker-payload featuring a AcLys-VC-PABC linker and a Dolastatin 10 analogue (PF-06380101) as a warhead which is attached to the engineered Q-tag via transglutaminase-mediated ligation. Auristatins, such as PF-06380101, are structurally based on Dolastatin 10, a natural product produced by the cyanobacteria Dolabella auricularia, which can inhibit tubulin polymerisation and tubulin dependent GTP hydrolysis, making them attractive molecules for use in cancer therapy (Maderna et al. Citation2014). Single doses of R927C at 0.75 − 1.5 mg/kg showed TROP-2-dependent tumour inhibition or regression in pancreatic, lung, ovarian, and breast cancer models, which was more efficacious than the standards of care, including paclitaxel and gemcitabine (Strop et al. Citation2016). Safety studies conducted in primates with doses up to 6 mg/kg showed reversible on-target epithelial toxicities, a finding that was surprising considering that peripheral neuropathy and other off-target effects are often associated with mAbs conjugated to MTIs, such as MMAE, due to loss of payload. The improved safety profile of R927 may be attributed, in part, to enhance ADC stability afforded by the peptide linkage, or may be due to slight differences in potency between the MMAE and Dolastatin-10 payloads. Based on these positive preclinical results, R927C progressed into phase I clinical trials (NCT02122146); however, the study was terminated due to toxicity concerns. Doses above 2.4 mg/kg led to rash, mucosal inflammation, and neutropenia. Furthermore, R927C showed only modest antitumor activity and no correlation between efficacy and TROP-2 expression.
Formylglycine-generating enzymes
Expanded amino acid chemistries have been demonstrated to provide unique handles for site-specific conjugation. Apart from genetic code expansion technologies capable of site specifically encoding non-natural amino acids (topic covered below), other means of achieving unique functional groups beyond the scope of the 20 natural amino acids includes the modification of natural amino acids post-translationally. One prominent example of this technology includes the use of formylglycine-generating enzymes that convert a single Cys residue within a recognition sequence to an aldehyde-bearing formyl glycine residue (FGly) via oxidation of the thiol side chain. The archetypical and ubiquitous formylglycine-generating enzyme (FGE) which utilises a pair of Cys residues within the active site to coordinate an O2-activating Cu(I) cofactor, is frequently exploited for this application. In the presence of a reducing agent such as DTT, FGE performs the oxidation of the target Cys with O2 as the electron acceptor () via a copper-oxygen mediated hydrogen atom abstraction (HAT) mechanism (Appel et al. Citation2019). FGE recognises a five-residue minimal sequence CXPXR, however, evidence has shown that reaction efficiency can be increased with the addition of a 4-mer hydrophobic segment followed by LTGR (i.e. CXPXRXXXXLTGR). Prokaryotic radical-S-adenosyl methionine (SAM) dehydrogenases, such as AtsB and anSMEcpe (Grove et al. Citation2013), are a second category of well-explored enzymes capable of performing this Cys to FGly transformation by using iron-sulphur clusters and SAM as cofactors; however, the need for anaerobic conditions to prevent oxidative damage of FeS clusters renders radical SAM dehydrogenases difficult to handle, making FGE the preferred enzyme of choice for industrial applications.
Figure 3. Overall scheme depicting O2-dependent conversion of Cys to formylglycine catalysed by formylglycine generating enzyme (FGE).
In nature, the conversion of Cys to FGly is particularly relevant for the activation of type I sulfatases, which require FGly for the hydrolysis of aryl- and alkylsulfate esters (Hanson et al. Citation2004); however, the versatility of the aldehyde functional group has rendered FGE as an attractive enzyme to be leveraged for ADC generation. To this end, the SMARTag technology developed by Redwood Biosciences and Catalent Pharma Solutions remains a staple for site-specific labelling of antibodies (Liu et al. Citation2019). This conjugation platform involves encoding the peptide sequence CXPXR into the antibody heavy or light chain, and co-expressed with FGE in mammalian production cells (e.g. Chinese Hamster Ovary (CHO) cells). Simultaneous expression of the tagged mAb and FGE enables Cys-to-FGly conversion as the antibody is produced in the culture. This aspect of SMARTag technology streamlines the conjugation process by obviating the need for purification of FGE separately. As an added benefit, another purification step to remove FGE is also avoided because this is accomplished when the mAb is captured from the expression culture. Following purification of the FGly-labeled mAb, an alkylhyrdrazine-functionalised indole is used to react with the aldehyde group to generate a hydrazonium ion followed by intramolecular alkylation in a Hydrazino-Iso-Pictet-Spangler (HIPS) fashion as mentioned above. This strategy has been showcased with the development of Catalent’s CD22-targeting ADC, Trph-222 (now licenced to Triphase Accelerator), which features a microtubule-inhibiting maytansinoid payload (RED-106) and a proprietary non-cleavable 4AP linker at a DAR of 1.8 (MacLaren et al. Citation2018). This synthetic linker-payload is attached to the mAb via HIPS chemistry enabled by an aldehyde-bearing SMARTag engineered at the heavy chain C-terminus (). Trph-222 completed phase I clinical studies, during which it demonstrated robust clinical activity in patients with non-Hodgkin’s lymphoma (NHL). Key findings of the study include responses in patients at a dose range from 0.6 to 10 mg/kg with minimal AEs at the highest dose, depicting a minimal toxicity profile and warranting further study of the drug in the clinic (Hernandez-Ilizaliturri et al. Citation2022).
Figure 4. Schematic overview for the generation of Trph-222. A Cys residue in the FGE-recognition sequence (CTPSR) engineered at the C-terminus of a CD22-targeting mAb is converted to FGly by FGE, which is coupled to an alkylhyrdrazine-functionalised indole payload, RED-106 (orange) in a HIPS reaction.
Oligosaccharide remodelling enzymes
Monoclonal antibodies feature a conserved N-linked glycan moiety attached to the side chain of Asn297 during a series of modifications by glycosidases and glycosyltransferases in the ER and Golgi (Marth and Grewal Citation2008). Antibodies expressed from cell hosts as well as those isolated from humans and animals exist as a mixture of different glycoforms (e.g. G0, G0F, and G1F, etc.), each having differences in the abundance and connectivity of monosaccharides composing the carbohydrate, namely mannose, galactose, fucose, N-acetylglucosamine (GlcNAc), and sialic acid. The carbohydrate has major implications on antibody effector functions, as it plays a role in modulating antibody-FcγR binding, recruiting complement proteins, activating inflammatory and anti-inflammatory properties, and regulating antibody-dependent cell-mediated cytotoxicity (ADCC) (Jefferis Citation2009).
The most prevalent strategy for antibody conjugation based on enzymatic oligosaccharide remodelling consists of Synaffix’s ‘GlycoConnect’ technology (Wijdeven et al. Citation2022). To briefly summarise the process, the glycan group of a mAb of interest is first homogenised by glycan trimming via endoglycosidases such as endo S or endo S2 (). The resulting core GlcNAc residue is then attached to a UDP-activated azide modified N-acetylgalactosamine monosaccaride (GalNac) via a β(1,4) bond-forming reaction catalysed by N-acetylgalactosaminyltransferase from Trichoplusia ni (TnGalNAc-T). The presence of the azide group enables conjugation to alkyne-carrying payloads via metal-free click chemistry (). A particularly attractive aspect of this strategy from a manufacturing standpoint is the fact that the glycan trimming and azidosugar conjugation steps can be run concurrently in one pot, as the endoglycosidases and glycosyl transferases used for this process do not interfere with one another. Furthermore, it has been demonstrated that His-tagged TnGalNAc-T can be expressed from mammalian cells and purified in high isolated yields using Ni Sepharose Excel (125–140 mg/L) with no need for refolding from inclusion bodies. In addition to this conjugation strategy, Synnafix has developed a proprietary linker known as HydraSpace, which consists of a negatively charged and highly polar sulfamide moeity that improves the overall hydrophobicity of GlycoConnect ADCs, resulting in improved efficacy, safety and PK (Verkade et al. Citation2018). Synnafix has paired their innovations in linker/spacer technology with a new payload platform, known as toxSYNTM, which consists of a collection of propriety cytotoxic linker-payloads spanning a wide range of mechanisms of action (MoAs), including DNA scission (SYNeamicinTM), topoisomerase 2 inhibition (SYN-38TM), and microtubule inhibition (SYNstatinTM and SYNtansineTM). With a variety of payload MoAs to choose from, a linker that improves the hydrophobicity, and a stable conjugation chemistry, the Synnafix platform offers a wholistic approach to ADC construction with a suite of technologies aimed at maximising the efficacy, improving the payload stability, and thus reducing off target toxicities, as well as expanding the pool of targets that can be selected with optimal matchmaking of tumour biology and cytotoxin MoA.
Figure 5. Schematic overview of the GlycoConnect technology. A) Target mAb is homogenised via treatment with Endo S or Endo S2, thereby cleaving the glycan moiety to core GlcNAc residue. In the same reaction mixture, this core residue is then coupled with a UDP-activated azidosugar via a β(1,4) linkage mediated by an N-acetylgalactosaminyltransferase (GalNAc-T). B) A payload functionalised with a strained alkyne, such as bicyclononyne (BCN, orange), is conjugated the azide group of the azidosugar in a metal-free SPAAC reaction to afford the final ADC product.
Five GlycoConnect ADCs have entered clinical trials, consisting of ADCT-601, XMT-1592, XMT1660, IBI343, and MRG004a. Of these, XMT1660, MRG004a, and IBI343 are still under clinical investigation, while more than a dozen additional ADCs are in preclinical development, making the GlycoConnect approach to ADC generation the most prevalent site-specific antibody modification strategy in the clinic. The wealth of ADCs constructed in this manner gives an abundance of insight regarding the advantages and disadvantages of the GlycoConnect approach. ADCs generated via GlycoConnect are often characterised with high stability and low hydrophobicity, likely due to the tendency of the N297 side chain to point towards the interstitial space in the Fc domain, leading to a shielding effect. Furthermore, the use of azides as functional handles for conjugation is advantageous as they are unreactive towards naturally abundant nucleophiles, such as amines and thiols, ensuring site-specific conjugation. On the other hand, glycan modification with GlycoConnect comes with some disadvantages as well. For example, GalT enzymes are not very efficient, requiring large enzyme-to-substrate weight ratios (up to 1:20) and long reaction times (overnight) to ensure completion. The process itself can be considered more work-intensive than most other conjugation techniques, as the need for three steps (homogenisation, azide labelling, drug conjugation) requires a notable amount of time, effort, and resources to complete, especially with additional purification steps. Homogenisation of mAbs via treatment with endoglycosidases presents its own disadvantages as well. Since the glycan moiety is cleaved and modified during this process, Fcγ Receptor (FcγR) binding and effector functions of the ADC are generally ablated when using the GlycoConnect approach, eliminating any cell killing caused by these effects. This aspect, however, could be advantageous depending on the application, as the possibility of off-target uptake by FcγR-expressing cells is also removed, which could have benefits in terms of toxicity and safety.
Prenyltransferase
Prenyltransferases (PTs) represent a class of enzymes that catalyse post-translational modification of Cys residues to isoprenyl pyrophosphate derivatives, a property that is attractive for site-selective bioconjugation (Marchwicka et al. Citation2022). Farnesyl transferase (FTase), a member of the PT family, recognises a CaaX peptide sequence preceded by a Gly spacer, where C is Cys, a is an aliphatic amino acid, and X is variable, typically Ser or Met (Roskoski Citation2003). FTase catalyses the site-specific isoprenylation of the thiol side-chain within this target sequence with a farnesyl pyrophosphate-derived substrate molecule, resulting in a thioether linkage (). The activity of FTase is dependent on the presence of zinc and magnesium ions, which highlights their importance in the reaction mechanism. It has been demonstrated that the zinc directly coordinates the Cys thiol resulting in a decrease in the pKa, suggesting that the role of the zinc is to enhance the nucleophilicity of the sulphur by maximising the amount of thiolate present at physiological pH. The magnesium ion, on the other hand, is believed to activate pyrophosphate leaving group by facilitating the formation of a positive charge on C1 of the farnesyl group and stabilising the resulting negative charge on pyrophosphate in the transition state. Farnesylation of proteins involved in signal transduction pathways such as Ras GTPases as well as nuclear lamins A and B, facilitates membrane association, and might be important for modulating protein-protein interactions (Long et al. Citation2002). In the case of Ras, farnesylation has also been shown to be critical for activity, as inhibition of FTases results in a blockade of signal transduction pathways leading to cell growth inhibition (Appels et al. Citation2005). Selectively inhibiting FTases, therefore, has become an established method for the treatment of cancers caused by deleterious Ras mutants in the ‘always-on’ state.
Figure 6. Farnesylation of a protein with an engineered CaaX tag (blue) catalysed by farnesyltransferase (FTase) with a farnesyl disphosphate substrate.
LegoChemBio have spearheaded the use of farnesyl transferases for ADC production and have validated their methodology in the clinic. The strategy, referred as their ConjuAllTM technology, involves engineering a CVIM recognition tag on the light-chain C-terminus of a MOI preceded by a (Gly)7 linker (Shin et al. Citation2021). A proprietary ketone-functionalised farnesyl-pyrophosphate substrate is conjugated to the Cys thiol side chain within the recognition sequence via FTase-mediated thioether bond formation. An oxime ligation between a hydroxyamine-functionalised toxic payload and ketone moiety of the isoprenoid linker affords the final ADC product. Using either an unbranched or branched linker on the synthetic payload, a DAR of 2 or 4 is achievable. Oxime linkages derived from aldehydes (aldoximes) can undergo hydrolysis leading to premature release of payload; however, this is circumvented with the use of a ketone moiety (ketoxime), which results in a comparatively more stable linkage. Additionally, oxime bonds have been demonstrated to be more stable than similar structures such as hydrazones and imines (Kalia and Raines Citation2010); however, the acidic conditions (pH 4 − 4.5) and long reaction times (≥24hr) required for complete oxime bond formation could create some manufacturing challenges and limit this approach to payloads and mAbs that are not acid-sensitive. The frontrunner ADC in LegoChem’s pipeline is LCB14-0110, which features a HER2-targeting mAb with a CVIM recognition tag conjugated to a glucoronide-linked MMAF at a DAR of 2, which is realised via enzymatic prenylation and oxime ligation (). In preclinical studies, LCB14-0110 showed dose-dependent cytotoxicity in HER2-positive cell lines and complete regression in moderate HER2-expressing breast cancer xenograft models. Moreover, the ADC displayed encouraging PK and high tolerability in rat and monkey (Lee et al. Citation2020). Currently, LCB14-0110 is undergoing a phase I clinical trial in China (NCT03944499).
Figure 7. Schematic overview for the generation of LCB14-0110. A HER2-targeting mAb is prenylated with a farnesyl diphosphate derivative bearing a ketone moiety via FTase at a CVIM tag preceded by a (Gly)7 spacer engineered at the light chain C-terminus. The prenylated mAb is then reacted with a linker-payload with a hydroxylamine group (orange) to form an oxime linkage.
Sortase
Sortase is a type of cysteine transpeptidase that performs site-specific attachment of biomolecules in prokaryotes for a variety of critical functions. In pathogenic bacteria, sortases perform covalent attachment of proteins to the cell wall of invading microbes, which promote cell adhesion, facilitate nutrient acquisition, and enable evasion of the host’s immune system (Susmitha et al. Citation2021). Moreover, sortases play a role in the assembly of pili via polymerisation of pili subunits. The activity of sortase is derived from a His-Cys-Arg active site, which recognises the sequence LPXTG (in which X is any amino acid) located at the C-terminus of a target protein. The side-chain thiol of the Cys residue within the sortase active site initiates a nucleophilic attack on the amide carbon located at the peptide linkage between Thr and Gly on the target protein, resulting in a tetrahedral intermediate, stabilised by the Arg residue () (Clancy et al. Citation2010). Reformation of the C = O bond and proton transfer by the active site His residue results in removal of the Gly residue on the target protein along with any following residues if present, leading to a thioester intermediate. A secondary substrate beginning with a pentaglycine sequence then attacks this thioester group from the N-terminal nitrogen, after which the tetrahedral oxyanion collapses to regenerate the sortase active site.
Figure 8. Sortase-catalysed conjugation scheme. A protein of interest with a C-terminal engineered LPXTG tag (blue) is coupled with the N-terminus of a multi-glycine motif (orange). The N-terminal amine attacks the carboxyl group located between the Thr and Gly residues of the LPXTG tag, leading to a new peptide linkage and loss of Gly as well as any following residues (if any). Peptide bonds formed by sortase in either direction are highlighted in bold.
NBE Therapeutics, now owned by Boehringer Ingelheim, developed a conjugation platform leveraging sortase A from Stapholococcus aureus for generating site-specific ADCs. Their antibody conjugation strategy, referred to as sortase mediated antibody conjugation (SMAC), involves engineering a sortase recognition sequence (LPXTG) at the C-terminus of the light chain and/or heavy chain of a mAb, which is covalently attached to a pentaglycine-containing synthetic payload via a sortase-mediated peptide linkage to afford DAR 2 or DAR 4 ADCs (Gébleux et al. Citation2019). NBE-Therapeutics has also developed its own linker-payload platform consisting of an anthracycline related to doxorubicin known as PNU, which has been conjugated to therapeutic mAbs using the SMAC technology. Anthracyclines consist of DNA damaging and intercalating toxins that show greater potency compared to commonly used microtubulin-inhibiting payloads, necessitating the highly stable peptide linkage generated by the sortase enzyme during conjugation to avoid toxic effects, namely cardiotoxicity (McGowan et al. Citation2017). Indeed, high stability is an advantage of this approach, as manufactured ADCs generated via SMAC-technology have been observed to have high serum stability and low toxicity at efficacious doses (Stefan et al. Citation2017). The main disadvantage of this approach, however, is that conjugation sites are limited to C-termini, as any residues following the sortase conjugation site are inevitably removed following ligation. This limitation unfortunately eliminates any possible benefits that can be achieved by changing the conjugation site, such as lowering hydrophobicity, reducing the risk of aggregation, and improving the therapeutic index (TI) and PK. Furthermore, the product of the reaction is susceptible to undergo an enzyme catalysed reverse reaction, thus requiring high amounts of linker payload to favour the forward reaction, especially in reaction mixtures with multiple primary amine donors.
Currently, the frontrunner ADC in NBE Therapeutics portfolio is NBE-002, which consists of an antibody (huXBR1) targeting receptor tyrosine kinase-like orphan receptor (ROR1) conjugated to the anthracycline analogue PNU159682 featuring a pentaglycine (G5) linker (). After promising preclinical results showing high safety and tolerability of the drug (Hu et al. Citation2021), NBE-002 entered a phase I/II clinical trial in 2020 for treatment of patients with advanced solid tumours, particularly triple-negative breast cancers (NCT04441099). In 2016, SOTIO Biotech announced a collaboration with NBE Therapeutics for the discovery, development, and manufacturing of ADCs against non-disclosed targets, leveraging NBE’s SMAC conjugation technology and ultra-potent anthracycline-based payload platform. From this partnership, a Claudin18.2-targeting ADC referred as SOT102 (formerly SO-N102), also consisting of a PNU159682-conjugated mAb, has emerged as the most promising drug candidate (Sadilkova et al. Citation2021) entering a phase I/II clinical trial in patients with gastric and pancreatic cancer in 2022 (NCT05525286).
Figure 9. Schematic overview for the generation of NBE002. A LEPTG peptide is engineered on the C-terminus of a ROR1-targeting antibody, which is conjugated to a pentaglycine-bearing payload via sortase-mediated peptide bond formation.
Genetic code expansion using non-natural amino acids for site-specific ADCs
While nucleophilic cysteine or lysine coupling and chemoenzymatic conjugations alike are limited to exposed sites and often require manipulation of the surrounding amino acid sequence, a technology that allows for encoding of non-natural amino acids (nnAAs) during the translation of a protein offers the same benefits of site-specific conjugation, with the advantage of limited impact on protein sequence and greater accessibility to sequestered sites (Fu et al. Citation2022). Genetic code expansion requires the function of an orthogonal aminoacyl tRNA synthetase (aaRS), with specificity for a non-canonical amino acid, and tRNA that suppress a stop codon during translation to incorporate the nnAA of choice into the resultant polypeptide chain with high fidelity and at user defined sites (Liu and Schultz Citation2010). Modifications of the aaRS have led to the incorporation of more than 200 amino acids analogues with a variety of useful chemical moieties in both E. coli and mammalian expression systems (Chin Citation2014; Wan et al. Citation2014; VanBrunt et al. Citation2015; Nödling et al. Citation2019). These reactive groups enable chemical functions like fluorescence, ligand biding, cross-linking and photocaging, but in medicinal applications they are most commonly used to enable bioconjugation. Typically, amino acids bearing azide or ketone functional groups that enable click cycloaddition chemistry or stable oxime bonds, respectively, are used to generate bioconjugates. Recently, a lysine analogue bearing a cyclopentadiene has shown to be reactive with both maleimide and tetrazine bearing molecules, generates stable adducts and looks to have promise for ADC development (St. Amant et al. Citation2018; St. Amant et al. Citation2019; Roy et al. Citation2020; Ting et al. Citation2022).
Genetic code expansion techniques are the most robust method for nnAA incorporation into recombinant proteins. Two methods have emerged for high-yield production of nnAA-containing antibodies for the construction of ADCs; cell-based expression and cell-free synthesis. (Zimmerman et al. Citation2014; Yin et al. Citation2017; Koehler et al. Citation2020; Roy et al. Citation2020). While productivity and manufacturing challenges vary between these approaches, the result has been an increase in nnAA-containing ADCs within the pre-clinical and clinical landscapes. Sutro Biopharma has developed cell-free expression systems, XpressCF® and XpressCF+®, in which bacterial lysates are employed for incorporation of para-azidophenlyalanine (pAzF), para-azidomethylphenylalanine (pAMF), or both into a-glycosylated antibodies (Zimmerman et al. Citation2014; Yin et al. Citation2017). Strained alkynes, such as DBCO-derived molecules, can be covalently linked to the azide groups in pAzF or pAMF via a metal-free SPAAC reaction (). Sutro and collaborators have used this platform to populate a strong portfolio led by STRO-002 (Luvelta), an anti-folate receptor alpha (FolRa) site-specific DAR4 ADC with novel cleavable 3-aminophenyl hemiasterlin linker-payload, SC239, conjugated to pAMF at sites Y180 and F404 on the heavy chain () (Li et al. Citation2023). This construction of a homogeneous, highly stable conjugate with desirable pharmacokinetics has proven advantageous in controlled delivery of their potent warhead, SC239, to an assortment of FolRa positive cell lines. The superior efficacy and reduced systemic toxicity showcased pre-clinically has translated to rapid progression of STRO-002 through clinical trials. In January 2023, Sutro announced results from a Phase 1 dose-expansion and plans to advance into phase 2/3 trial REFRaME for platinum resistant ovarian cancer patients. STRO-002 is also in phase 1 as a monotherapy for endometrial cancer and CBF/GLIS2 paediatric acute myeloid lymphoma, and as a combination therapy for ovarian cancer. These, alongside the CD74 targeting ADC STRO-001 for B-cell malignancies, total five nnAA containing ADCs to reach the clinic within Sutro’s robust pipeline.
Figure 10. (A) Conjugation of a DBCO-functionalised payload to pAzF or pAMF via copper-free strain-promoted azide alkyne cycloaddition. (B) Structural diagram for STRO-002, a homogenous DAR 4 ADC featuring a FRα-targeting mAb with two engineered pAMF substitutions at HC-Y180 and HC-F404. Conjugation of a hemiasterlin-derived payload (SC239) bearing a DBCO handle to the pAMF azide group occurs via SPAAC chemistry.
However, Sutro was not the first nnAA ADC company to reach the clinic. Prior to Sutro, Ambrx Biopharma developed and revealed their own nnAA-based conjugation platform, involving covalent attachment of hydroxylamine payloads to the non-natural residue para-acetyl-phenylalanine (pAF) via oxime ligation (). In December 2020, Ambrx and collaborator NovoCodex Biopharmaceuticals received Fast-Track Designation for their HER2-targeting ADC ARX788 for the treatment of metastatic breast cancer. ARX788 is an ADC in which the nnAA para-acetyl-phenylalanine (pAF) is incorporated for site-specific conjugation of a proprietary hydroxylamine-functionalised, non-cleavable Amberstatin (a tubulin inhibitor derived from auristatin), AS269 at a DAR of 1.9 () (Skidmore et al. Citation2020). In contrast to Sutro’s cell-free system, Ambrx has developed an expression system using Chinese hamster ovary (CHO) cells engineered to contain an orthogonal aaRS/tRNA pair for nnAA incorporation (Axup et al. Citation2012; Tian et al. Citation2014). With the use of their nnAA platform, Ambrx has demonstrated precise control over site of conjugation and high stability of the oxime linkage (Axup et al. Citation2012; Tian et al. Citation2014; Skidmore et al. Citation2020). These attributes led to the generation of an ADC with high serum stability, low hydrophobicity, and relatively long half-life (12.5 days) that contributed to superior efficacy of ARX788 in both breast and gastric xenograft models compared to DM1-conjugated controls in pre-clinical evaluations. In March 2023 ARX788 reported greater progression free survival over controls in phase 3 trials earning Breakthrough Therapy Designation by the National Medical Products Administration. ARX788 also received Orphan Drug Designation from the FDA for the treatment of HER2+ metastatic gastric/gastroesophageal junction (GEJ) cancer and, after positive reports upon completion of phase 1 clinical trial ACE-Gastric-01, phase 2/3 (ACE-Gastric-02) dosing commenced in August 2021. In addition to ARX788, Ambrx’s pipeline boasts an anti-PSMA ADC (ARX517) and an anti-CD70 ADC (ARX305), both currently in phase 1 clinical trials.
Figure 11. (A) Conjugation scheme for the oxime-bond forming reaction between pAF and a hydroxylamine-functionalised payload. (B) Structural diagram for ARX788, a homogeneous DAR 2 ADC featuring a HER2-targeting mAb with an engineered pAF substitution at HC-A121. Conjugation of the hydroxylamine-functionalised payload (AS269) to pAF side-chain results in an oxime linkage.
More recently, Veraxa, a company founded in 2019 in Heidelberg, Germany, has utilised genetic code expansion techniques for incorporation of a proprietary trans-cyclooctene (TCO) nnAA enabling strain-promoted inverse electron-demand Diels − Alder cycloaddition (SPIEDAC) conjugation to tetrazine functionalised linker-payloads (Koehler et al. Citation2023) (). While all molecules showcased in Veraxa’s pipeline are still in discovery and pre-clinical stages, they span multiple indications across both lymphoma and solid tumour landscapes. Significantly, these next-generation molecules contribute to a general trend in the industry towards more homogeneous and more stable ADCs.
Figure 12. General scheme for conjugating engineered mAbs featuring trans-cyclooctene substitution(s) with tetrazene derivatives via inverse electron demand Diels-Alder reaction.
Dual-payload ADCs deliver multiple cargos to address mechanisms of drug resistance
The adaptability of cancer and their ability to develop resistance to drugs persists as a critical limitation to effective cancer treatment. Mechanisms of drug resistance include, but are not limited to, metabolic irregularities, downregulation of the target antigen, endogenous expression and upregulation of efflux pumps, adventitious genetic mutations or some combination of these obstacles represented in heterogeneous cell populations comprising cancerous tumours. (Housman et al. Citation2014; McGranahan and Swanton Citation2015; Nilchan et al. Citation2019). To combat this, the administration of combination therapies has become a highly exercised approach to cancer treatment. First demonstrated in 1965 by Emil Frei, James F. Holland, and Emil J. Freireich as a treatment strategy for acute leukaemia in children, combination therapy has repeatedly proven more efficacious than monotherapy regimes (Frei et al. Citation1965; DeVita and Chu Citation2008; Mokhtari et al. Citation2017). Most commonly, two or more small molecules with varying mechanisms of action have been dosed simultaneously or sequentially. The rationale is that while cancer cells may be able to resist one challenge it is less likely to develop resistance to two. While effectivity is applaudable, treatment is often discontinued due to serious adverse effects that diminish patients’ quality of life (Gajra et al. Citation2018; Komiya et al. Citation2019; Ingrand et al. Citation2020) As targeted drug delivery methods have become more successful, ADCs too have been paired with chemotherapy, endocrine therapy, targeted therapies, immunotherapies, or radiotherapy in an attempt to treat refractory patients (Sun et al. Citation2017; Daver et al. Citation2020, Mamounas et al. Citation2021, Salvestrini et al. Citation2023, Nicolò et al. Citation2022, Wei et al. Citation2016). This has even led to combinations of multiple ADCs in clinical trials. For example, phase 1 results from Double Antibody-Drug Conjugate (DAD) trial (NCT04724018) were recently disclosed at the 2023 ESMO Congress. Although this combination of Trodelvy and Padcev has highlighted the potential for safe co-dosing of ADCs, it had an overall response rate (ORR) of 70%, fortifying the notion that coadministration is not a simple fix for drug resistance.
Recent advancements in conjugation technologies enabling precision engineering have led to the emergence of a new approach to combination therapy – dual-payload ADCs (dpADCs). Conventional monotherapy ADCs transport a single linker-payload to the target antigen of choice with the goal of increasing on-target efficacy of the chemotherapeutic cargo whilst minimising non-specific interactions, thus reducing off-target toxicities. While administering two ADCs in combination holds the advantage of using clinically validated molecules with existing manufacturing infrastructure, the biology of antibody-cell interactions stands as a potential pitfall. Antibody-mediated drug delivery relies on the premise of binding an antigen on the surface of the tumour cell. If two ADCs that target the same antigen are dosed together, they may compete for binding sites, reducing the chances of effectively delivering both drugs (Yamazaki et al. Citation2021). Furthermore, two monotherapy ADCs with distinct biophysical properties may suffer from unaligned pharmacokinetics, resulting in detrimental dissimilarities in exposure that limit the desired effect (Hamblett et al. Citation2004; Yamazaki et al. Citation2021). However, if two warheads, with different but complementary mechanisms of action, are tethered to the same antibody via stable chemistry, co-delivery is ensured (Levengood et al. Citation2017; Nilchan et al. Citation2019). This, in combination with streamlined product validation, more straightforward regulatory processes for the approval of a single molecule, and the potential for cost savings makes dpADCs an appealing approach to multi-drug therapy.
While numerous techniques have been disclosed for dpADC construction, very few have resulted in a molecule with suitable biophysical properties for clinical, or even pre-clinical, success. One example, Sutro Biopharma, the aforementioned nnAA-focused company, has demonstrated incorporation of two different nnAAs, one in the light chain and one in the heavy chain, for the use of distinct click chemistries to attach a combination of cargos. Other site-specific approaches have been showcased by Huang and co-workers, who developed a one-pot assembly method combining LacNAc-based linker-payloads for glycoengineering with their ‘traceless’ FcBP conjugation at site HC-K248 (Tang et al. Citation2023), and GeneQuantum, who has exploited the combinability of two chemoenzymatic techniques; a sortase A recognition site on the light chain for directed ligation, and glycoengineering with an Endo-S2 mutant on the heavy chain. Much like monotherapy ADCs, site-specific conjugation affords these molecules greater stability, improved homogeneity and superior pharmacokinetics (Tang et al. Citation2023; Xiong et al. Citation2023). However, to achieve this, two orthogonal chemistries are required. This can drastically limit manufacturability, with bottlenecks in both the material generation and product validation steps of drug development. More commonly, companies such as Seattle Genetics, Immunwork and collaborators, as well as academic labs, have employed branched linkers for tethering different blends, or ‘bundles’ of payloads (Levengood et al. Citation2017; Nilchan et al. Citation2019; Boschanski et al. Citation2021; Yamazaki et al. Citation2021). At first glance, this approach may seem more straightforward, but conjugation of bulky payload bundles can be limited by sterics. If exposed, cytotoxins, clustered or otherwise, can have detrimental impacts on physiochemical properties such as hydrophobicity, leading to more off-target interactions, rapid clearance and increased hepatic uptake (Shen et al. Citation2012; Strop et al. Citation2013; Lyon et al. Citation2015). Only time will tell if these ambitious molecules can successfully be engineered to co-deliver a pair of complementary cytotoxins for combatting drug resistance in cancer. This technology is not limited to the attachment of cytotoxins. One can foresee combinations of a cytotoxin with a hormone, anti-apoptosis inhibitors, or immune modulating compounds to enhance efficacy and engage the immune system for more durable antitumor activity.
Bispecific ADCs
As discussed above, protein engineering advances are being explored to improve the functional characteristics of ADCs. The potent activity of ADCs requires careful selection of tumour specific antigens, at sufficient density on target cells, and limited expression on normal cells to enable a wide therapeutic index. In addition, surface expression, internalisation rate and pathway, and cross species homology need to be evaluated for ADC targets (reviewed in Esapa et al. (Citation2023)). Most cancer antigens are not tumour specific, but rather overexpressed in malignant cells. ADCs are effective when a large disparity of expression occurs between diseased and normal cells, as this limits on-target, off-tumour activity. Bi-specific ADCs (bsADCs) were developed to target tumour cells where this antigen expression disparity does not occur, by requiring a cell to express two different antigens to achieve effective delivery. bsADCs are a new generation of ADCs with the potential to target two antigens (e.g. M1231, an EGFR/MUC1 bsADC developed by EMD Serono Merck and Sutro Pharma), or two non-overlapping epitopes on the same antigen (aka biparatopic ADC) (e.g. REGN5093-M114 developed by Regeneron targeting METxMET currently in Phase I/II clinical trial; ZW49 by Zymeworks targeting HER2xHER2; IMGN151 by Immunogen targeting folate receptor alpha (FRα) which is currently in Phase I) (Ab et al. Citation2020; Knuehl et al. Citation2022; Barnscher et al. Citation2023; Oh et al. Citation2023), and thereby enhance specificity, improve efficacy and reduce off-target toxicity. So far, it seems that this approach is bearing fruit, the bsADC, IMGN151 which carries the maytansinoid derivative DM21 payload has shown potent antitumor activity against a broad range FRα expressing tumours compared to Elahere (IMGN853) an FDA-approved monospecific ADC (Ab et al. Citation2020).
Like other ADCs, bsADCs come with several design challenges that impact their heterogeneity, linker-payload stability, and manufacturability. Well studied and developed protein engineering strategies including the bipod (Nesspor et al. Citation2020) and knob-in-hole (K-I-H) (Ridgway et al. Citation1996) technology, the use of common light chains, and genetic recombination of binding domains into a target IgG have enabled the generation of bi-specific antibodies (Brinkmann and Kontermann Citation2017). The commonly used bispecific antibody formats adapted for bsADC generation are shown in , and described in Tsuchikama et al. (Citation2024).
Figure 13. Schematic representation of various bi-specific antibody drug conjugates showing different specificities (blue and red) for two different target antigens or epitopes of the same antigen. The heterodimeric pairing of heavy chain component of the antibody is accomplished through ‘knob-into-hole’ (K-I-H) design strategy. Cytotoxic payloads are conjugated (site-specific or stochastic) via linkers as described for conventional ADCs. Created with BioRender.com.
Table 2. Bispecific ADCs in clinical use.
Selecting the right combination of antigen binding arms for two different antigens on the same tumour can be complex because binding affinities need to be fine-tuned to ensure tumour specific delivery. For example, AZD9592 (AstraZeneca), is a first-in-class bsADC designed to target EGFR and cMET and conjugated to a topoisomerase I inhibitor (Comer et al. Citation2023). Strategically this bsADC was designed to include a higher affinity (>15 fold) cMET antibody arm in combination with a lower affinity EGFR targeted arm. This design strategy was successful in mitigating EGFR-driven toxicity in normal tissues as shown in preclinical evaluations. In another example, YH013, a fully human EGFR x MET bsADC generated using a proprietary common light chain RenLiteTM mouse platform (Biocytogen) conjugated with MMAE via a protease-cleavable linker exhibited superior and durable efficacy at 3 mg/kg, and overcame MET-driven EGFR-TKI resistance, thus, outperforming benchmark antibodies (Han et al. Citation2023). This design was extended to a HER2 x TROP2 bsADC (YH012) which showed increased potency and tissue specificity in both HER2-positive and HER2-low PDX models indicating broad therapeutic efficacy (Shang et al. Citation2023). BL-B01D1 (Sichuan Baili Pharmaceutical/SystImmune-) is an example of tetravalent bsADC containing a topoisomerase 1 inhibitor targeting EGFR and HER3 antigens with a cleavable linker (DAR8) which is currently in phase I and demonstrated encouraging efficacy as well as tolerability in heavily pre-treated metastatic or locally advanced solid tumours, especially in patients with EGFR mutant NSCLC (Zhang, Ma et al. Citation2023).
The construction of a bispecific antibody introduces mutations into conserved domains of human immunoglobulin genes that enable the assembly of asymmetric antibodies. These digressions can lead to higher immunogenic potential and the development of immunogenicity and anti-drug antibodies that impact their pharmacokinetics (clearance) and therapeutic efficacy (Staton et al. Citation2019; Cohen et al. Citation2021). Furthermore, bsADCs are also subject to the design constraints of ADCs described above. Site of conjugation, stability of the payloads, linker chemistry, and hydrophobicity profiles all impact the in vivo properties of these molecules. Beyond this, bsADCs must consider several different bispecific modalities including monovalent and bivalent bispecific formats that impact tumour avidity and residence time at antigen bearing cells. Thus, identifying robust conjugation methods is critical to optimise bsADC activity, maximise manufacturability, and decrease production costs. While most bsADC utilise thiol-maleimide conjugations (targeting reduced cysteines) for payload attachment, we must consider that reducing the interchain disulphides may affect the heavy chain-light chain pairing of some bispecifics. More precise conjugation methods using enzymatic methods or non-natural amino acids may be preferred to generate more stable, homogeneous and robust molecules. But, while the design principles are complex, bsADCs have been shown to have either improved binding affinity towards a broad range of tumour expressing antigens or overcome drug resistance and tumour heterogeneity over their parental monospecific ADCs and thus are becoming a valuable tool in our antitumor armamentarium.
Radio-immunoconjugates return to the spotlight
Radioimmunoconjugates (RIC) are re-emerging as a treatment modality for cancer (Nasr et al. Citation2022). Like ADCs, radioimmunoconjugates use targeting agents to deliver radioactive isotopes to diseased cells. But unlike ADCs, radionuclides that emit alpha, beta, or gamma particles, do not require internalisation, lysosomal function for efficient payload release, and due to their large decay sphere, they are not subject to efflux pumps. That said, internalisation of a RIC, while not essential, is preferred, as it leads to concentration enrichment and elongated tumour retention times that improve RIC activity. These significant functional differences thus dictate their design criteria. The utility of RICs was first demonstrated with two CD20 targeted immunotherapies that were approved for the treatment of non-Hodgkins lymphoma, 131I -Tositumomab (Bexxar, GlaxoSmithKline) and 90Y – Ibritumomab tiuxetan (Zevalin; Spectrum Pharmaceuticals). Bexxar was generated by direct iodination of the antibody and approved by the FDA in 2003. Despite promising efficacy with 65% response rates in patients resistant to chemotherapy and rituximab, and favourable safety profiles, the drug was discontinued in 2014 due to the emergence of other effective non-radioactive treatments, and the requirement for on-site iodination that required specialised facilities for its preparation (Kraeber-Bodéré et al. Citation2016). Protein engineering and bioconjugation techniques improved the utility of radioimmunoconjugates with Zevalin, a CD20 targeted RIC that is constructed by conjugating a chelator to surface exposed lysines of the antibody (Emmanouilides Citation2009). The radionuclide was added last in the sequence (trapped by the chelator) and the final product purified. The pre-formation of the mAb-Chelator simplified production of the final drug. Zevalin was approved by the FDA in 2002 for NHL and is still available now. Since, RICs have relied on the use of conjugated chelators like DTPA or DOTA with an expanded panel of radionuclides (In-111, Ac-225 and Lu-177).
The same enzymatic and engineering site-specific conjugation methods described for ADCs have been applied to the construction of RICs and the consensus is that site-specific conjugation methods improve their binding affinity over stochastic methods (e.g. lysine) likely by avoiding regions necessary for antigen binding (Junutula et al. Citation2008; Boswell et al. Citation2011; Morais and Ma Citation2018). But the long half-life and broad biodistribution of antibodies leads to prolonged exposure of normal tissues to the radionuclide. The ideal RIC should have a short half-life, appropriate penetration range and high linear energy transfer. When targeting solid tumours, smaller targeting fragments are more desirable as they can penetrate solid tumours more efficiently, but more importantly, the fragments that do not target can be rapidly cleared from circulation (Sun et al. Citation2021). Smaller targeting agents also have less liver uptake and more rapid kidney secretion, thus sparing excessive exposure of critical organs. This ‘hit and stick -or- miss and clear’ approach may be beneficial in limiting off target toxicity, but retaining activity to tumour sites (Ahmadzadehfar et al. Citation2016; Kratochwil et al. Citation2016). Indeed, Lu-177 Vipivotide tetraxetan (Pluvicto, developed by Novartis), a PSMA targeting peptide-DOTA complexed with Lu-177 has shown promising results in castration resistant prostate cancer patients and was approved by the US FDA in 2022. In clinical trials, Pluvicto, when combined with the best standard of care, demonstrated a 62% overall survival (15.3 months median) and 66% progression free survival (for 8.7 months median) with an acceptable safety profile for this life-threatening condition in a patient population with high unmet medical need (Fallah et al. Citation2023). Similarly, a radiolabeled somatostatin analogue has shown efficacy in treating neuroendocrine tumours in the gastrointestinal tract, pancreas (Strosberg et al. Citation2017) and prostate (Ichikawa et al. Citation2022). The treatment developed by Novartis combines a targeting peptide-DOTA with Lu-177 (known as Lutathera or Lu-177 dotate) that showed clinical benefit in the NETTER-1 phase III trials. In this study patients with gastrointestinal neuroendocrine tumours experienced a progression free survival rate of 65.2% and 18% response rate, a significant improvement over the control arm (treated with long-acting octreotide alone where the progression free survival rate was 10.8% and the response rate 3%). The success of Pluvicto and Lutathera will undoubtedly spur the development of other small targeting formats for targeted radiotherapy. Small format antibodies like VHH, scFvs, and FAbs fit the design criteria, but their small size also limits the conjugation positions available which do not alter antigen binding properties. Thus, site specific conjugation methods will be needed to generate homogeneous conjugates that retain full binding activity. The conjugation methods described above can be readily adapted for the construction of stable chelator conjugates for targeted delivery.
Nanoparticle-based technologies
The clinical successes of ADCs have fuelled continued interest in developing methods for targeted drug delivery. As described above new and emerging technologies are being implemented to improve the therapeutic index of ADCs. This includes the development of attenuated cytotoxic warheads that aim to reduce off-target toxicities. However, this necessitates high drug loads to retain on-target efficacy, which can negatively impact stability, developability, aggregation propensity, and pharmacokinetics. Moreover, if exposed, the hydrophobic linker-payloads may succumb to deconjugation or premature linker cleavage. As an alternative, nanoparticle (NP) drug delivery platforms are being developed to overcome some of these limitations.
NPs are a diverse class of small materials ranging between 8 and 100 nm in size. They have a large surface area to volume ratio and can encapsulate and protect drugs for controlled release. NPs can be lipid-based (liposomes, lipid NP, emulsion), polymeric (dendrimer, micelles, nanospheres, polymerosome), inorganic (silica NP, Fe oxide, gold NP) or protein-based (virus like particles, ferritins, albumin) () (Hou et al. Citation2021; Puri et al. Citation2023). The key advantages of these technologies are (1) NPs can encapsulate and transport a variety of payloads including cytotoxic chemicals, or nucleic acids (e.g. mRNA, DNA, siRNA, and ASO), as used in genomic medicine. The protected payload is less subject to degradation, or interactions that could influence biodistribution and pharmacokinetics. (2) The retention mechanisms of NP are governed by the nanoparticle size and surface properties, not by the Fc-FcRN interaction seen in ADCs. This gives nanoparticles unique pharmacokinetic properties that can be modulated for improved activity. (3) The synthetic nature of NPs allows for functionalization to enable targeting through attachment of ligands (Florinas et al. Citation2016; Chen et al. Citation2018). A variety of ligands ranging from small molecules to antibody fragments can be used to influence NP biodistribution and tissue retention. In the case of cancer, targeting agents can be used to facilitate tumour accumulation, retention, internalisation, and ultimately delivery of therapeutic drugs.
Figure 14. A wide variety of nanoparticles, using different materials, have been generated. Each nanoparticle is optimised for different payloads, and surface modified for tumour targeting (image created in BioRender.com).
Over the past decade, numerous NP delivery technologies have been reported (Puri et al. Citation2023), and more than 30 NP formulations have been approved by the FDA, many of which are targeted for cancer therapy (Cabral et al. Citation2018; Anselmo and Mitragotri Citation2019). Even though tumours are permeable to NPs because of their extravasation, enhanced permeability and retention (EPR) characteristics, untargeted NPs have a propensity for accumulating in the liver or spleen in a size dependent manner (Cabral et al. Citation2011). Hence, current NP technologies are focused on modifying NP surfaces (e.g. with PEG) or using conjugation chemistry to functionalise the NP with tumour targeting agents (peptides (e.g. arginylglycyclaspartic acid (RGD), scFvs, Fabs, or even small molecule ligands such as folate, GalNAc and manose-6-phosphate) that direct them towards specific cell surface proteins (Friedman et al. Citation2013).
One recent example comes from the laboratories of Ulrich Weisner and Michelle Bradbury (formerly at Memorial Sloan Kettering Cancer Center) at Cornell University who developed a targeted NP technology utilising the Cornell prime dots, or C’dots. These consist of an inorganic silica core coordinated around a fluorescent dye and functionalised with a polyethylene glycol (PEG) coat (Phillips et al. Citation2014). PEG components can be modified to contain a radionuclide chelator and a targeting moiety (e.g. RGB peptide). Initially developed as diagnostic agent, the small size of the NP (6 nm diameter) allowed for effective tumour penetration and retention as well as rapid clearance through kidney excretion. The accumulation of the NP in solid tumours combined with a rapid loss of background signal improved tumour visualisation and detection by PET at time scales that were more convenient for patients than antibody-based diagnostics. To improve tumour targeting, scFvs containing a nnAA (azidolysine) at a permissive position were conjugated to DBCO-modified PEG on the C’dot coat. This allowed for a stable conjugation of a Her2/neu targeting scFv on the surface of the C’dot that demonstrated effective tumour targeting to Her2 expressing tumours in vivo (Chen et al. Citation2018). Indeed, over 12% of the injected C’dots localised to tumours in mouse studies with minimal (<5%) accumulation in non-target tissues including liver, spleen, and kidney. The efficiency of targeting, and the rapid excretion of the unbound C’dots, led to the generation of C’dots armed with a cytotoxic TopoIi inhibitor. Tumour bearing mice treated with these NPs (containing ∼30 toxins) showed complete tumour regression (Zhang et al. Citation2023a). C’dots have a carrying capacity of 80 cytotoxins per particle, a drug load not achievable by ADCs, that opens the door to the use of less potent cytotoxins, or cell modifying agents (inhibitors or apoptotic inducers) that require higher drug loads. Targeted C’dots are currently being developed by Elucida Oncology Inc. for commercial applications. Phase1 clinical studies examining a FRa-targeted C’dots armed with 20 exatecan molecules showed positive initial data in a dose escalation trial in patients with brain metastases (Ma et al. Citation2023). Further functionalization of C’dots with imaging agents is an attractive proposition as it would allow physicians to both treat disease, while assessing effectivity to better personalise patient treatments.
Other NP platforms have been modified to include targeting ligands such as antibodies, antibody fragments, folate, aptamer, transferrin, hyaluronic acid, and others. (Wong et al. Citation2014; Liang et al. Citation2018; Bhattacharya Citation2021). Beyond the targeting capabilities, the interest in NP technologies stems from the unique functionality that distinguishes them from ADCs. One preclinical example includes a fucoidan-based nanocarrier targeting P-selectin that enables transcytosis across the blood brain barrier (Tylawsky et al. Citation2023). The BBB has historically been difficult to access for conventional antibodies and ADCs, but the small hydrodynamic radius and surface chemistry of this NP enables access to the brain tumour microenvironment. Another example comes from the use of naturally occurring nanoparticles like ferritins that can be engineered to carry therapeutic disease modifying agents like nucleic acids (mRNA, siRNA, ASOs and DNA) that are less amenable to conventional antibody conjugates (Lee et al. Citation2015). This is an exciting proposition as they open the door to therapeutic solutions outside of cytotoxins and may extend to the treatment of the underlying genetic conditions associated with cancer.
Conclusions
Cancer is both multifactorial and adaptive, and as such, has proven an elusive foe. Blunt weapons like systemically administered chemotherapeutics have a history of moderate success. However, their efficacy has consistently been limited by their toxicity. This realisation has given rise to the next generation of anti-cancer therapeutics, a collection of molecules designed to direct cytotoxic cargos to tumour cells. The expectation has been that this would effectively improve on-target potency whilst limiting off-target toxicities. Alas, this too has proven anything but straightforward, and thus the scientific community has spent the last two decades developing a toolbox for next-generation engineering. New warheads of varying degrees of potency with added layers of safety, new linkers for greater control over drug release, and as highlighted in this review, new conjugation chemistries enabling superior stability, homogeneity and enhanced biophysical properties have not only changed the way we approach drug design but also enabled a deeper, more comprehensive understanding of how these molecules impact cancer biology.
This brings us to the obvious question; if the technologies detailed in this review have proven so advantageous in improving the efficacy and safety of ADCs, why have we not seen more of them in clinical trials? The answer is both simple and complex: the covalent attachment of drugs to antibodies is straightforward using conventional conjugation methods, and these ADCs have performed well in clinical settings. New conjugation methods, like those described here, need to demonstrate a substantial improvement in performance in order to warrant the risks, and costs, associated with any new technology entering the clinic. Preclinical data does suggest that more precisely built ADCs offer advantages, but whether this translates to human patients remains to be seen. As the technologies mature, and are tested in clinical trials, the verdict will be revealed.
These technologies will also need to overcome a second hurdle, their manufacturability. For example, nnAA containing proteins are produced either by cell-based expression systems that suffer from poor titres, or cell-free systems that are bacterial derived and require facilities adept at handling bacterial cell culture without contamination and endotoxin liabilities. Enzymatic conjugation techniques have been hindered by the need to produce not one but two proteins, the therapeutic and the enzyme required for conjugation. Bispecific antibodies are notorious for mAb assembly issues; mispairing of light chains is one of many complications that chronically lead to low yields; For radioimmunoconjugates, popular alpha-therapy radionuclide Ac-225 is now facing global supply chain issues, resulting in increased competition for raw materials enabling alpha-targeted therapy development. Additionally, unique manufacturing facilities are required for any future approved targeted radiotherapy agents. These challenges may seem daunting, but it is important to remember that these technologies are still in their infancy. Already nnAA companies have made significant strides in their production capabilities with cell-based and cell-free systems achieving gram-scale yields (Axup et al. Citation2012; Zimmerman et al. Citation2014; Roy et al. Citation2020). For enzymatic conjugations, co-expressing enzymes and engineered mAbs (SMARTag) as well as performing multiple reactions in one pot (GlycoConnect) are examples of effective strategies for circumventing manufacturing obstacles. Furthermore, clever protein and cell line engineering solutions have been implemented to improve bispecific antibody expression, light chain pairing, and ultimately the fidelity and yields that have enabled the manufacture of therapeutic bi-specific antibodies.
With these new technologies driving innovation we are entering an exciting period in medicine where physicians have sufficient tools to not only treat and manage cancer, but also drive towards a cure. While the vast majority of ADC on the marker today are conventionally constructed, there is mounting evidence that site-specific ADCs provide better outcomes for patients. As discussed here, there are numerous methods for constructing site-specific ADCs, and it is our challenge to understand the limitations and advantages of each technology, to design therapies with optimal properties.
Acknowledgments
The authors are indebted to Ronald James Christie and Asya Grinberg for their valuable input, critical review, and helpful comments in the preparation of this manuscript.
Disclosure statement
In accordance with Taylor & Francis policy and our ethical obligation as researchers, we are reporting that the authors E. Moore, M. Rice, G. Roy, W. Zhang and M. Marelli are employees of AstraZeneca and receive financial compensation and stock from their employer.
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References
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