Comprehensive Overview of Antibody-Drug Conjugates (ADCs)

Antibody-drug conjugates (ADCs) are formed by linking monoclonal antibodies targeting specific antigens with small molecule cytotoxic drugs through linkers, combining the powerful killing effects of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs. ADCs consist of three main components: the antibody responsible for selectively recognizing cancer cell surface antigens, the drug payload responsible for killing cancer cells, and the linker that connects the antibody and payload.

ADC’s recognition of antigens leads to their entry into cells via endocytosis. After degradation in lysosomes, the drug payload is released in a bioactive form and exerts its effect, leading to cancer cell death. The amount of payload inside the cell is determined by the number of surface antigens on each cell, the number of drug payload molecules per ADC (also known as drug-to-antibody ratio, DAR), and the time required for antigens to return to the cell surface. Payloads may escape after cancer cell death and degradation or may transmembrane out of the cytoplasm. The consequences of this release may be beneficial (also known as the bystander effect) or harmful, leading to systemic toxicity.

The first ADC (Mylotarg) was approved in 2000, and since 2019, the number of approved ADCs has more than doubled, with five ADCs approved from 2019 to 2020, indicating a continued hot trend in the ADC field.

There are several variable components in ADCs, and there is clearly no universal formula for success. Therefore, how to select the appropriate antibody, the mechanism of antibody endocytosis, where and how to attach the linker to the antibody, how many drug molecules to attach to each antibody, how to connect the linker and drug payload, and what the optimal drug payload looks like? These questions require a deep understanding of the biological and chemical characteristics of each component of ADCs to obtain satisfactory answers.

Payloads

1. Microtubule Disruptors

Monomethyl auristatin E (MMAE)

Auristatins are important payloads used in ADCs, with the most notable family member MMAE present in two marketed drugs, Adcetris and Polivy. Currently, over ten ADCs utilizing auristatin derivatives (such as MMAE) or MMAF as payloads are undergoing clinical trials.

The above figure describes the structure-activity relationship (SAR) of auristatin and its commonly used linker site, which has been extensively studied, primarily focusing on the terminal subunits: P1 (N-terminal) and P5 (C-terminal), with the most common method being the introduction of carbamate functionalities at P1.

In 2015, researchers at Seattle Genetics expanded the range of ADC payloads to include tertiary amines, particularly N,N-dimethyl auristatin, which was the first to covalently link the drug to monoclonal antibodies via ammonium bonds. The resulting ADCs demonstrated stability under physiological conditions, high in vitro and in vivo activity, and strong immunospecificity. These results broadened the range of drugs that can be used for targeted delivery via ADCs.

Recently, Agensys introduced azide groups into the P2 and P4 subunits by modulating the central subunits P2-P3-P4, producing hydrophilic derivatives with improved potency both in vitro and in vivo after coupling with protease-cleavable linkers, providing a new avenue for linker attachment.

Generally, in auristatins containing both amine and alcohol reactions, the preferred attachment point is the amine through carbamate bonds. Seattle Genetics developed a new strategy to couple alcohol-containing payloads with methylene ether carbamate (MAC). To stabilize the MAC bond, basic and electron-withdrawing groups were placed near the amine bond, resulting in the coupling being stable under physiological conditions, with high efficacy and strong immunospecificity both in vitro and in vivo.

Additionally, researchers at Uppsala University developed AZASTATIN, a new class of potent auristatin derivatives containing an antibody binding site with a central amine side chain. Their findings confirmed that these auristatin derivatives are a new class of cytotoxic payloads suitable for ADC development.

Maytansine Derivatives (DM2, DM4)

Maytansine is a highly effective microtubule assembly inhibitor that can induce mitotic arrest in cells. However, this structure is difficult to conjugate due to the lack of reactive functional groups. To overcome this issue, a series of highly effective derivatives containing SMe groups were created. The first examples of this class of molecules are DM1 and DM4, which carry a methoxy propanoyl group instead of the natural N-acetyl group.

Payloads DM1 and DM4 are coupled with linkers using disulfide bonds. Stable disulfide-linked payloads exhibit good stability in the bloodstream while maintaining effective division within cells.

Additionally, several maytansine-based ADCs utilize the same secondary hydroxyl as an attachment point, and in most cases carry protease-cleavable linkers. For example, an ADC based on Daratumumab has been shown to specifically deliver DM4 to CD38-overexpressing cancer cells.

Recently, ImmunoGen developed a new type of ADC that includes a sulfur-containing maytansinoid linked to the antibody via a highly stable tripeptide linker, with the attachment point being the same as the aforementioned hydroxyl. Compared to previous maytansine ADCs, increasing the number of methylene units in the linker increased bystander killing activity and improved efficacy in mouse models. In a similar approach, researchers from Regeneron and Abzena studied the effects of nitrogen substitution on N-methylpropanamide and also altered the lengths of side chains on the macrocycle, as well as the linkers connected via primary and secondary amines.

Microtubule Disruptors

Tubulysins are effective inhibitors of microtubule polymerization that lead to rapid disintegration of the cytoskeleton in dividing cells, resulting in apoptosis. They are a naturally occurring family of tetrapeptides containing Mep, Ile, Tuv, and Tut, where R3=OH or Tup, R3=H.

Utilizing tubulysins as ADC payloads, their extensive attachment points have been well developed. A notable attachment point in this structure is the carboxylic acid of the Tut or Tup module, as seen in Endocyte’s EC1428, where the carboxylic acid is linked to the linker via acylhydrazone. Oncomatryx has also adopted the same approach, installing cleavable PABAValCit maleimide linkers in the same manner.

Another method used by AstraZeneca, Bristol-Myers Squibb, and Pfizer relies on the derivatization of the phenyl ring in Tup or Tut.

Linkers with Mep groups have also been extensively studied. Researchers at Ingenica reported that demethyl Mep analogs retained strong cytotoxic activity and could be considered valuable payloads, allowing for the introduction of non-cleavable maleimide hexyl linkers on secondary amines. Oncomatryx’s research showed that when Mep is replaced by another sequence with a secondary amine, introducing a cleavable linker is an effective way to produce ADCs. Particularly interesting is Genentech’s work on linking linkers to tertiary amine-containing payloads via quaternary ammonium groups. Introducing mc-Val-Cit-PABA linkers on the payload led to more hydrophilic conjugates and improved stability in the bloodstream. Seattle Genetics also adopted a similar approach, demonstrating that by overexpressing glucuronic acid, glucuronic acid linkers could also improve hydrophilicity and selective intracellular cleavage via β-glucuronidase overexpressed in cancer cells.

Cryptomycins

Cryptomycins (CR) are a family of hexacyclic dipeptides with antitumor activity. Results from existing clinical trials indicate that at the doses required for therapeutic effect, their toxicity levels are unacceptable.

Several groups have attempted to use CR for ADCs, but due to the lack of coupling sites in CR, two different methods have been developed to introduce other groups, allowing for the coupling of ADC linkers. One involves converting phenyl into benzyl to produce an effective payload that can be linked via carbamate bonds. In the second approach, researchers from Sichuan University utilized a prodrug form of cryptomycin-52 (CR55), which can cyclize back to CR52 under physiological conditions.

Mitotic EG5 Inhibitors

Kinesin spindle protein (KSP, also known as Eg5 or KIF11) is an ATP-dependent motor protein involved in the separation of centrosomes during the cell cycle. Therefore, blocking this important event in mitosis with KSP inhibitors (KSPis) can produce antitumor efficacy.

Bayer discovered a new class of KSPis based on pyrrole derivatives, studying the compatibility of different positions of these molecules with linkers that maintain strong affinity for KSP.

Similarly, researchers at Novartis used imidazole-containing KSP inhibitors as Eg5 ADCs. Using primary alcohol or secondary amide portions, they installed non-cleavable linkers with maleimide end groups. When coupled with antibodies targeting HER2 and c-KIT, the resulting ADCs demonstrated superior in vivo efficacy compared to Kadcyla.

2. DNA-Damaging Drugs

Pyrrolobenzodiazepines and Indole Chlorobenzodiazepines

Pyrrolobenzodiazepines (PBDs) are a class of natural products with antitumor activity. Their mechanism of action involves selective alkylation in the minor groove of DNA, where the N2 of guanine forms a covalent bond with the electrophilic N10/C11 imine on the PBD.

Seattle Genetics used the aniline of SGD1882 as an attachment point, mimicking the commonly used PAB unit in cleavable linkers, releasing free PBD payloads. StemCentrx collaborated with Spirogen to utilize the N-10 position of PBD to connect a carbamate linker. The same carbamate bond was also employed by Immunogen for structurally similar indole chlorobenzodiazepine (IBD) payloads. They also reported different methods for the same class of IBD, where a substituted phenyl ring was used as a linker between two IBD monomers at the C8/C8′ positions.

In a similar manner, Spirogen and Genentech designed a PBD linked via iodophenyl that allows for the introduction of different linkers in transition metal-catalyzed reactions. By using Sonogashira coupling, Buchwald–Hartwig coupling, or azide-alkyne click reactions, linker payload conjugates were obtained with alkynes, piperazines, or triazoles, respectively.

Doxorubicin

Doxorubicin is a potent cytotoxic agent that binds to the minor groove of DNA through its highly active cyclopropane ring and alkylates adenine at the N3 position. Non-cyclized, halomethyl forms of doxorubicin exhibit significantly reduced cytotoxic activity. The phenolic group in the molecule can serve as an internal racemization activator, forming an electrophilic cyclopropane. Therefore, the linker strategies in doxorubicin ADC development focus on the attachment of the phenolic functional group.

In SYD985 developed by Synthon, the phenolic group is linked through a biscarbamate linker to the Mc-val-cit-PABC payload. After cleavage by cathepsin B, the free phenol promotes intramolecular rearrangement into the electrophilic cyclopropyl form. Medarex adopted a different approach by linking the linker to the non-alkylated portion of the aromatic amine, masking the phenolic prodrug with an N-methylpiperazine carbamate portion. In vivo, the phenol will be released, and the active cyclopropane will form under the action of carboxylesterase.

Camptothecin

Camptothecin (CPT) and its derivatives are classic examples of topoisomerase I inhibitors. They stabilize topoisomerase-induced single-strand breaks in DNA, leading to double-strand breaks when the ternary DNA-TOP1-inhibitor complex encounters a replication fork. Natural camptothecin has a pentacyclic structure, and its extremely low solubility prevents its widespread application as an anticancer drug. Its water-soluble prodrug irinotecan has been approved for metastatic colorectal cancer. SN-38 is the active metabolite of irinotecan, formed in vivo through the action of carboxylesterase in the human liver, which can be inactivated by opening the lactone ring.

Immunomedics established two different strategies to couple SN-38 using its hydroxyl portion. In one example, the linker was connected via a more reactive C-10 phenolic group, leading to stable carbamate bonds, while in another example, the C-20 hydroxyl was used to stabilize the lactone form, with the C-20 hydroxyl being critical for in vivo efficacy.

Another highly effective drug suitable for ADCs is exatecan (DDX-8951f). This camptothecin analogue has amine substituents on its cyclohexane ring bridging positions 7 and 9. The amine in exatecan contributes to its water solubility, while the rigidity imparted by the cyclohexane ring is believed to favor the balance between the active lactone form and the inactive hydrolyzed hydroxyl acid. Aminoacetylation generates DXd (1), while 4-amino-butyrylation generates DXd (2), both of which retain the biological activity of exatecan.

Hanging hydroxyl and amino groups are obvious attachment points that can be linked to payloads using enzyme-cleavable Gly-Gly-Phe-Gly linkers. The resulting ADCs, when coupled with anti-HER2 antibodies, show great potential against HER2-expressing cancers in clinical settings.

Although the cyclohexyl ring of DXd is believed to stabilize the biologically active lactone form, it carries a chiral center, complicating synthetic work and SAR studies. To overcome this difficulty, researchers at Immunogen studied a new set of camptothecin analogues that could couple with monoclonal antibodies. Here, the ring is opened, and the additional chiral center is eliminated. Starting from a common intermediate, researchers attempted three types of structures and subsequently used different polypeptide linkers to attach the payloads. When coupled with antibodies targeting human epidermal growth factor receptor (HuEGFR), the resulting ADCs were effective in EGFR-positive HSC-2 tumor xenograft models.

Calicheamicin

Calicheamicin is a class of extensively studied enediyne antibiotics, whose structure and mechanism of action are particularly interesting and complex, making them a class of antibiotics in the field of ADC payloads. The strategy of linking calicheamicin in ADCs is exemplified by the market ADC Mylotarg and Besponsa.

The release of the payload occurs in two steps: sensitive cleavage of the hydrazone in the acidic intracellular environment, followed by the reduction of disulfide bonds by intracellular glutathione. The released thiol undergoes intramolecular 1,4-addition of the enediyne, triggering a Bergman cyclization reaction that generates a diradical. This active intermediate can extract hydrogen atoms from the deoxyribose backbone, resulting in double-strand DNA breaks and subsequent cell death.

Recently, a new enediyne natural product called uncialamycin was isolated from a strain of Streptomyces discovered in British Columbia. This structure was confirmed by total synthesis, and since then, several effective synthetic analogs have been prepared as potential ADC payloads.

BMS researchers have shown that due to low reactivity under various peptide coupling conditions, the secondary amine of uncialamycin is not a suitable attachment point. They synthesized an analog where one amine was directly introduced into the aromatic ring, but the reactivity of this phenyl amine was also too weak to serve as a linker. On the other hand, using an aminoethyl extension provided a suitable attachment point for the linker.

From the latter payload, they prepared precursors using protease-cleavable dipeptides and non-cleavable linkers. The CD70-ADC exhibited highly specific cytotoxic activity against renal cancer cell lines with a cleavable linker, while the corresponding non-cleavable ADC showed no activity against the same cell line.

Recently, to continue this work, BMS researchers used phenolic groups as attachment points in designed, efficient, and chemically stable uncialamycin analogs. Using newly developed phenolic alkylation, a classical cleavable linker was added to the phenolic group of the payload. The resulting payload was coupled with antibodies, showing antigen-specific antitumor activity both in vitro and in vivo.

3. Innovative Drugs

Apoptosis Inducers (Bcl-xL Inhibitors)

Overexpression of anti-apoptotic Bcl-2 family members (including Bcl-xL) is one of the mechanisms by which cancer cells acquire resistance to apoptosis. Drugs capable of blocking the BH3 binding domain on Bcl-xL can trigger apoptosis in cancer cells. In 2017, AbbVie first demonstrated the efficacy of BcL-xL inhibitors as payloads in the form of ADCs, targeting specific cells or tissues expressing EGFR. Interestingly, researchers utilized three different attachment points on the payload to connect cleavable linkers. Aminoalkyl extensions were used for core modifications to establish suitable attachment points as needed.

Thailanstatin A and Its Analogues

Targeting spliceosomes represents a promising therapeutic option for cancer treatment. Several natural products can inhibit RNA splicing by binding to different spliceosome subunits. The most representative is thailanstatin A, which can bind to the SF3b subunit of the spliceosome, thereby blocking RNA splicing.

Thailanstatin A lacks a suitable group for linker attachment. To address this issue, carboxylic acids were coupled with ethylenediamine to introduce an amine spacer, which is commonly used for linker installation.

Using this natural product for ADCs is complicated by the presence of multiple reactive functionalities. For instance, the diene in the central core can react with the maleimide moiety used for bioconjugation through a Diels–Alder reaction. This issue was addressed by using another conjugated moiety, halogenated acetamides. The combination of these two modifications and the inclusion of cleavable linkers in ADCs was first reported in patent literature, claiming moderate activity in several HER2-expressing cell lines.

Recently, Pfizer reported that direct coupling of carboxylic acids with the effective surface lysines of antibodies (without linkers) led to the most effective Thailanstatin ADC to date. The activity of these lysine conjugates is related to the drug load, while other classes of payloads typically do not exhibit this characteristic. ADCs showed good action in gastric cancer xenograft models.

Amanitins

In the field of ADC technology, the use of transcription inhibitors similar to amatoxins is a relatively new approach. Nine naturally occurring amatoxin derivatives share the same scaffold structure, a large ring composed of eight L-configured amino acids, linked through a sulfoxide moiety between tryptophan and cysteine residues. Three side chains of amatoxins are hydroxylated, and the OH groups provide good solubility and bind to target molecules. Two peptides, α-amanitin and β-amanitin, account for 90% of all toxins.

Three attachment points have been used to generate ADCs based on amatoxins. The first attempt involved coupling the carboxyl group of β-amanitin to the amino group of lysine on IgG, which exhibited good plasma stability and high cytotoxicity, but this bioconjugation had a low yield. The hydroxyl group of dihydroisoleucine was also considered as an attachment point, introducing glutathione as a linker, which then coupled through lysine, yielding ADCs with excellent in vitro cytotoxicity and in vivo antitumor activity, but unfortunately, poor circulation stability due to serum carboxylesterase cleavage of the linker.

The third method, attaching to the 6-hydroxyl of tryptophan, represents the current standard procedure, where phenols and various linkers form ethers. This approach has been used to generate ADCs based on amanitins. Representative ADCs based on amanitin include HDP-101. The payload itself is a synthetic adamantanol derivative optimized for stability. Compared to natural amanitin, two differences are that the tryptophan lacks a 6′-OH and the thioether chain replaces the sulfoxide. By forming amides on the aspartic acid side chain, protease-cleavable linkers are introduced.

Recently, Park and collaborators designed a new linker motif (OHPAS) for payloads containing phenolic groups. It is a di-aryl sulfonate, with one aryl part derived from the payload and the other from the potential phenolic group of the linker motif. Applying this technology to the a-amanitin in trastuzumab ADC demonstrated strong cytotoxic activity both in vitro and in vivo.

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1. Microtubule Disruptors

2. DNA-Damaging Drugs

3. Innovative Drugs

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