Recent Advances in ADCs

Antibody Drug Conjugates (ADCs) are an innovative class of targeted cancer therapies, that combine the specificity of monoclonal antibodies (mAbs) with potent anti-cancer drugs to specifically attack tumor cells, while minimizing damage to healthy tissues and reducing side effects. The comprehensive review below is periodically updated with the latest advancements in the ADC sector and covers all major aspects of antibody-drug conjugates, encompassing the mechanism of action, bioconjugation methods, types of linkers, cytotoxin selection, linker-payload selection, mechanism of ADC toxicity, ADCs approved and in clinical trials, ADC conjugation technologies, discontinued ADC projects, and notable business deal, mergers & acquisitions in the ADC space.

Introduction

Despite numerous setbacks including multiple project discontinuations and clinical withdrawals, the approval of third-generation antibody-drug conjugates (ADCs) such as Enhertu® and Trodelvy® has sparked hope for broader therapeutic applications of ADCs in oncology. Notably, Enhertu® demonstrated a remarkable 36% reduction in the risk of death for patients with HER2-positive metastatic breast cancer (Astrazeneca, Press Release, Dec 2022). This resurgence in the field of Antibody-Drug Conjugates (ADCs) is highlighted by a significant rise in the number of ADCs (~233) undergoing clinical trials (Beacon Intelligence Database, Mar 2024), substantial business investments and a wave of mergers and acquisitions (M&A). In terms of revenue, the global ADC market reached an estimated value of $9.7 billion in 2023 and is projected to exhibit a CAGR of 15.2% from 2023 to 2028 (MarketsandMarkets™, Oct 2023).

However, amidst these triumphs, the field of antibody-drug conjugates faces persistent challenges, particularly regarding high discontinuation rates observed across multiple payload mechanisms, notably auristatins, maytansinoids, and PBD-dimers. Despite the approval of 14 ADCs and multiple ongoing clinical investigations, the landscape is marked by 68 documented discontinued ADC projects and several inactive projects, reflecting the complexities of ADC development. Serious adverse events resulting from off-site toxicity limits doses below the levels necessary for optimal anti-cancer efficacy. Even among FDA-approved ADCs, a considerable fraction of treated patients requires supportive care to manage ADC-associated toxicities, often leading to dose reductions, treatment delays, or discontinuation. The conventional “one-size-fits-all” or “copy-paste” approach, which focuses on repurposing established targets and linker-payload combinations, can frequently be unsuccessful. Considering the array of advancements and resources in the field, it is advisable to explore novel drug targets and linker-payload options. The emergence of site-specific conjugation technologies and innovative payload-linker strategies, coupled with the identification of new target compounds, has fueled the evolution of ADCs over the past two decades, and is helping move the field to second- and third-generation successes.

To address the challenge of high project discontinuation rates, a “data-driven approach” is essential. A thorough understanding of the mechanism of action, advancements, and obstacles in the field of ADCs is crucial for making informed decisions and progressing effectively. It is imperative to embrace innovative approaches based on years of research data and leverage technological advancements to develop next-generation ADCs.

In this review, we offer an in-depth exploration of recent advancements in the field while covering essential aspects such as the mechanism of action, a summary of approved drugs and ongoing clinical studies, the underlying mechanisms of ADC toxicity, current research trends, and insights into the business landscape of ADCs. The technological progress discussed here holds promise for addressing challenges in drug discovery and advancing ADC development across established and emerging therapeutic areas.

Mechanism of action of Antibody-Drug Conjugates

The combination of a monoclonal antibody and cytotoxic payload allows the delivery of tailor-made chemotherapeutics preferentially to cancer cells while largely sparing normal cells. A well-designed ADC increases the therapeutic index by lowering the toxicity through limiting the systemic circulation of cytotoxic agents without compromising their activity on the tumor tissue. Together, this ultimately allows to treat patients who would not tolerate systemic chemotherapies because of their large side effects. An ADC is composed of three elements (Figure 1): the antibody, the linker and the cytotoxin (payload), which are all very important to obtain the best therapy.¹ This discussion will go over the vital aspects of ADC design and their elements.

Figure 1: Anatomy of an ADC²

One could ask, how does one even start developing an ADC? The starting point is the antigen that will be recognized by the antibody for delivering the cytotoxin. How much of the antigen is expressed on cancer versus normal cells? How many copies are found on these cancer cells? How quickly does the ADC internalize? Is the indication a liquid or a solid tumor? Is the antigen expressed on 100% of the cancer cells or only on 50% of the cancer cells? All these questions help guide the selection of the ADC’s individual elements. ³

Figure 2 below describes the basic steps that lead to ADC activity. First, the ADC must have a high enough plasma concentration to get into the tumor tissue. Second, the ADC must have enough binding, internalization, and processing events to release enough active catabolite to cause apoptosis. This sounds simple enough but is much more complicated to practically develop. Keeping this mechanism in mind, different elements can be optimized to find the right balance of activity versus toxicity.³

Figure 2: Mechanism of action of an ADC. ²

The following discussion will focus on the conjugation and linker-payload portion of ADCs rather than on the design of the antibody itself. The antibody used in ADCs can have a variety of different modifications to modulate ADC activity such as extending half-life, binding two different epitopes, and having engineered conjugation sites. We will start our discussion with conjugation, followed by linkers and cytotoxins, and finish with the entire ADC construct.⁴

Bioconjugation: Attaching Cytotoxins to Antibodies

Attaching a linker-payload to an antibody might seem simple, but there are many different considerations and methods that need to be carefully balanced. The first concept is a measurement of how many conjugations occurred on the antibody. Any ADC that is prepared will have a drug-to-antibody ratio (DAR), which is the average number of cytotoxins found per antibody (Figure 3). This ratio is measured by a variety of analytical techniques, which generally all give similar results but almost never the same number.⁵ That is why NJ Bio recommends using two different methods to determine the DAR. As the field evolves, finding orthogonal methods for DAR determination is becoming more routine.⁶ The distribution of stochastic conjugations is usually referred to as heterogeneous, that is a mixture of populations (DAR 0 – 8) is obtained to give an average DAR (e.g., DAR 4), or homogeneous when the population is mostly one species (e.g., mostly DAR 2).³

Figure 3: DAR Distribution between Heterogeneous (Stochastic) and Homogeneous Conjugations.²

The most common methods to attach linkers to antibodies utilize the natural nucleophilic amino acids found on the antibody, with lysine and cysteine being by far the two residues of choice. Lysine conjugations are typically accomplished by mixing the antibody with an activated ester. Lysine conjugation has the advantages of being operationally simple and forming a stable amide bond between the antibody and the linker-payload. However, linker attachment to lysine can change the overall charge of the antibody. Lysine-based conjugations result in a DAR distribution between 0 and 9 when an average DAR of 3.5 is targeted.

The other nucleophilic amino acids used for conjugation are the interchain cysteines which requires initial manipulation of the antibody. The reactive thiols are masked as interchain disulfides between the heavy-light and heavy-heavy chains and must be released with a reducing agent (e.g., TCEP, DTT). Once the antibody is reduced, the thiols are available to react with an electrophile, such as a maleimide or haloacetamide. The DAR is controlled by the amount of reducing agent used at the reduction stage. The standard conjugation using cysteine aims for a DAR around 4 with a DAR distribution between 0-8 for IgG1 antibodies. Cysteine conjugation is fast, reliable and does not alter the charge of the antibody but can with certain motifs undergo a reverse reaction that releases the linker-payload into circulation.⁷ Currently, most ADCs employ cysteine conjugation for the attachment of the linker-payloads.³

Over the years many new technologies and protocols have been developed to construct ADCs with a homogeneous DAR. Examples of these technologies include engineered cysteines, non-natural amino acids, bridging linker groups and the use of enzymes to control the distribution of the linker-payload. These technologies improve the pre-clinical therapeutic index3 of the antibody-drug conjugate and are discussed in detail below.

Types of Linkers

Cytotoxins generally do not have a conjugatable group and thus a spacer or linker must be added to allow the cytotoxin to be attached to the antibody. This spacer is called the linker and comes generally in two flavors: cleavable (releasable) and non-cleavable (non-releasable).⁸

A non-cleavable linker is a linker that does not contain any biologically or chemically labile bond and an active catabolite is released by the complete degradation of the antibody. The released catabolite will contain the amino acid from the antibody to which the linker-payload was conjugated. Catabolites from non-cleavable linkers do not cross membranes passively and thus do not distribute within a tissue. The two most common non-cleavable linkers that are used are maleimidocaproyl (Mc) and succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC). In general, a non-cleavable linker is better tolerated but less efficacious.⁹

Cleavable linkers come in a variety of motifs and release mechanisms, but all have a bond that is custom designed to be broken at either a lower pH, with an enzyme or in the presence of thiols. The cleavable linkers will release a metabolite that can have membrane permeability. Membrane permeability usually will mean that the cytotoxin will have bystander activity, which is the ability to kill neighboring cells through passive diffusion.

The most popular linker is the protease cleavable linker that contains a valine-citrulline-para-aminobenzyl-carbamate moiety (vc-PABC).¹⁰ This is a traceless linker that allows the release of amine containing cytotoxins. Many di-, tri-, and tetra-peptide sequences are cleaved by proteases and changing the sequence can facilitate the synthesis of the linker-payload and improve the properties of the ADC construct. There are also traceless cleavable linkers that get cleaved by glucuronidases that offer higher water solubility.¹¹ The advantages of cleavable linkers are that they have good plasma stability and robust activity in a variety of cell lines and preclinical models.¹²

Another means of releasing a cytotoxin is to use the acidic environment found in the tumor microenvironment and in the lysosome. Hydrazones and carbonates are two commonly used motifs for pH-sensitive linkers. These acid labile linkers do shed cytotoxins in circulation but are nonetheless still powerful linker motifs.⁸

Finally, the last type of cleavable linker discussed will be the one containing reducible bonds. These are usually identified by their disulfide bond which breaks in half in the presence of cysteine or glutathione. The advantages of disulfide linkers are that the kinetics of release can be controlled by steric bulk.¹³

Selection of Cytotoxin for Antibody-Drug Conjugates

A variety of different cytotoxins can be combined and matched with different linkers. ADC cytotoxins commonly have activity in the sub-nanomolar range by disrupting tubulin, damaging DNA, inhibiting topoisomerases, and preventing other essential cell processes. These cytotoxins will have different potencies, permeabilities, and hydrophobicities. The selection of the exact cytotoxin will be dependent on the linker type and how sensitive the tumor is to the active catabolite. The major classes of cytotoxins are the following: auristatins,¹⁴ maytansinoids,¹⁵ calicheamicins,¹⁶ pyrrolidinobenzodiazepines (PBDs),¹⁷ indolinobenzodiazepines (IGNs),¹⁸ duocarmycins,¹⁸ camptothecins,¹⁹ alpha-amanitins,²⁰ and protein degraders.²¹ The validated cytotoxins in approved ADCs consist of calicheamicins, maytansinoids, auristatins and camptothecins. All these cytotoxins can be further derivatized to have the best possible properties.

Figure 4: Aspects of ADC design.

Selection of the Linker-Payloads for ADCs

Now comes the important question of how to select the linker, the cytotoxin, the conjugation method, and the DAR. This usually involves several rounds of optimization but understanding the biology can help decide the best starting point. If, for example, the tumor has consistent expression throughout the tissue then one could consider non-cleavable linkers to lower toxicity. If there is heterogeneous expression, then a cleavable linker generating a membrane permeable catabolite (with bystander activity) would be advantageous. Knowing the internalization rate and the number of antigens per cell can help determine how potent the cytotoxin will need to be. If the tumor cells have high expression of the antigen, then a less potent cytotoxin could be used. But if there is low expression, then a potent cytotoxin will be required. With this information in hand, one can start testing the linker-payload that has the best chance of success and begin refining the ADC from the data generated. Another widely used method for the selection of a linker-payload is to start with a representative model and screen all advanced linker-payloads. The standard linker-payloads are MC-MMAF, MC-VCPAB-MMAE, SMCC-DM1, SPDB-DM4, Dxd(1), CL2A-SN38, Tesirine, and DGN549. These cytotoxins are available from commercial sources and their clinical doses are already established.³

When it comes to the selection of the entire construct – which involves optimizing DAR, hydrophobicity, and pharmacokinetics – it will depend on what the expected clinical dose will be.²² To overcome the tumor antigen barrier, maximize tumor penetration and increase uptake in the tumor versus normal tissue, dosing in the linear PK range should be the objective.²² This information can be extrapolated from pre-clinical studies in non-human primates. Toxicity is driven by the linker-payload and not the target, thus selecting the right linker-payload platform is of great importance.²³ Having a proper linker-payload for an indication is key for clinical success.

To conclude, there are many parameters that need to be optimized for ADCs and there are no hard and fast rules to follow. This synopsis highlights considerations that can help guide linker-payload selection. A well-designed ADC can have a tremendous clinical benefit but, in the end, it is not a one-size-fits-all approach.

Mechanism of ADC Toxicity

Despite being designed to enhance the targeted delivery of cytotoxic payloads to specific cancer cells, ADCs often encounter challenges related to toxicity. First-generation ADC data revealed that only a small fraction (~0.003 – 0.08%) of the injected dose reached the intended tumor cell ²⁴ leading to the distribution of a significant amount of cytotoxin to non-target tissues and subsequent adverse events.

The toxicity associated with ADCs can be broadly classified into on-target and off-target toxicity. On-target toxicity occurs when the ADC binds to the intended cell surface protein on healthy cells, leading to adverse effects (Figure 5A). Off-target toxicity involves non-specific endocytosis, binding of the ADC to Fc/C-type lectin receptors, or internalization of free payload by passive diffusion across the cell membrane (Figure 5B and 5C). ²⁵ The bystander toxicity effect is observed when cleavage of the payload from ADCs leads to the diffusion of free payload into neighbouring healthy cells.²⁶

While ADCs with the same class of payload-linkers share similar toxicity profiles, using the same ADC to treat different cancers may result in varied toxicities. Common adverse events associated in late-stage clinical and marketed ADCs outlined in Table 1, can vary in severity, with some reaching grade 3 or higher. Among commonly used payload classes, peripheral neutropenia or thrombocytopenia is reported as the most common toxicity, followed by peripheral neuropathy, hepatotoxicity, skin rash, ocular toxicity, and toxicity of the gastrointestinal tract.²⁷

Various strategies have been explored recently to address these challenges and reduce ADC-associated toxicities. These strategies include modifying conjugation technology or payload-linker chemistry, making antibody modifications, adjusting dosage regimens, and implementing inverse targeting strategies.²⁵

Antibody-Drug Conjugates that are approved or undergoing clinical trials²⁸

Several ADCs are currently approved or undergoing clinical trials as summarized in Table 2 and 3. The first ADC to receive market approval, gemtuzumab ozagamicin, was first introduced in 2001 by Pfizer, withdrawn in 2010, and re-introduced in 2017 along with inotuzumab ozogamicin. Brentuximab vedotin, the second ADC launched in 2011 by SeaGen and Millenium Pharmaceuticals/Takeda, achieved $751 million in sales in 2023 (3Q). Trastuzumab deruxtecan, launched by Daiichi Sankyo and trastuzumab emtansine, launched by Roche, continue to enjoy blockbuster status with $2.556 billion and $2.22 billion in sales respectively in 2023. Between 2019 and 2022, additional ADCs have received approval, bringing the total number of approved ADCs to 14, as summarized in Table 2. The importance of ADCs as a key therapeutic modality is demonstrated by their clinical and market success, as well as the size and number of ADC business deals, described in a later section.

ADC Conjugation Technologies

The most commonly employed conjugation approach in ADC development is chemical stochastic conjugation, which involves random modification, with either native lysine on the antibody surface, or cysteine generated from partial reduction of the interchain disulfide bonds of the antibody.  However, this approach often leads to the generation of a heterogenous mixture of different species with a widely distributed drug-to-antibody ratio (DAR), which is measured by averaging the different species. This heterogeneity can lead to the formation of a subpopulation with a lower DAR than optimal which can lower the efficacy and another one with a higher DAR than optimal which can increase side effects/toxicity issues. Since, each of these generated subpopulations have different PK and efficacy profiles it could be hard to predict the effect of all the different species during clinical trials.  A small population of such species could be generating most of the toxicity observed, due to extensive antibody modification, which may cause structural changes affecting its biological function.²⁹

To address these challenges, several site-specific conjugation technologies have been developed. These strategies can create a more homogeneous ADC population with improved plasma stability, heightened tumor uptake, and enhanced binding efficiency.²⁹ The case of homogeneous ADCs into the clinic involved the modification of each interchain disulfide bonds (DAR8), enabling precise and site-specific conjugation, as demonstrated by trastuzumab deruxtecan (Enhertu®). This advancement allowed for more consistent DARs and improved therapeutic outcomes. In the past few years, numerous site-specific conjugation technologies have been developed. Table 4 provides an overview of commonly employed strategies in developing ADC constructs with improved homogeneity and therapeutic index.

Discontinued Antibody-Drug Conjugates

Although the field of ADCs has had many successes and new approvals in the last few years, there are many discontinued programs that provide important information. Most of these ADCs have been discontinued due to the lack of a favorable therapeutic index, or in other words, a lack of efficacy at a tolerable dose. Some ADCs have also been discontinued due to pipeline reprioritization or due to the competitive landscape. Most ADCs have used auristatin- and maytansinoid-based ADCs, and in many cases, there may have been the wrong selection of drug-linker for the indication. Some antibodies targeting Her2+ cancers have not progressed in the clinic or did not show meaningful improvements to trastuzumab emtansine, but careful selection of new drug-linkers led to fam-trastuzumab deruxtecan-nxki, a very promising new ADC. Table 5 displays targets and ADCs that have entered the clinic and have not proceeded, and a new generation of ADCs can be developed with this knowledge.

Business Deals in the Antibody-Drug Conjugate space

A flurry of deal-making activity is occurring in the ADC space, with ADCs once again emerging as a leading field of interest. These deals show the value that ADCs can offer, and the ADC platform is becoming an important therapeutic modality. With blockbuster deals from Gilead’s acquisition of Immunomedics for $21 billion in September 2020 to Pfizer’s acquisition of Seagen for $43 billion in 2023, and multiple licensing agreements and strategic collaborations, ADC deals range from early to late-stage to marketed products and include a variety of oncology targets and indications.

The tables below summarize recent partnership deals, venture capital funding events, and successful IPOs in the ADC space. Table 6 lists key licensing deals and mergers and acquisitions (M&As) in the ADC space since January 2020. Table 7 lists key venture capital funding events and IPOs in the ADC space since January 2020.