Building Safe and Effective Antibody-Drug Conjugates

October 2, 2015
Adeline Siew, PhD

Adeline Siew is editor for Pharmaceutical Technology Europe. She is also science editor for Pharmaceutical Technology.

The development of successful ADCs involves careful selection of drug, antibody, and linker, as well as choosing the right attachment chemistry to link the cytotoxic to the antibody.


Antibody-drug conjugates (ADCs) have revolutionized the field of cancer treatment by enhancing the selectivity of traditional chemotherapeutic agents while limiting systemic exposure. “ADCs synergistically combine a specific anticancer antibody or antibody fragment linked to a cytotoxic drug,” explains Dave Simpson, PhD, CEO of biotech company Glythera. “The aim is to achieve better tumour penetration and killing properties with lower side effects for cancer patients.”

Despite the rapidly growing clinical pipeline of these targeted therapies, developing an ADC is no easy task. The challenge of optimal ADC design lies in the overall complexity of these molecules and the increasingly clear requirement to optimize each aspect relative to any given antibody–toxin pair and its target, according to Jennifer L. Mitcham, PhD, SMARTag Business Development, Catalent Biologics. “Conceptually, combining the targeting power of a monoclonal antibody (mAb) with the potency of a cytotoxic molecule should produce a drug with high efficacy relative to its toxicity (known as the therapeutic index),” she says. “However, there are significant hurdles to overcome when developing effective and safe ADCs. The first ADC was generated more than 30 years ago, yet today, there are only two FDA-approved ADCs on the market.”

There are a number of parameters that must be taken into account when designing an ADC, observes Albert Garofalo, PhD, group leader, Chemistry, Catalent Biologics. “The antibody is set based on the target antigen, and the payload might be set based on intellectual property considerations or the desired mechanism of action. This leaves the linker as the one area of the ADC that can be chemically manipulated to affect physicochemical properties such as solubility, protein aggregation, therapeutic index, and efficacy,” he remarks. 

Target antigen and antibody selection

To begin, the choice of target for the antibody is a key consideration, notes Cynthia Wooge, PhD, Global Strategic Marketing at SAFC. “The chosen cell-surface antigen should be over-expressed in the tumour cell and relatively non-expressed in normal tissue,” she highlights. This is because the tolerability of an ADC is affected by the specificity of antigen expression in cancerous tissue versus normal tissue, Simpson adds. “Non-specific expression results in toxicity and reduced efficacy due to a reduction in the dose of conjugate available to the tumour,” he explains. “Therefore, the ideal tumour antigen must be specifically localized to the tumour cell surface to allow ADC binding and, preferably, display differential expression between tumour and normal tissue, with increased expression in cancer cells.”

Ian Evetts, PhD, commercial director of Glythera, points out that antigen choice is also dictated by its ability to internalize upon ADC binding. “Internalization of an ADC occurs through receptor-mediated endocytosis, followed by ADC degradation in the lysosome, which leads to free drug release for effective cell killing,” he says. “However, endocytosis is not guaranteed for all cell-surface antigens, and the rate of internalization can vary considerably. Optimal drug release into the cell requires minimal recycling of the ADC to the cell surface as well as enhanced delivery of an internalized antigen/ADC to the lysosome. The ideal tumour antigen then should be cell-surface expressed, highly upregulated in cancer tissue, internalized upon ADC binding, and able to release the cytotoxic agent inside the cell.”

“As the cytotoxic payloads need to be internalized, the conjugated antibody should be amenable to this [prerequisite], as well as exhibiting any other effector functions desired,” Wooge reiterates. “The payload needs to deliver the desired cytotoxic effect once released within the cell, [which means that] the linker needs to have the necessary stability to maintain the payload until delivered to the target cell,” she asserts.

Once the target antigen has been identified, it is important to select a suitable antibody. Antibody selection plays a crucial part in the successful development of an ADC, observes Evetts. “Among the key attributes, high specificity for the tumour antigen is essential because an antibody that binds non-specifically or cross-reacts with other antigens can be taken up in normal tissues, thereby, inducing toxicity as well as elimination of the ADC before it can reach the tumour.” He adds that efficiency of uptake requires high-affinity binding to the target antigen (Kd < 10 nM). “Antibodies should also be minimally immunogenic,” Evetts highlights. “And ideally, antibodies should possess optimal pharmacokinetic properties including relatively long half-lives and slower clearance in the plasma.”

Drug selection

Currently, the drugs being used to construct ADCs generally fall into two categories: the microtubule inhibitors and the DNA-damaging agents, says Simpson, although he notes that other drugs such as polymerase II inhibitors are also being investigated. “Microtubule inhibitors bind tubulin, destabilize microtubules, and cause G2/M phase cell-cycle arrest,” Simpson explains. “DNA-damaging agents include anthracyclines, calicheamicins, duocarmycins, and pyrrolobenzodiazepines (PBDs). These drugs function by binding the minor groove of DNA and causing DNA stand scission, alkylation, or cross-linking. The cytotoxins are highly potent, with free drug IC50 of <10-9 M.” He cautions that drugs associated with neurotoxicity (such as platinum-based therapies or vinca alkaloids and first-generation taxanes) should be avoided.

A key consideration, according to Simpson, is that the drug selected for construction of an ADC must contain a suitable functional group for conjugation and be stable under physiological condition. “It is important that therapeutically appropriate amounts of the cytotoxic are linked to the mAb to optimize the potential effectiveness of the ADC once it reaches the target cancer cell,” he stresses. “The cytotoxic should also be chemically tractable and stable while bound to the mAb.” Simpson lists the following criteria as attributes of an ideal cytotoxic drug: 

  • High in-vitro potency (preferably picomolar)

  • Sensitivity of the cancer type to the class of the cytotoxic (e.g., antitubulin agents are frequently used in breast cancer)

  • Activity in multidrug-resistant models

  • Ability to induce cell death-high toxicity.


The crucial role of linkers

A crucial part in ADC development is choosing the right linker and method of attachment. The linker connects the small-molecule API to the large-molecule antibody that is engineered to bind to the target antigens on specific cell types.

To be effective, the ADC must remain stable in the bloodstream until it reaches the target cell, whereupon following internalization, the cytotoxic payload is delivered. Any premature drug release will be detrimental to normal cells. The first-generation ADCs had limitations because the early linkers used were either too stable, resulting in low potency and reduced efficacy, or suffered instability while in circulation, causing poor targeting and high systemic toxicity similar to that of an unconjugated chemotherapeutic.

“Ultimately, the main role of linkers is to ensure specific release of free drug in the cancer cells,” Simpson adds. “Linker properties are, therefore, crucial for controlling the toxicity of the highly potent drugs used to construct ADCs.” The linker can influence the distribution and pharmacokinetics of an ADC, and hence, its safety and efficacy, Wooge explains. “Depending on the preferred site of attachment on the antibody, linkers utilizing different functional groups or chemistries can be employed.”

The selection of suitable linkers, however, remains a key challenge in ADC design and manufacture, according to Garofalo. “The long circulating half-life of an ADC demands linkers that have high plasma stability in order to minimize off-target effects,” Mitcham says. “Yet, balancing the need for plasma stability with the need for selective intracellular drug release can be quite challenging.” 

Cleavable and non-cleavable linkers

There are two types of linkers used in ADC development-cleavable and non-cleavable. “As the cytotoxic payload is delivered into the cell, it can either be freed by digestion of the whole antibody, or it can be released by selective cleavage of the linker,” notes Vivek Sharma, CEO, Pharma Solutions, Piramal Enterprises. “You can tailor the drug release inside the target cell through design of the linker.”

The majority of ADCs in the clinic use cleavable linkers, observes Evetts, in particular, the protease-cleavable valine-citrulline dipeptide linker, originally designed to balance high plasma stability with intracellular protease cleavage. “Protease-sensitive linkers take advantage of the high levels of protease activity inside the lysosomes and include a peptide sequence that is recognized and cleaved by these proteases,” he explains. “Acid-sensitive linkers exploit the low pH within the lysosomal compartment to trigger hydrolysis of an acid-labile group within the linker, such as a hydrazone, to release the drug payload,” Evetts continues, but points out that ADCs using these linkers have yet to deliver successful clinical profiles due to instability-associated toxicity or lack of clinical efficacy. “Glutathione sensitive linkers (e.g., disulfides) utilize the higher concentration of thiols inside the cell relative to the bloodstream,” says Evetts. “Disulfide bonds within the linker are relatively stable while in circulation but are reduced by intracellular glutathione to release the free drug.”

Cleavable linkers release the free drug in a state that is more likely to permeate from the cell and kill surrounding cells-the by-stander killing effect, observes Mark Wright, site lead at Piramal Healthcare UK. “This comes down to the design of the conjugate,” he adds. “Should a conjugate be required to kill not only the highest expressing cells in the tumour, but also the surrounding ones, a cleavable linker is preferred.”

The downside is that with cleavable linkers, there is a higher chance of the payload being released into the circulation and causing off-target toxicity, Sharma asserts. “However, larger molecules with a significant charge can limit this off-target toxicity, as the drug is unable to cross through cell membranes and damage other cells,” he says.

On the other hand, non-cleavable linkers (e.g., thioethers) are more stable in the blood than cleavable linkers, according to Simpson, but they are wholly dependent on internalization, lysosomal delivery, and ADC degradation to release drug payload. “They cannot kill neighbouring tumour cells through the by-stander effect,” he says.

“Non-cleavable linkers release the drug with the linker still attached to the amino acid,” explains Wright. “These kind of linkers tend to generate active payloads that are charged and can’t escape from the cell easily.”

“With this approach, the target cell is killed, but nothing leaks out into the general circulation,” adds Sharma. “This approach delivers more tolerable conjugates, which work well with tumours that have very homogenous expression of antigen (although this is not always the case due to different cell classes making up tumours).”

Currently, there are two marketed ADCs-Roche’s Kadcyla, which is manufactured by Lonza, and Adcetris, jointly developed by Seattle Genetics and Takeda and produced by Piramal. Each of these ADCs use different linker systems, Mitcham and Garofalo remark. “Ado-trastuzumab emtansine (Kadcyla) consists of a maytansinoid payload randomly conjugated to the trastuzumab antibody using a 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimate (SMCC) linker,” says Mitcham. “This linker is considered to be a non-cleavable linker and as such, once the ADC is internalized within the target cancer cell, the antibody is degraded by proteolysis, leaving the maytansinoid payload still attached to a lysine residue via the SMCC linker. In this case, the payload remains effective because it can still bind to its intracellular target and induce cell death.”

Brentuximab vedotin (Adcetris) is the other marketed ADC, as Garofalo highlights. “It consists of an auristatin payload randomly conjugated to an anti-CD30 antibody using an enzymatically-cleavable dipeptide linker. This linker is proteolytically degraded in the cell and releases the payload as an unmodified drug. In this case, the cleavable linker was selected because chemical modification of the toxin interferes with its ability to bind its intracellular target.”

“Adcetris uses partial reduction chemistry, which has proven to be robust and generates consistent products. From a manufacturing perspective, this chemistry remains a good option for simple, trouble-free manufacturing of ADCs,” says Wright. “Partial reduction conjugation may have some drawbacks therapeutically in terms of stability and off target toxicity, but we expect to see more advanced conjugation technologies in future.”

There are some interesting developments in the newer generation of linkers, according to Sharma, such as the ability to incorporate more payloads, enabling a linker to hold more than one payload, and combining multiple effects from a particular payload. A lot of work is being done on developing the linker itself, observes Wright, adding that the linker choice can have a dramatic effect on the amount of payload that can be loaded onto the antibody. “There is some really interesting new science coming through where changes in the linker can make the payload significantly more effective in terms of killing the target cells, for example, by overcoming multidrug resistance or through enhanced killing of lower antigen-expressing cancer cells,” Sharma and Wright highlight. 


Attachment chemistry

While linkers are generally selected based on the preferred method of drug release from the antibody, Simpson points out that the attachment chemistry used to link both drug and antibody will determine the overall stability and clinical performance of the ADC.

Attachment to the polypeptide of the antibody is carried out via reaction with amino acids-cysteine being the most common, followed by lysine. According to Simpson, most approaches use maleimide chemistry, whereby maleimide selectively attaches to exposed or engineered cysteines via a covalent Michael addition. “However, maleimide is inherently associated with (i) deconjugation in physiological conditions via the reverse-Michael reaction, resulting in free drug in circulation and (ii) hydrolysis of the succinimide ring,” he explains, stressing that these factors hamper conjugate stability, ADC stability, and consequently, the therapeutic activity.

Simpson notes that several groups have focused on enhancing ADC stability through stabilizing conjugation although most have concentrated on next-generation maleimide chemistry approaches. Methods include (i) controlling the microenvironment around the succinimide ether to slow down the retro-Michael reaction and (ii) shifting the equilibrium through hydrolysis and developing more stable ring-opened thioethers.

“Through controlling microenvironment around the succinimide thioether, stability of maleimide conjugation can be improved since solvent accessible sites slow down retro-Michael. If cysteine is close to positively charged entities, rapid succinimide hydrolysis occurs, preventing further linker-maleimide exchange,” Simpson continues. “Also, modifying maleimide with the insertion of an amine group can shift the reaction through hydrolysis.” Nonetheless, although improved conjugation stability has been demonstrated, Simpson highlights that these approaches have not yet led to better clinical profiles, at least for cleavable linker systems. “Moreover, the processes required are not amenable to large-scale conjugation.”

Simpson believes that non-maleimide approaches may, therefore, offer a better option for delivering stable conjugation of drug to antibody. Highly stable constructs allow the incorporation of even more potent payloads. According to Simpson, Glythera’s vinyl-pyrridine-based chemistry platform, PermaLink, is associated with more stable ADCs compared with maleimide equivalents, with the potential for delivering superior, better tolerated next-wave options.

Mitcham and Garofalo from Catalent emphasize that the chemistry employed for conjugation must be compatible with conditions suitable for handling antibodies. “The reaction must proceed in an aqueous environment and must demonstrate chemical selectivity in the context of complex biomolecules that present a number of functional groups,” they say. “For this reason, there are relatively few reaction types that can be used for chemical ligation and even fewer that afford the potential for site-specific placement of the payload and the formation of a stable carbon–carbon bond. The Hydrazino-iso-Pictet-Spengler (HIPS) conjugation chemistry, developed by Catalent and used in conjunction with Catalent’s SMARTag technology, delivers on both of those needs.”

Recently, there has been an expanded use of a variety of linking technologies that improve the bioavailability and/or stability of the conjugate, according to Wooge. “This is reflected in technologies such as Mersana with its Fleximer backbone, which uses a polymer as a drug carrier,” she says. “Alternatively, there are multiple approaches that synthetically build the linker into the potent payload such as with the first-generation auristatin-peptide drug-linkers of Seattle Genetics and newer ones such as the Polytherics ThioBridge linker.” Wooge notes that Immunogen has remained on the forefront of linker technology, with active development of molecules with differing charges and spacers to allow the optimal match between the linker, payload, and antibody.

There is a growing body of evidence that a structure–activity relationship approach is required for optimal ADC design, observes Garofalo. “Catalent’s SMARTag technology is one of the newer technologies that allows for a structure–activity relationship study approach to building and testing each design component of the ADC,” Mitcham adds. “These components include programmable toxin placement, controlled drug-to-antibody ratio, stable conjugation chemistry, and novel linker technologies as applied to any given mAb–toxin pair.”

Given that different ADCs have different characteristics, it is necessary to evaluate the optimal mAb-linker-cytotoxic combination for each cancer type and target antigen under investigation, Simpson reminds. Successful ADC development clearly depends on the optimization and combination of all components-antibodies, payloads, linkers, and the attachment chemistries. 


Article DetailsPharmaceutical Technology Europe
Vol. 27, No. 10
Pages: 12–18

Citation: When referring to this article, please cite it as A Siew, “Building Safe and Effective Antibody-Drug Conjugates,” Pharmaceutical Technology Europe, 27 (10) 12–18 (2015).