Introduction to ADCs

By: Julien Dugal-Tessier, Srinath Thirumalairajan, and Ralf Mueller
NJ Bio, 675 US Highway 1, Suite B129, North Brunswick, NJ 08902, U.S.A.

Antibody-Drug Conjugates: Mechanism

An antibody-drug conjugate (ADC) is a molecule that combines the specificity of a monoclonal antibody and the cell killing ability of cytotoxic agents. This combination 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 that 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 sufficient 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 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

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 require 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 give ADCs with a homogeneous DAR. Examples of these technologies include engineered cysteines, non-natural amino acids, bridging linker groups and use of enzymes to control the distribution of the linker-payload.3 These technologies improve the pre-clinical therapeutic index but are beyond the scope of this discussion.3

Types of Linkers for an Antibody-Drug Conjugate

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 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 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 an Antibody-Drug Conjugate

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,16pyrrolidinobenzodiazepines (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-Payload

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 heterogenous expression, then a cleavable linker generating a membrane permeable catabolite (have 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.

List of ADCs that are approved or undergoing pivotal trials24

INN (Isotype) Drug Linker Target Indication Stage
Brevituximab vedotin (IgG1) MMAE Cleavable CD30 HL Approved
Trastuzumab emtansine (IgG1) DM1 Non-cleavable Her2 Breast Cancer Approved
Gemtuzumab ozagamicin (IgG4) Calicheamicin pH Sensitive CD33 AML Approved
Inotuzumab ozagamicin (IgG4) Calicheamicin pH Sensitive CD22 NHL Approved
Polatuzumab vedotin (IgG1) MMAE Cleavable CD79b B-Cell lymphoma Approved
Enfortumab vedotin (IgG1) MMAE Cleavable Nectin4 Bladder Cancer Approved
Trastuzumab deruxtecan (IgG1) DXd Cleavable Her2 Breast Cancer Approved
Sacituzumab govitecan (IgG1) SN-38 pH Sensitive Trop 2 Breast Cancer Approved
Belantamab mafodotin MMAF Non-Cleavable BCMA Multiple Myeloma Approved
Trastuzumab duocarmazine Duocarmycin Cleavable Her2 Breast Cancer PhIII
BAT8001 (IgG1) Maytansinoid Non-Cleavable Her2 Breast Cancer PhIII
Mirvetuximab soravtansine (IgG1) DM4 Cleavable Folate R1 Ovarian Cancer PHIII
Loncastuximab tesirine (IgG1) PBD Cleavable CD19 B-Cell Lymphoma PhII
Camidanlumab tesirine (IgG1) PBD Cleavable CD25 HL PhII

INN = International Nonproprietary Name; HL = Hodgkin Lymphoma; NHL = non-Hodgkin Lymphoma; AML = Acute Myeloid Lymphoma; PhII = Phase 2 clinical trial; PhIII = Phase 3 clinical trial.


Review Articles for Antibody-Drug Conjugates

(1)        Pysz, I.; Jackson, P. J. M.; Thurston, D. E. CHAPTER 1. Introduction to Antibody–Drug Conjugates (ADCs). In Cytotoxic Payloads for Antibody–Drug Conjugates; 2019; pp 1–30.

(2)        Jain, N.; Smith, S. W.; Ghone, S.; Tomczuk, B. Current ADC Linker Chemistry. Pharmaceutical Research 2015, 32, 3526–3540.

(3)        Beck, A.; Goetsch, L.; Dumontet, C.; Corvaïa, N. Strategies and Challenges for the next Generation of Antibody-Drug Conjugates. Nature Reviews Drug Discovery 2017, 16 (5), 315–337.

(4)        Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Antibody-Drug Conjugates: An Emerging Concept in Cancer Therapy. Angewandte Chemie International Edition 2014, 53 (15), 3796–3827.

Other Articles

(5)        Matsuda, Y.; Robles, V.; Malinao, M. C.; Song, J.; Mendelsohn, B. A. Comparison of Analytical Methods for Antibody-Drug Conjugates Produced by Chemical Site-Specific Conjugation: First-Generation AJICAP. Analytical Chemistry 2019, 91 (20), 12724–12732.

(6)        Sarrut, M.; Fekete, S.; Janin-Bussat, M.-C.; Colas, O.; Guillarme, D.; Beck, A.; Heinisch, S. Analysis of Antibody-Drug Conjugates by Comprehensive on-Line Two-Dimensional Hydrophobic Interaction Chromatography x Reversed Phase Liquid Chromatography Hyphenated to High Resolution Mass Spectrometry. II- Identification of Sub-Units for the Characterization of even and odd load drug species. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2016, 1032, 91–102.

(7)        Tumey, L. N.; Charati, M.; He, T.; Sousa, E.; Ma, D.; Han, X.; Clark, T.; Casavant, J.; Loganzo, F.; Barletta, F.; Lucas, J.; Graziani, E. I. Mild Method for Succinimide Hydrolysis on ADCs: Impact on ADC Potency, Stability, Exposure, and Efficacy. Bioconjugate chemistry 2014, 25 (10), 1871–1880.

(8)        Sievers, E. L.; Senter, P. D. Antibody-Drug Conjugates in Cancer Therapy. Annual Review of Medicine 2013, 64 (1), 15–29.

(9)        Alley, S. C.; Okeley, N. M.; Senter, P. D. Antibody–Drug Conjugates: Targeted Drug Delivery for Cancer. Current Opinion in Chemical Biology 2010, 14 (4), 529–537.

(10)       Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Chace, D. F.; Deblanc, R. L.; Gearing, R. P.; Bovee, T. D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer, D. L.; Senter, P. D.; Zhang, L.; Miles, M. F.; Aldape, K. D. Development of Potent Monoclonal Antibody Auristatin Conjugates for Cancer Therapy Corrigendum : A Model of Molecular Interactions on Short Oligonucleotide Microarrays. Nat. Biotech. 2003, 21 (8), 2003.

(11)       Jeffrey, S. C.; de Brabander, J.; Miyamoto, J.; Senter, P. D. Expanded Utility of the β-Glucuronide Linker: ADCs That Deliver Phenolic Cytotoxic Agents. ACS Medicinal Chemistry Letters 2010, 1 (6), 277–280.

(12)       Lyon, R. P.; Bovee, T. D.; Doronina, S. O.; Burke, P. J.; Hunter, J. H.; Neff-Laford, H. D.; Jonas, M.; Anderson, M. E.; Setter, J. R.; Senter, P. D. Reducing Hydrophobicity of Homogeneous Antibody-Drug Conjugates Improves Pharmacokinetics and Therapeutic Index. Nature Biotechnology 2015, 33 (7), 733–735.

(13)       Lewis Phillips, G. D.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons, K. L.; Mai, E.; Blättler, W. A.; Lambert, J. M.; Chari, R. V. J.; Lutz, R. J.; Wong, W. L. T.; Jacobson, F. S.; Koeppen, H.; Schwall, R. H.; Kenkare-Mitra, S. R.; Spencer, S. D.; Sliwkowski, M. X. Targeting HER2-Positive Breast Cancer with Trastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate. Cancer Research 2008, 68 (22), 9280–9290.

(14)       Akaiwa, M.; Dugal-Tessier, J.; Mendelsohn, B. A. Antibody–Drug Conjugate Payloads; Study of Auristatin Derivatives. Chemical and Pharmaceutical Bulletin 2020, 68 (3), 201–211.

(15)       Costoplus, J. A.; Veale, K. H.; Qiu, Q.; Ponte, J. F.; Lanieri, L.; Setiady, Y.; Dong, L.; Skaletskaya, A.; Bartle, L. M.; Salomon, P.; Wu, R.; Maloney, E. K.; Kovtun, Y. v.; Ab, O.; Lai, K.; Chari, R. V. J.; Widdison, W. C. Peptide-Cleavable Self-Immolative Maytansinoid Antibody-Drug Conjugates Designed to Provide Improved Bystander Killing. ACS Medicinal Chemistry Letters 2019, 10 (10), 1393–1399.

(16)       Shor, B.; Gerber, H.-P.; Sapra, P. Preclinical and Clinical Development of Inotuzumab-Ozogamicin in Hematological Malignancies. Molecular immunology 2015, 67 (2 Pt A), 107–116.

(17)       Mantaj, J.; Jackson, P. J. M.; Rahman, K. M.; Thurston, D. E. From Anthramycin to Pyrrolobenzodiazepine (PBD)-Containing Antibody-Drug Conjugates (ADCs). Angewandte Chemie International Edition 2017, 56 (2), 462–488.

(18)       Elgersma, R. C.; Coumans, R. G. E. E.; Huijbregts, T.; Menge, W. M. P. B. P. B.; Joosten, J. A. F. F.; Spijker, H. J.; de Groot, F. M. H. H.; van der Lee, M. M. C. C.; Ubink, R.; van den Dobbelsteen, D. J.; Egging, D. F.; Dokter, W. H. A. A.; Verheijden, G. F. M. M.; Lemmens, J. M.; Timmers, C. M.; Beusker, P. H. Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985. Molecular Pharmaceutics 2015, 12 (6), 1813–1835.

(19)       Li, W.; Veale, K. H.; Qiu, Q.; Sinkevicius, K. W.; Maloney, E. K.; Costoplus, J. A.; Lau, J.; Evans, H. L.; Setiady, Y.; Ab, O.; Abbott, S. M.; Lee, J.; Wisitpitthaya, S.; Skaletskaya, A.; Wang, L.; Keating, T. A.; Chari, R. V. J.; Widdison, W. C. Synthesis and Evaluation of Camptothecin Antibody-Drug Conjugates. ACS Medicinal Chemistry Letters 2019, 10 (10), 1386–1392.

(20)       Pahl, A.; Lutz, C.; Hechler, T. Amanitins and Their Development as a Payload for Antibody-Drug Conjugates. Drug Discovery Today: Technologies 2018, 30, 85–89.

(21)       Pillow, T. H.; Adhikari, P.; Blake, R. A.; Chen, J.; del Rosario, G.; Deshmukh, G.; Figueroa, I.; Gascoigne, K. E.; Kamath, A. v.; Kaufman, S.; Kleinheinz, T.; Kozak, K. R.; Latifi, B.; Leipold, D. D.; Sing Li, C.; Li, R.; Mulvihill, M. M.; O’Donohue, A.; Rowntree, R. K.; Sadowsky, J. D.; Wai, J.; Wang, X.; Wu, C.; Xu, Z.; Yao, H.; Yu, S. F.; Zhang, D.; Zang, R.; Zhang, H.; Zhou, H.; Zhu, X.; Dragovich, P. S. Antibody Conjugation of a Chimeric BET Degrader Enables in Vivo Activity. ChemMedChem 2020, 15 (1), 17–25.

(22)       Cilliers, C.; Menezes, B.; Nessler, I.; Linderman, J.; Thurber, G. M. Improved Tumor Penetration and Single-Cell Targeting of Antibody–Drug Conjugates Increases Anticancer Efficacy and Host Survival. Cancer Research 2018, 78 (3), 758–768.

(23)       Mahalingaiah, P. K.; Ciurlionis, R.; Durbin, K. R.; Yeager, R. L.; Philip, B. K.; Bawa, B.; Mantena, S. R.; Enright, B. P.; Liguori, M. J.; van Vleet, T. R. Potential Mechanisms of Target-Independent Uptake and Toxicity of Antibody-Drug Conjugates. Pharmacology & Therapeutics 2019, 200, 110–125.

(24)       Khongorzul, P.; Ling, C. J.; Khan, F. U.; Ihsan, A. U.; Zhang, J. Antibody-Drug Conjugates: A Comprehensive Review; 2020; Vol. 18.