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Antibody — Drug Conjugates (ADCs), a Growing Class of Targeted Cancer Therapeutics
Despite of disappointing clinical results and withdraw for the first antibody-drug conjugate (ADC) Gemtuzumab ozogamicin, tremendous ADC development on modification and optimization has been attempted to improve clinical efficacy and minimize toxicity. After decades of dynamic research, these efforts are now bearing fruit with about a dozen of new ADC approvals in the past 10 years (Table 1). In 2017, a lower and fractionated dose of Gemtuzumab ozogamicin was approved too. Most recently, the phenomenal clinical results of Trastuzumab deruxtecan used in the treatment of previously treated HER2-low advanced breast cancer ignite more enthusiasm in the field and will certainly boost exponential research and growth in the development of ADCs for more approvals.
Drug Maker Indications Trade name Target Antigen Approval Year
Gemtuzumab ozogamicin Pfizer/Wyeth Relapsed acute myelogenous leukemia (AML) Mylotarg CD33 2017;2000
Brentuximab vedotin Seattle Genetics, Millennium/Takeda Relapsed HL and relapsed sALCL Adcetris CD30 2011
Trastuzumab emtansine Genentech, Roche HER2-positive metastatic breast cancer (mBC) following treatment with trastuzumab and a maytansinoid Kadcyla HER2 2013
Inotuzumab ozogamicin Pfizer/Wyeth Relapsed or refractory CD22-positive B-cell precursor acute lymphoblastic leukemia Besponsa CD22 2017
Moxetumomab pasudotox Astrazeneca Adults with relapsed or refractory hairy cell leukemia (HCL) Lumoxiti CD22 2018
Polatuzumab vedotin-piiq Genentech, Roche Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) Polivy CD79 2019
Enfortumab vedotin Astellas/Seattle Genetics Adult patients with locally advanced or metastatic urothelial cancer who have received a PD-1 or PD-L1 inhibitor, and a Pt-containing therapy Padcev Nectin-4 2019
Trastuzumab deruxtecan AstraZeneca/Daiichi Sankyo Adult patients with unresectable or metastatic HER2-positive breast cancer who have received two or more prior anti-HER2 based regimens Enhertu HER2 2019
Sacituzumab govitecan Immunomedics Adult patients with metastatic triple-negative breast cancer (mTNBC) who have received at least two prior therapies for patients with relapsed or refractory metastatic disease Trodelvy Trop-2 2020
Belantamab mafodotin-blmf GlaxoSmithKline (GSK) Adult patients with relapsed or refractory multiple myeloma Blenrep BCMA 2020
Loncastuximab tesirine-lpyl ADC Therapeutics Large B-cell lymphoma Zynlonta CD19 2021
Tisotumab vedotin-tftv Seagen Inc Recurrent or metastatic cervical cancer Tivdak Tissue factor 2021
Table 1. FDA-approved ADC drugs

The concept of ADC can be traced back to the early 1900s, when German physician and scientist Paul Ehrlich proposed a visionary “magic bullet” that could deliver a toxic drug to certain malignant cells without affecting other normal tissues.

In the second half of last century, advances in chemistry for the linkage between cytotoxic agents and antibodies, as well as new techniques in hybridoma technology enabling the production of homogenous and target-accurate mAbs, led to the generation of ADCs with promising results. Now at a seemingly golden age of ADC drug development, the global market sales for ADC drugs are projected to exceed $ 16.4 billion in the next five years.[1] A scheme of the brief history of ADC development is shown in Fig 1 and the structures of some selected FDA-approved ADCs are listed in Fig 2.

Figure 1. Brief History of ADC development
Figure 1. Brief History of ADC development [2]
Figure 2. Structures of selected FDA-approved ADCs
Figure 2. Structures of selected FDA-approved ADCs [3]
(Orange: cytotoxin agents; blue: linkers; purple: antibodies)
Structure and Mechanism of Action of ADC

Different from traditional chemotherapeutics, all ADCs consist of three core components: a monoclonal antibody that can binds to a tumor-associated antigen, a cytotoxic agent (payload) and a cleavable or uncleavable linker that covalently connects antibody and payload. After ADC enters blood circulation system, the antibody component of ADC recognizes and binds to the cell-surface antigens on the targeted cancer cells. Upon internalization of the ADC-antigen complex through endocytosis, payload component is released into cytosol after cleavage by lysosome degradation pathway. The released bioactive payload binds to its targets, resulting in cancer cell death. [4]

The targeted delivery cytotoxic payload by ADC is expected to increase payload concentration in tumor cells, thus minimizing the required effective dose. The therapeutic window is narrow for early ADCs due to their off-target toxicity linked to unstable conjugation, competition with unconjugated antibody, and aggregation or fast clearance of conjugates. Although the basic approach of design and construction of ADCs remain constant, the selection of three components significantly affects the pharmacokinetic, pharmacodynamic, and clinical outcomes among different ADCs.

The latest developments in new payload discovery, linker optimization, antibody engineering, and advances of conjugation chemistry have led to the third generation of ADCs with improved therapeutic window (Fig 3).

Figure 3. Therapeutic window of ADCs
Figure 3. Therapeutic window of ADCs [5]
Target Antigen Selection

The features of an ideal target antigen includes:
1) Predominantly expressed on the surface of target tumor cells with limited heterogeneity compared to normal tissues;
2) Minimal antigen shedding to avoid antibody binding within the circulation;
3) Well internalizing ADC through receptor-mediated endocytosis and should not be modulated during endocytosis;
4) Antigen levels remain constant after ADC treatment.

Target antigens in stroma and vasculature in solid tumor is another approach. Additionally, targeting antigens in cancer stem cells has also been investigated.

Figure 4. Target antigens for ADCs in solid tumors
Figure 4. Target antigens for ADCs in solid tumors [6]
Selection of Antibodies
The antibody component of ADC functions as a vehicle, responsible for selectively delivering the cytotoxic payload to the target cancer cells. Ideal antibodies have high specificity and affinity to tumor associated antigens, good stability, low immunogenicity, low cross reaction, long circulating half-life, and efficient internalization. Currently, human IgG isotypes, particularly IgG1, are predominantly used as antibody backbone in the construction of ADCs. Four subtypes of human IgG differ in their constant domains and hinge regions with different solubility and half-life as well as their different affinity for Fcγ receptors (FcγR) expressed on immune effector cells (Fig 5).
Figure 5. Summary of IgG subtypes for potential use in ADCs
Figure 5. Summary of IgG subtypes for potential use in ADCs [3]
Selection of Payloads

Studies have shown that only a small fraction of cytotoxic payload with about 1-2% of administered dose can reach the tumor cells. Therefore, high potency of cytotoxic payloads is required to achieve therapeutic efficacy, with IC50 in sub-nanomolar or picomolar range (Fig 6). Payloads are normally small molecules and exert their activity by binding to intracellular targets (Fig 7).

Other favorable features of desired payloads include acceptable aqueous solubility, sufficient stability as conjugates, low immunogenicity, and a long half-life. The payload should retain its potency when modified for linkage. In addition to prevention of antibody aggregation and clearance, a balanced hydrophobic/hydrophilic physicochemical property of payload could lead to bystander effects on killing surrounding cells.

Figure 6. Potency of selected payloads
Figure 6. Potency of selected payloads [7]
Figure 7. Payloads for ADC drugs
Figure 7. Payloads for ADC drugs [8]
Selection of ADC Linkers

The linkers covalently tethering antibody and payload moieties play critical roles in the control of pharmacokinetic and pharmacodynamic (PK/PD) properties, therapeutic window, and ultimately the efficacy of ADC. The linkers should be metabolically stable in blood, thus preventing premature cleavage and ensuring sufficient delivery of ADC to the target tumor cells. Furthermore, a desired linker is able to facilitate rapid release of free and cytotoxic payload after internalization of ADC inside the tumor cells. The linkers with calibrated hydrophobicity possess capabilities to induce bystander effects for ADC to kill additional tumor cells in vicinity, irrespective of the expression of the target antigens on their surface. Therefore, linkers consist of three moieties: a suitable functional group for conjugating to the antibody, a spacer unit containing hydrophilic elements, and a trigger for releasing the cytotoxic payload.

There are two types of linkers: cleavable and non-cleavable. Cleavable linkers can be divided into acid cleavable, reducible and protease cleavable. The most frequently used linkers are maleimidocaproyl (MC), N-succinimidyl 4-(maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio) butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), peptides, hydrazones, and disulfides.

Figure 8. Comparison of different linkers
Figure 8. Comparison of different linkers [9]
Figure 9. Cleavage of linkers
Figure 9. Cleavage of linkers [8]
Conjugation for ADC Construction

The stoichiometry of the linker-payloads on the antibody (drug-to-antibody ratio, DAR) is an important factor for the efficacy and safety profile of the ADC. Since most payloads are hydrophobic species. High DAR with too many payloads attached to the antibody will cause an increase in protein aggregation, ADC clearance in blood, and off-target side effects. A controlled and homogenous DAR should be optimized with maximized PK/PD profile, safety, and efficacy. Novel approaches using site-specific conjugation (SSC) aim to minimize heterogeneity and produce more homogenous ADCs, thus expanding therapeutic window. These controlled conjugation strategies include engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases.

Selection of the attachment site of linker-payload to the antibody is also crucial. The selected site should not interfere antibody-antigen binding and leave internalization process unaffected. Additionally, the attachment site could have an impact on linker stability, subsequently affecting drug release rate.

Figure 10. The therapeutic effects of DAR and attachment sites on ADCs
Figure 10. The therapeutic effects of DAR and attachment sites on ADCs [3]
Summary and Prospective
A widespread interest in the development of ADC drugs for targeted cancer treatment in the past decade has led to a dozen of FDA-approved ADC drugs. Extensive research on selection of antigen targets and payloads, antibody engineering, linker optimization, and conjugation chemistry enable the construction of homogenous, effective, and safe ADCs with wider therapeutic windows. The rapid growth of ADC development warrants more innovative ADCs in the near future.
Strength of MCE Services
We have extensive experiences in research and development of ADC products. Having strong technical teams and state-of-the-art instruments, MCE is proud to partner with clients including academic research laboratories and international pharmaceutical companies, such as Abbie and AstraZeneca. Efficient and prompt services with high-quality products are guaranteed.
Wide-Range of Diversified Products
With breakthroughs and innovations on payload synthesis, diversified linkers, and conjugation chemistry, we offer customer synthesis of the most comprehensive, integrated portfolio of ADC products in response to clients’ needs. MCE serves global customers with 1000+ ADC related products.
Wide-Range of Diversified Products
One-stop Services for ADCs
With strong teams of experienced biochemists, synthetic and analytical chemists, MCE can provide one-stop services for the design, synthesis, analysis, purification, optimization, detection, and evaluation of ADC-related products (antibodies, payloads, linkers, drug-linker conjugates, and ADC drugs).
One-stop Services for ADCs
Related Products
  Product Name Description
ADC Cytotoxin Mertansine (DM1) A microtubulin maytansinoid inhibitor. To overcome systemic toxicity and enhance tumor-specic delivery.
Calicheamicin An antitumor antibiotic. A DNA synthesis inhibitor. To cause double-strand DNA breaks.
ADC Linker MC-Val-Cit-PAB A cathepsin cleavable ADC linker that is used for making antibody-drug conjugate.
SMCC A non-cleavable ADC linker that is used for making antibody-drug conjugate.
Drug-Linker Conjugates for ADC SMCC-DM1 (DM1-SMCC) SMCC-DM1 (DM1-SMCC) is a drug-linker conjugate composed of a potent microtubule-disrupting agent DM1 and an SMCC linker to make antibody drug conjugate (ADC).
MC-Val-Cit-PAB-duocarmycin MC-Val-Cit-PAB-duocarmycin is a drug-linker conjugate for ADC with potent antitumor activity using Duocarmycin (a DNA minor groove binding alkylating agent), connected via the ADC linker MC-Val-Cit-PAB.
Antibody-drug Conjugates (ADCs) Trastuzumab emtansine Trastuzumab emtansine (Ado-Trastuzumab emtansine) is an antibody-drug conjugate (ADC) that incorporates the HER2-targeted antitumor properties of trastuzumab with the cytotoxic activity of the microtubule-inhibitory agent DM1 (derivative of maytansine).
Trastuzumab deruxtecan Trastuzumab deruxtecan (DS-8201a) is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugate (ADC). Trastuzumab deruxtecan is composed of a humanized anti-HER2 antibody, an enzymatically cleavable peptide-linker, and a topoisomerase I inhibitor. References
References
[1]. do Pazo C, Nawaz K, Webster RM, et al. The oncology market for antibody-drug conjugates. Nat Rev Drug Discov. 2021 Aug;20(8):583-584.
[2]. David E Thurston, Paul J M Jackson, et al. Cytotoxic Payloads for Antibody–Drug Conjugates[M]. The Royal Society of Chemistry, 2019.
[3]. Walsh SJ, Bargh JD, Dannheim FM, Hanby AR, Seki H, Counsell AJ, Ou X, Fowler E, Ashman N, Takada Y, Isidro-Llobet A, Parker JS, Carroll JS, Spring DR. Site-selective modification strategies in antibody-drug conjugates. Chem Soc Rev. 2021 Jan 21;50(2):1305-1353.
[4]. Chau CH, Steeg PS, Figg WD, et al. Antibody-drug conjugates for cancer. Lancet. 2019 Aug 31;394(10200):793-804.
[5]. Beck A, Goetsch L, Dumontet C, Corvaïa N, et al. Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017 May;16(5):315-337.
[6]. Diamantis N, Banerji U, et al. Antibody-drug conjugates--an emerging class of cancer treatment. Br J Cancer. 2016 Feb 16;114(4):362-7.
[7]. Nakada T, Sugihara K, Jikoh T, Abe Y, Agatsuma T, et al. The Latest Research and Development into the Antibody-Drug Conjugate, [fam-] Trastuzumab Deruxtecan (DS-8201a), for HER2 Cancer Therapy. Chem Pharm Bull (Tokyo). 2019;67(3):173-185.
[8]. Drago JZ, Modi S, Chandarlapaty S, et al. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol. 2021 Jun;18(6):327-344.
[9]. Tsuchikama K, An Z, et al. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018 Jan;9(1):33-46.