V38 N2 / 2023 ©2023 American Medical Writers Association. All rights reserved. ISSN 2163-5315 AMWAJournal.org 28 ABSTRACT Antibody-drug conjugates (ADCs) are currently among the fastest growing drug classes in oncology, combining the specificity and targeting capabilities of monoclonal anti- bodies (mAbs) with the potent cytotoxicity of small mole- cule drugs. Considered the “biological missiles” of cancer therapy, ADCs are composed of 3 key elements: (1) a mAb framework that selectively binds to an antigen on the tumor cell surface, (2) a cytotoxic drug payload, and (3) a chemical linker attaching the 2 entities. Because each of these com- ponents can vary widely among ADCs, the associated drug design is relatively complex, with subtle differences leading to immense diversity in the overall drug structure and asso- ciated pharmacological and clinical properties. As medi- cal communication experts, it is essential to have a basic understanding of the various components of ADC design and their potential impact on drug efficacy, safety, and capability in targeting certain degrees of antigen expression and tumor types. This review aims to provide a basic under- standing of each component related to ADC design and the role they play in defining the pharmacological properties of a particular ADC. BACKGROUND First proposed by Paul Ehrlich in the early 1900s, the foun- dational concept of a “magic bullet” as a way to selectively transport cytotoxic drugs to a specific target tissue has become an ever-closer reality, passing through key mile- stones over the last century.1,2 The development of chemo- therapy in the 1940s was a first major step in the transition from concept to reality.3 However, the lack of high-level specificity and targeting capabilities with cytotoxic agents led to a high degree of systemic toxicities and has remained an ongoing challenge. The advent of hybridoma technology and the development of monoclonal antibodies (mAbs) in the 1970s established a highly effective method for target- ing specific antigens expressed on the tumor cell surface.1,4,5 This led to the development of targeted therapeutics that have become an attractive method for improving tumor selectivity and reducing the systemic toxicity associated with traditional chemotherapy.6 Combining these 2 tech- nologies enabled the development of the first antibody-drug conjugate (ADC). In recent decades, ADCs have become a rapidly expanding therapeutic drug class specifically designed to overcome the shortfalls associated with chemo- therapeutic agents.6,7 Currently, there are 13 ADCs that have received US Food and Drug Administration approval for various hematological and solid tumor cancers.1,8-20 ADC MECHANISM OF ACTION ADCs are a group of tripartite drugs made up of a tumor- specific mAb conjugated via a stable linker to a potent cytotoxic payload.21,22 The core concept of an ADC is to use the specific recognition between an antibody and antigen to selectively deliver cytotoxic drugs to the tumor site, after which the payload is released in the tumor via a specific release mechanism.23 The general mechanism of action for an ADC can vary depending on the inherent design (Figure 1, next page). ADC DESIGN The clinical success achieved with a particular ADC is con- tingent upon several key factors: (1) target antigen, (2) anti- body framework, (3) method of conjugation, (4) chemical linker, and (5) cytotoxic payload (Table 1, next page). Target Antigen To achieve a favorable therapeutic index and reduce the potential for off-target toxicity, the selected target anti- gen should be tumor-specific or tumor-associated with a high level of expression in tumor cells and minimal to no expression in healthy tissues.24,28 Following ADC binding, target antigens should internalize efficiently via endocyto- sis to enable ADC entry into the cell and subsequent cel- lular transport and payload release.1,21,29 The target antigen should also undergo efficient recycling or replenishment on the cell surface with no associated antigen shedding into Jason R. Lewis, PharmD, MS, MBA, BCACP / LRx Precision Health Consulting and Medical Communication Services, Nephi, UT Antibody-Drug Conjugates: Understanding Associated Drug Design and Pharmacology SCIENCE SERIES http://www.amwajournal.org AMWAJournal.org 29Antibody-Drug Conjugates Figure 1. General mechanism of action of ADCs with and without bystander killing effect.5,21-23 Following introduction of the antibody-drug conjugate (ADC) into the plasma circulation by intravenous injection, the target antigen is recognized on the tumor cell surface, leading to subsequent binding and formation of an antigen–ADC complex (ADCc). The complex is then internalized via receptor-mediated endocytosis. For ADCs with cleavable linker design, some initial release of drug payload may occur prior to internalization. Once inside the cytosol, endosome, or lysosome, the chemical characteristics of these environments (eg, low pH, high glutathione/thiol levels, proteolytic enzymes) allow for further payload release. In the case of ADCs with noncleavable linker design, compete degradation of the ADC is typically required for payload release. The resulting free cytotoxic drug payload then exerts its cellular destruction via a pathway-specific mechanism (eg, microtubule disruption, DNA intercalation). Hydrophobic or nonpolar payloads are capable of crossing cell membranes, thereby exerting a so-called bystander effect. The bystander killing effect involves the diffusion of free drug across cell membranes from the target tumor cell and into neighboring tumor cells, thereby expanding antitumor activity to tumor cells with low or no target antigen expression, or those that are less accessible directly from the circulatory system, eg, solid tumor cells. Ag, antigen. Table 1. Components of ADC Drug Design1,7,25,26Table 1. Components of ADC Drug Design1,7,25,26 Target antigen Antibody framework Method of conjugation Chemical linker Cytotoxic payload Ideal characteristics • Highly expressed in tumor cells • Homogeneous expression in tumor cells • Minimal presence in circulation and healthy cells Ideal Characteristics • Minimal cross reactivity with healthy tissues • Low immunogenicity • Strong binding affinity for the target antigen • Long PK half-life Conventional/stochastic • Lysine sites • Reduced cysteine sites Site-specific • Engineered reactive cysteine residues • Disulfide re-bridging • Unnatural amino acids • Enzyme assisted ligation • Glycan remodeling and glycoconjugation • pClick technology Cleavable linkers Chemically cleavable • Acid sensitive • Glutathione sensitive Enzymatically cleavable • Peptide based (protease sensitive) • Β-glucuronide based • Phosphate based Noncleavable linkers • Thioether (SMCC) • Maleimido propionyl • Maleimido caproyl Tubulin Inhibitors • Auristatins (eg, MMAE, MMAF) • Maytansinoids (eg, DM1, DM4) • Tubulysins (eg, tubulysin A) DNA damaging agents • Calicheamicins (eg, ozogamicin) • Duocarmycins (eg, duocarmazine) • Pyrrolbenzodiazepines • Camptothecin analogues (eg, govitecan, DXd) Immunomodulators • TLR agonists • STING agonists Abbreviations: Ag, antigen; DM1, mertansine; DM4, ravtansine; DXd, deruxtecan; Fab, fragment antigen binding; Fc, fragment crystallized; MMAE, monomethyl auristatin E; MMAF, monomethyl auristatin F; PK, pharmacokinetic; SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbocylate; STING, stimulator of interferon genes; TLR, toll-like receptor. http://www.amwajournal.org AMWAJournal.org 30Antibody-Drug Conjugates circulation to improve tumor cell targeting and reduce the risk of toxicity.30,31 Solid tumors present a challenge in terms of both the drug accessibility and intratumoral heterogene- ity of the target antigen. Therefore, certain characteristics of ADC design, such as cleavable linkers or nonpolarized payloads, which permit the release or distribution of drug to neighboring tumor cells with low or no antigen expression (the so-called bystander effect), may be advantageous in ADCs targeting these tumor types.31 Antibody Framework The antibody framework used in the ADC structure is essen- tial for facilitating selective binding of target antigens and can significantly influence the overall efficacy, therapeutic index, and pharmacokinetic and pharmacodynamic char- acteristics of the end drug product.1,21 Ideally, the selected mAb should possess a strong binding affinity with high target specificity and minimal cross-reactivity with healthy tissues.24 The selected mAb should also facilitate efficient internalization, demonstrate low immunogenicity, and have a long half-life. Among the 5 major subtypes of human anti- bodies (eg, immunoglobulin [Ig]A, IgD, IgE, IgG, and IgM), IgG antibodies are most often used for ADC development because of their plasma stability and strong binding affin- ity for the fragment crystallized (Fc) receptor.1,21,32 Moreover, because of their decreased potential for immunogenicity, humanized or fully human mAbs are typically favored in ADC design as opposed to murine or chimeric mAbs. Method of Conjugation: Conventional The method of conjugation in ADC design refers to the approach used to connect the linker and payload to the mAb framework.1 Historically, conventional conjugation methods have been the most widely used in ADC design. Conventional conjugation entails either the alkylation or acylation of lysine side chains or the reduction of disulfide bonds to liberate cysteine residues for conjugation sites.21,33 Notably, mAbs such as IgG contain a natural abundance of lysine (80-100) and cysteine (32-40) residues, which offer ideal sites for linker attachment via conventional conjuga- tion involving coupling reactions.1,27,33-36 Conventional conjugation using lysine and cysteine residues is stochastic. This can lead to heterogeneous mix- tures of ADC species with varying sites of conjugation and drug-to-antibody ratios (DARs), defined as the number of drug payloads conjugated to each mAb.37 DARs associated with ADCs produced by conventional conjugation methods often vary, ranging from 0 to 8 or more.38 DAR-associated variability can potentially impact certain ADC character- istics, such as hydrophobicity, charge, polarity, pharma- cokinetics, and thermostability of mAbs.38-40 In some cases this may result in insufficient stability, causing premature payload release and increased potential for off-target toxici- ties.1 Elevated DARs can also lead to aggregation, increased metabolism, or disruptive coupling within the antigen bind- ing region of the mAb.21,41,42 Because lysine residues are dis- tributed throughout both heavy and light chain regions of the antibody, coupling reactions resulting in payload con- jugation near antibody–antigen recognition sites could interfere with ADC target binding.1,39 Moreover, ADCs with cytotoxic payloads conjugated to heavy chain regions of the mAb have lower in vivo efficacy compared with light chain conjugates.24,44 Consequently, novel site-specific conju- gation strategies have been developed that produce more homogeneous ADC products with favorable pharmacoki- netic and antigen binding properties.1,37,41 Method of Conjugation: Site-Specific Site-specific conjugation strategies are categorized accord- ing to their associated methodology and generally include the following: (1) engineered cysteine residues, (2) disulfide rebridging, (3) engineered unnatural amino acids, (4) enzy- matic assisted ligation (5) glycan remodeling/glycoconju- gation, and (6) proximity-induced antibody conjugation method (pClick) technology.1,21,45,46 Site-specific cysteine residues are commonly engineered using THIOMAB tech- nology, which allows specific positioning of conjugation sites within both heavy chain and light chain regions of the antibody.47 Drug conjugates created using THIOMAB technology are typically referred to as THIOMAB-drug conjugates and are shown to have improved safety and ther- apeutic indexes.46 Disulfide rebridging is a process in which 4 interchain disulfide bonds in an IgG antibody are reduced and sub- sequently treated with a cysteine-selective cross-linking reagent.48 This rebridging process enables simultaneous reattachment of polypeptide chain and installation of drug molecules or function groups that may be further mod- ified. Genetically encoded unnatural amino acids, such as p-acetylphenylalanine and p-azidophenylalanine, are other site-specific conjugation methods that have demon- strated specific advantages in terms of optimizing physical properties of the ADC, and improved associated efficacy, pharmacokinetic, and safety profiles.41,49 Compared with conventional cysteine conjugated ADCs, those using unnat- ural amino acids for site-specific conjugation demonstrate superior in vitro selectivity and efficacy, particularly in low antigen expressing tumor cells.50 Enzyme-assisted liga- tion techniques use enzymes such as transglutaminase to conjugate specific amino acid sequences or tags that are genetically engineered and artificially induced to express in the antibody.1,21,51 Glycan remodeling or glycoconjuga- http://www.amwajournal.org AMWAJournal.org 31Antibody-Drug Conjugates tion methods exploit naturally-occurring glycosylation sites at the N297 residue in the CH2 domain of IgG antibod- ies.52 However, relative to other site-specific conjugation approaches, glycan remodeling has limited control over site placement. A site-selective conjugation method using pClick has more recently emerged and uses a proximity activated crosslinker to covalently attach to a specific anti- body site, which eliminates the need for additional antibody engineering or post-synthesis treatments.53 Chemical Linker The primary function of chemical linkers in ADC design is to effectively bridge the mAb to the cytotoxic drug pay- load.1 ADC specificity, potency, safety, and overall activity are greatly influenced by the associated linker chemis- try.24 Linkers are generally designed to remain stable in cir- culation and release the drug payload once the ADC has reached the target tumor site.2,24 Chemical linkers com- monly employed in ADC design are classified into 2 main categories: cleavable and noncleavable linkers.1,24 Cleavable Linkers Cleavable linkers are designed to exploit cancer-specific cellular conditions and are generally divided into 3 pri- mary categories: acid or pH sensitive, glutathione or redox sensitive, and enzymatically cleavable (eg, protease sen- sitive).21,24,28 Acid-sensitive linkers, such as hydrazone, are designed to release the drug payload within acidic environ- ments, such as lysosomal or endosomal cellular compart- ments, while remaining stable within the blood’s neutral pH environment.1,31 Glutathione-sensitive or disulfide linkers exploit the high levels of glutathione and other thiols pres- ent in the cytosol of cancer cells, thereby enabling selec- tive cleavage of the cytotoxic payload at the tumor site.24,28,54 Protease sensitive or peptide-based linkers most often consist of dipeptide linkers (eg, valine-citrulline), which require enzymatic cleavage of peptide bonds for payload release.28,31,55 Compared with chemically liable linkers (eg, hydrazone and disulfide), peptide-based linker tech- nologies allow for greater control of drug delivery, with improved systemic stability and rapid enzyme-mediated release of the drug payload within the target cell.31 Noncleavable Linkers In contrast with cleavable linkers that rely on tumor-specific cellular conditions for payload release, noncleavable linkers require internalization via antigen-mediated endocytosis and lysosome-mediated proteolytic degradation for payload release.24,31,56 Generally, noncleavable linkers are associated with greater plasma stability, longer half-lives, and pose a reduced risk for off-target side effects. However, the actions of ADCs with noncleavable links are typically restricted to the target tumor cell. Moreover, amino acid-drug metab- olites resulting from the drug payload release tend to be more hydrophilic and have greater intrinsic polarity, which reduces bystander effect. Commonly used noncleavable linkers in ADC design include the thioether linker succin- imidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate or maleimide moieties, such as maleimdopropionyl and maleimdocaproyl.25 Cytotoxic Drug Payload The drug payloads employed in the design of ADCs are typ- ically 100 to 1,000 times more potent than the cytotoxic agents used in traditional chemotherapy.24,57 Cytotoxic pay- loads are generally subdivided into 2 main categories: (1) DNA-damaging agents, including calicheamicins, duocar- mycins, pyrrolobenzodiazepines and camptothecin ana- logues; and (2) anti-tubulin agents, including auristatins, maytansinoids, and tubulysins.1,24,28 Calicheamicins (eg, ozogamicin) were first isolated from the actinomycete Micromonospora echinospora in the mid- 1980s, and act by binding the minor groove of DNA, causing site-specific, double-stranded DNA breaks.31,58-60 Notably, calicheamicins are highly hydrophobic, which limits the DAR or number of payload molecules able to be attached per mAb. Duocarmycins are potent alkylating compounds derived from the bacteria species Streptomyces zelensis. Duocarmycin and its derivatives, such as duocarmazine, act by binding to and alkylating adenine within the minor groove of DNA, leading to subsequent DNA strand cleavage and cellular apoptosis.61-64 Pyrrolobenzodiazepine (PBD) dimers, such as tesirine, are DNA-damaging agents derived from anthramycin, an antitumor antibiotic isolated from Streptomyces.65-68 PBD dimers bind the DNA minor groove, forming covalent interstrand DNA crosslinks within tumor cells that lead to cytotoxicity and cell death.28,69,70 The forma- tion of crosslinks resulting from PBD activity, which occurs rapidly and with minimal DNA distortion, is thought to contribute to their persistence and evasion of DNA repair mechanisms. Campothecin is a pentacyclic alkaloid iso- lated from the stem wood of Camototheca acuminata, a tree indigenous to China.71-73 Campothecin and its derivatives (eg, govitecan, deruxtecan) act by inhibiting topoisomerase I enzyme activity, which results in double-stranded DNA breaks and subsequent cell death.72-75 Auristatins commonly used as ADC payloads include monomethyl auristatin E and monomethyl auristatin F, which are both synthetic analogues of dolastatin 10, a natu- rally-occurring antimitotic drug isolated from the sea hare Dolabella auricularia.28,76,77 Auristatins inhibit tubulin polym- erization by attaching to the same binding site as vinca alka- http://www.amwajournal.org AMWAJournal.org 32Antibody-Drug Conjugates loids (eg, vincristine, vinblastine), which are frequently used as traditional chemotherapy agents.78-80 Maytansinoids are benzoansamacrolides derived from the bark of an African shrub, Maytenus ovatus.81 Similar to auristatins, maytansi- noids bind at or near the vinca-binding site, thereby inhib- iting microtubule assembly and inducing mitotic arrest.31,82 Maytansinoids commonly used in ADC design include 2 thiomethyl derivatives of maytansine: mertansine and ravtansine.1,31,83 Tubulysins comprise a family of more recently discovered cytostatic peptides isolated from the myxobacteria species, Archangium gephyra and Angiococcus disciformis, that act by inhibiting microtubule polymerization during mitosis, thereby inducing apoptosis.28,84,85 DISCUSSION Rapid advances in molecular and genomic technologies over the last several decades have ushered in a new era of precision medicine and led to the increased use of these technologies as important clinical tools for the diagnosis, classification, and treatment of disease.86 As the use of pre- cision-based treatment strategies has expanded, biomarker testing has become an increasingly important prognostic and predictive tool to improve disease management and enabled the development of numerous targeted thera- pies.88,89 The effect of precision-based approaches on the treatment landscape has been especially profound in the oncology space, as reflected the growing number of bio- marker-driven clinical trials, which increased from 15% in 2000 to 55% in 2018.89 Although targeted therapies have led to major improvements in progression-free and over- all survival, acquired drug resistance has led to associated therapeutic limitations and the need for other treatment options.86 ADCs in particular represent a unique treatment approach that combines precision-based technology used in targeted therapy approaches with chemotherapy-based strategies using cytotoxic agents with greater potency. Since the approval of the first ADC in 2000, continued technological advancements have led to an explosion in the number of ADCs under clinical development over the last few decades. As of 2022, a total of 12 ADCs with 9 asso- ciated biomarker targets have been approved for use in the treatment of both solid tumor and hematological malignan- cies.7,90 In addition, over 80 ADCs are currently being eval- uated in clinical trials, suggesting that utilization of these novel agents will continue to increase. Most recently, the clinical development of novel ADCs has had a profound impact on the treatment landscape for solid tumors. The DESTINY series of clinical trials, inves- tigated the human epidermal growth factor receptor 2 (HER2)-directed ADC, trastuzumab deruxtecan (T-DXd) in a number of solid tumor types.91 Resulting data from these trials, led to approved indications in HER2-positive meta- static breast cancer (mBC), HER2-low mBC, and gastric or gastroesophageal cancer, as well as accelerated approval in non-small cell lung cancer.16,92-95 Notably, DESTINY trial data surrounding T-DXd in mBC were not only unprece- dented, but also transformative, and have played a major in reshaping how HER2 expression in tumors is assessed and managed. Historically, HER2 expression assessed via immunohistochemistry methods has been associated with a binary classification, namely HER2-positive or HER2- negative.96 Early targeted therapies directed toward the HER2 receptor only demonstrated efficacy in HER2-positive mBC, with no effect in those with HER2-negative status.96 However, the unique ADC design associated with T-DXd and the resulting bystander effect have expanded observed therapeutic responses to patients with lower levels of HER2 expression.97 CONCLUSION As the incidence of cancer increases worldwide, growing demand for safer, more personalized cancer therapies with fewer side effects will undoubtedly propel further advances in ADC technology. Over the last 5 years alone, 39 clinical trials have investigated over 19 ADCs.98,99 Given the pace at which ADC therapeutics are being introduced, the need for clear and accurate communication of information regard- ing these sophisticated treatments will also continue to grow. We as medical communicators must make it our mis- sion to educate ourselves and our readers so that they can approach the literature critically and make informed deci- sions about their use. Acknowledgments We thank Yuqian Lu, MA, for her design of the accompany- ing figure and table. Author declaration and disclosures: The author notes no commercial associations that may pose a conflict of interest in relation to this article. Author contact: lrxprecsionhealth@gmail.com References 1. Fu Z, Li S, Han S, Shi C, Zhang Y. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduct Target Ther. 2022;7(1):93. doi:10.1038/s41392-022-00947-7 2. Feld J, Barta SK, Schinke C, Braunschweig I, Zhou Y, Verma AK. Linked-in: design and efficacy of antibody drug conjugates in oncology. Oncotarget. 2013;4(3):397-412. doi: 10.18632/ oncotarget.924 3. DeVita VT Jr, Chu E. A history of cancer chemotherapy. Cancer Res. 2008;68(21):8643-8653. doi:10.1158/0008-5472.CAN-07-6611 4. Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495- 497. doi:10.1038/256495a0 http://www.amwajournal.org AMWAJournal.org 33Antibody-Drug Conjugates 5. Tong JTW, Harris PWR, Brimble MA, Kavianinia I. An insight into FDA approved antibody-drug conjugates for cancer therapy. Molecules. 2021;26(19):5847. doi:10.3390/molecules26195847 6. LoRusso PM, Weiss D, Guardino E, Girish S, Sliwkowski MX. Trastuzumab emtansine: a unique antibody-drug conjugate in development for human epidermal growth factor receptor 2-positive cancer. Clin Cancer Res. 2011;17(20):6437-6447. doi:10.1158/1078-0432.CCR-11-0762 7. Dean AQ, Luo S, Twomey JD, Zhang B. Targeting cancer with antibody-drug conjugates: promises and challenges. MAbs. 2021;13(1):1951427. doi:10.1080/19420862.2021.1951427 8. Adcetris prescribing information. Seagen Inc. Published 2022. Accessed November 1, 2022. https://www.adcetrispro.com/ 9. Besponsa prescribing information. Pfizer Inc. Published 2018. Accessed November 1, 2022. https://besponsa.pfizerpro.com/ 10. Blenrep prescribing information. GlaxoSmithKline. Published 2022. Accessed November 1, 2022. https://gskpro.com/content/ dam/global/hcpportal/en_US/Prescribing_Information/Blenrep/ pdf/BLENREP-PI-MG.PDF 11. Lumoxiti prescribing information. AstraZeneca Pharmaceuticals LP. Published 2022. Accessed November 1, 2022. https://www. lumoxiti.com/ 12. Mylotarg prescribing information. Pfizer Inc. Published 2021. Accessed November 1, 2022. https://mylotarg.pfizerpro.com/ 13. Polivy prescribing information. Genentech Inc. Published 2020. Accessed November 1, 2022. https://www.polivy.com/hcp.html 14. Zynlonta prescribing information. ADC Therapeutics SA. Published 2022. Accessed November 1, 2022. https://www. zynlontahcp.com/ 15. Elahere prescribing information. ImmunoGen, Inc. Published 2022. Accessed November 23, 2022. https://www.immunogen. com/wp-content/uploads/2022/11/ELAHERE_PI.pdf 16. Enhertu prescribing information. Daiichi Sankyo, Inc. Published 2022. Accessed November 1, 2022. https://www.enhertuhcp.com/ en/breast 17. Kadcyla prescribing information. Genentech Inc. Published 2022. Accessed November 1, 2022. https://www.kadcyla-hcp.com/ 18. Padcev prescribing information. Seagen Inc. Published 2022. Accessed November 1, 2022. https://www.padcev.com/hcp 19. Tivdak prescribing information. Seagen Inc. Published 2022. Accessed November 1, 2022. https://www.tivdakhcp.com/ 20. Trodelvy prescribing information. Gilead Sciences, Inc. Published 2022. Accessed November 1, 2022. https://www.trodelvyhcp.com/ 21. Hafeez U, Parakh S, Gan HK, Scott AM. Antibody-drug conjugates for cancer therapy. Molecules. 2020;25(20):4764. doi:10.3390/ molecules25204764 22. Giugliano F, Corti C, Tarantino P, Michelini F, Curigliano G. Bystander effect of antibody-drug conjugates: fact or fiction? Curr Oncol Rep. 2022;24(7):809-817. doi:10.1007/s11912-022-01266-4 23. Trail PA, Dubowchik GM, Lowinger TB. Antibody drug conjugates for treatment of breast cancer: novel targets and diverse approaches in ADC design. Pharmacol Ther. 2018;181:126-142. doi:10.1016/j.pharmthera.2017.07.013 24. Peters C, Brown S. Antibody-drug conjugates as novel anti-cancer chemotherapeutics. Biosci Rep. 2015;35(4):e00225. doi:10.1042/ BSR20150089 25. Singh D, Dheer D, Samykutty A, Shankar R. Antibody drug conjugates in gastrointestinal cancer: from lab to clinical development. J Control Release. 2021;340:1-34. doi:10.1016/j. jconrel.2021.10.006 26. Sheyi R, de la Torre BG, Albericio F. Linkers: an assurance for controlled delivery of antibody-drug conjugate. Pharmaceutics. 2022;14(2):396. doi:10.3390/pharmaceutics14020396 27. You J, Zhang J, Wang J, Jin M. Cysteine-based coupling: challenges and solutions. Bioconjug Chem. 2021;32(8):1525-1534. doi:10.1021/acs.bioconjchem.1c00213 28. Beck A, Goetsch L, Dumontet C, Corvaïa N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017;16(5):315-337. doi:10.1038/ nrd.2016.268 29. Damelin M, Zhong W, Myers J, Sapra P. Evolving strategies for target selection for antibody-drug conjugates. Pharm Res. 2015;32(11):3494-3507. doi:10.1007/s11095-015-1624-3 30. Carter PJ, Senter PD. Antibody-drug conjugates for cancer therapy. Cancer J. 2008;14(3):154-169. doi:10.1097/ PPO.0b013e318172d704 31. Nolting B. Linker technologies for antibody-drug conjugates. Methods Mol Biol. 2013;1045:71-100. doi:10.1007/978-1-62703- 541-5_5 32. Wang W, Wang EQ, Balthasar JP. Monoclonal antibody pharmacokinetics and pharmacodynamics. Clin Pharmacol Ther. 2008;84(5):548-558. doi:10.1038/clpt.2008.170 33. Brun MP, Gauzy-Lazo L. Protocols for lysine conjugation. Methods Mol Biol. 2013;1045:173-187. doi:10.1007/978-1-62703-541-5_10 34. Chumsae C, Gaza-Bulseco G, Liu H. Identification and localization of unpaired cysteine residues in monoclonal antibodies by fluorescence labeling and mass spectrometry. Anal Chem. 2009;81(15):6449-6457. doi:10.1021/ac900815z 35. Adumeau P, Sharma SK, Brent C, Zeglis BM. Site-specifically labeled immunoconjugates for molecular imaging—part 1: cysteine residues and glycans. Mol Imaging Biol. 2016;18(1):1-17. doi:10.1007/s11307-015-0919-4 36. Padlan EA. Anatomy of the antibody molecule. Mol Immunol. 1994;31(3):169-217. doi:10.1016/0161-5890(94)90001-9 37. Shefet-Carasso L, Benhar I. Antibody-targeted drugs and drug resistance—challenges and solutions. Drug Resist Updat. 2015;18:36-46. doi:10.1016/j.drup.2014.11.001 38. Boylan NJ, Zhou W, Proos RJ, Tolbert TJ, Wolfe JL, Laurence JS. Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates. Bioconjug Chem. 2013;24(6):1008- 1016. doi:10.1021/bc4000564 39. Wakankar AA, Feeney MB, Rivera J, et al. Physicochemical stability of the antibody-drug conjugate trastuzumab-DM1: changes due to modification and conjugation processes. Bioconjug Chem. 2010;21(9):1588-1595. doi:10.1021/bc900434c 40. Acchione M, Kwon H, Jochheim CM, Atkins WM. Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates. MAbs. 2012;4(3):362-372. doi:10.4161/mabs.19449 41. Hallam TJ, Wold E, Wahl A, Smider VV. Antibody conjugates with unnatural amino acids. Molecular Pharmaceutics. 2015;12:1848- 1862. doi: 10.1021/acs.molpharmaceut.5b00082 42. Hamblett KJ, Senter PD, Chace DF, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004;10(20):7063-7070. doi:10.1158/1078-0432. CCR-04-0789 43. Fukunaga A, Maeta S, Reema B, Nakakido M, Tsumoto K. Improvement of antibody affinity by introduction of basic amino acid residues into the framework region. Biochem Biophys Rep. 2018;15:81-85. doi:10.1016/j.bbrep.2018.07.005 44. Shen BQ, Xu K, Liu L, et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol. 2012;30(2):184-189. doi:10.1038/nbt.2108 45. Chen XN, Nguyen M, Jacobson F, Ouyang J. Charge-based analysis of antibodies with engineered cysteines: from multiple peaks to a single main peak. MAbs. 2009;1(6):563-571. doi:10.4161/ mabs.1.6.10058 46. Junutula JR, Raab H, Clark S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol. 2008;26(8):925-932. doi:10.1038/nbt.1480 47. Zhou Q. Site-specific antibody conjugation for ADC and beyond. Biomedicines. 2017;5(4):64. doi:10.3390/biomedicines5040064 http://www.amwajournal.org https://www.adcetrispro.com/ https://besponsa.pfizerpro.com/ https://gskpro.com/content/dam/global/hcpportal/en_US/Prescribing_Information/Blenrep/pdf/BLENREP-PI-MG.PDF https://gskpro.com/content/dam/global/hcpportal/en_US/Prescribing_Information/Blenrep/pdf/BLENREP-PI-MG.PDF https://gskpro.com/content/dam/global/hcpportal/en_US/Prescribing_Information/Blenrep/pdf/BLENREP-PI-MG.PDF https://www.lumoxiti.com/ https://www.lumoxiti.com/ https://mylotarg.pfizerpro.com/ https://www.polivy.com/hcp.html https://www.zynlontahcp.com/ https://www.zynlontahcp.com/ https://www.immunogen.com/wp-content/uploads/2022/11/ELAHERE_PI.pdf https://www.immunogen.com/wp-content/uploads/2022/11/ELAHERE_PI.pdf https://www.enhertuhcp.com/en/breast https://www.enhertuhcp.com/en/breast https://www.kadcyla-hcp.com/ https://www.padcev.com/hcp https://www.tivdakhcp.com/ https://www.trodelvyhcp.com/ AMWAJournal.org 34Antibody-Drug Conjugates 48. Walsh SJ, Bargh JD, Dannheim FM, et al. Site-selective modification strategies in antibody-drug conjugates. Chem Soc Rev. 2021;50:1305-1353. doi: 10.1039/d0cs00310g 49. Axup JY, Bajjuri KM, Ritland M, et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci USA. 2012;109(40):16101-16106. doi:10.1073/ pnas.1211023109 50. Tian F, Lu Y, Manibusan A, et al. A general approach to site- specific antibody drug conjugates. Proc Natl Acad Sci USA. 2014;111(5):1766-1771. doi:10.1073/pnas.1321237111 51. Kim EG, Kim KM. Strategies and advancement in antibody- drug conjugate optimization for targeted cancer therapeutics. Biomol Ther (Seoul). 2015;23(6):493-509. doi:10.4062/ biomolther.2015.116 52. Agarwal P, Bertozzi CR. Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug Chem. 2015;26(2):176-192. doi:10.1021/bc5004982 53. Cao YJ, Yu C, Wu KL, et al. Synthesis of precision antibody conjugates using proximity-induced chemistry. Theranostics. 2021;11(18):9107-9117. doi:10.7150/thno.62444 54. Balendiran GK, Dabur R, Fraser D. The role of glutathione in cancer. Cell Biochem Funct. 2004;22(6):343-352. doi:10.1002/ cbf.1149 55. Sanderson RJ, Hering MA, James SF, et al. In vivo drug- linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res. 2005;11(2 Pt 1):843-852. 56. Erickson HK, Park PU, Widdison WC, et al. Antibody- maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006;66(8):4426-4433. doi:10.1158/0008- 5472.CAN-05-4489 57. Pietersz GA, Krauer K. Antibody-targeted drugs for the therapy of cancer. J Drug Target. 1994;2(3):183-215. doi:10.3109/10611869408996804 58. Shor B, Gerber HP, Sapra P. Preclinical and clinical development of inotuzumab-ozogamicin in hematological malignancies. Mol Immunol. 2015;67(2 Pt A):107-116. doi:10.1016/j. molimm.2014.09.014 59. Walker S, Landovitz R, Ding WD, Ellestad GA, Kahne D. Cleavage behavior of calicheamicin gamma 1 and calicheamicin T. Proc Natl Acad Sci USA. 1992;89(10):4608-4612. doi:10.1073/ pnas.89.10.4608 60. Elmroth K, Nygren J, Mårtensson S, Ismail IH, Hammarsten O. Cleavage of cellular DNA by calicheamicin gamma1. DNA Repair (Amst). 2003;2(4):363-374. doi:10.1016/s1568-7864(02)00235-5 61. Yao HP, Zhao H, Hudson R, Tong XM, Wang MH. Duocarmycin- based antibody-drug conjugates as an emerging biotherapeutic entity for targeted cancer therapy: pharmaceutical strategy and clinical progress. Drug Discov Today. 2021;26(8):1857-1874. doi:10.1016/j.drudis.2021.06.012 62. Hanka LJ, Dietz A, Gerpheide SA, Kuentzel SL, Martin DG. CC-1065 (NSC-298223), a new antitumor antibiotic. Production, in vitro biological activity, microbiological assays and taxonomy of the producing microorganism. J Antibiot (Tokyo). 1978;31(12):1211-1217. doi:10.7164/antibiotics.31.1211 63. Menderes G, Bonazzoli E, Bellone S, et al. SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows promising antitumor activity in epithelial ovarian carcinoma with HER2/Neu expression. Gynecol Oncol. 2017;146(1):179-186. doi: 10.1016/j.ygyno.2017.04.023. 64. Boger DL. The duocarmycins: synthetic and mechanistic studies. Acc Chem Res. 1995;28(1):20-29. doi:10.1021/ar00049a004 65. Gerratana B. Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. Med Res Rev. 2012;32(2):254-293. doi:10.1002/med.20212 66. Tiberghien AC, Levy JN, Masterson LA, et al. Design and synthesis of tesirine, a clinical antibody-drug conjugate pyrrolobenzodiazepine dimer payload. ACS Med Chem Lett. 2016;7(11):983-987. doi: 10.1021/acsmedchemlett.6b00062. 67. Tendler MD, Korman S. 'Refuin': a non-cytotoxic carcinostatic compound proliferated by a thermophilic actinomycete. Nature. 1963;199:501. doi:10.1038/199501a0 68. Leimgruber W, Stefanović V, Schenker F, Karr A, Berger J. Isolation and characterization of anthramycin, a new antitumor antibiotic. J Am Chem Soc. 1965;87(24):5791-5793. doi:10.1021/ja00952a050 69. Hartley JA, Flynn MJ, Bingham JP, et al. Pre-clinical pharmacology and mechanism of action of SG3199, the pyrrolobenzodiazepine (PBD) dimer warhead component of antibody-drug conjugate (ADC) payload tesirine. Sci Rep. 2018;8(1):10479. doi:10.1038/ s41598-018-28533-4 70. Caimi PF, Ai W, Alderuccio JP, et al. Loncastuximab tesirine in relapsed or refractory diffuse large B-cell lymphoma (LOTIS-2): a multicentre, open-label, single-arm, phase 2 trial. Lancet Oncol. 2021;22(6):790-800. doi:10.1016/S1470-2045(21)00139-X 71. Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. Plant Antitumor Agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from camptotheca acuminata 1,2. J Am Chem Soc. 1966;88(16):3888-3890. doi:10.1021/ja00968a057 72. Li W, Veale KH, Qiu Q, et al. Synthesis and evaluation of camptothecin antibody-drug conjugates. ACS Med Chem Lett. 2019;10(10):1386-1392. doi:10.1021/acsmedchemlett.9b00301 73. Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985;260(27):14873-14878. doi:10.1016/S0021- 9258(17)38654-4 74. Ogitani Y, Aida T, Hagihara K, et al. DS-8201a, a novel HER2- targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a promising antitumor efficacy with differentiation from T-DM1. Clin Cancer Res. 2016;22(20):5097-5108. doi:10.1158/1078-0432.CCR-15-2822 75. Goldenberg DM, Sharkey RM. Antibody-drug conjugates targeting TROP-2 and incorporating SN-38: A case study of anti-TROP-2 sacituzumab govitecan. MAbs. 2019;11(6):987-995. doi:10.1080/19 420862.2019.1632115 76. Senter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol. 2012;30(7):631-637. doi:10.1038/nbt.2289 77. Pettit GR, Kamano Y, Herald CL, et al. The isolation and structure of a remarkable marine animal antineoplastic constituent: dolastatin 10. J Am Chem Soc. 1987;109(22):6883-6885. doi:10.1021/ja00256a070 78. Bai R, Pettit GR, Hamel E. Dolastatin 10, a powerful cytostatic peptide derived from a marine animal. Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain. Biochem Pharmacol. 1990;39(12):1941-1949. doi:10.1016/0006-2952(90)90613-p 79. Bai RL, Pettit GR, Hamel E. Structure-activity studies with chiral isomers and with segments of the antimitotic marine peptide dolastatin 10. Biochem Pharmacol. 1990;40(8):1859-1864. doi:10.1016/0006-2952(90)90367-t 80. Waight AB, Bargsten K, Doronina S, Steinmetz MO, Sussman D, Prota AE. Structural basis of microtubule destabilization by potent auristatin anti-mitotics. PLoS One. 2016;11(8):e0160890. doi: 10.1371/journal.pone.0160890. 81. Kupchan SM, Komoda Y, Branfman AR, et al. The maytansinoids. Isolation, structural elucidation, and chemical interrelation of novel ansa macrolides. J Org Chem. 1977;42(14):2349-2357. doi:10.1021/jo00434a001 82. Lambert JM, Chari RV. Ado-trastuzumab Emtansine (T-DM1): an http://www.amwajournal.org AMWAJournal.org 35Antibody-Drug Conjugates antibody-drug conjugate (ADC) for HER2-positive breast cancer. J Med Chem. 2014;57(16):6949-6964. doi:10.1021/jm500766w 83. Chen H, Lin Z, Arnst KE, Miller DD, Li W. Tubulin inhibitor-based antibody-drug conjugates for cancer therapy. Molecules. 2017 Aug 1;22(8):1281. doi: 10.3390/molecules22081281. PMID: 28763044; PMCID: PMC6152078. 84. Pegram MD, Hamilton EP, Tan AR, et al. First-in-human, phase 1 dose-escalation study of biparatopic anti-HER2 antibody-drug conjugate MEDI4276 in patients with HER2-positive advanced breast or gastric cancer. Mol Cancer Ther. 2021;20(8):1442-1453. doi:10.1158/1535-7163.MCT-20-0014 85. Sasse F, Steinmetz H, Heil J, Höfle G, Reichenbach H. Tubulysins, new cytostatic peptides from myxobacteria acting on microtubuli. Production, isolation, physico-chemical and biological properties. J Antibiot (Tokyo). 2000;53(9):879-885. doi:10.7164/ antibiotics.53.879 86. Peters S, Mok T, Passaro A, Jänne PA. The promising evolution of targeted therapeutic strategies in cancer. Cancer Discov. 2021;11(4):810-814. doi:10.1158/2159-8290.CD-21-0124 87. El-Deiry WS, Goldberg RM, Lenz HJ, et al. The current state of molecular testing in the treatment of patients with solid tumors, 2019. CA Cancer J Clin. 2019;69(4):305-343. doi:10.3322/ caac.21560 88. Seebacher NA, Stacy AE, Porter GM, Merlot AM. Clinical development of targeted and immune based anti-cancer therapies. J Exp Clin Cancer Res. 2019;38(1):156. doi:10.1186/ s13046-019-1094-2 89. Vadas A, Bilodeau TJ, Oza C. The evolution of biomarker use in clinical trials for cancer treatments. PMC; 2019. Accessed February 7, 2022. http://www.personalizedmedicinecoalition. org/Userfiles/PMC-Corporate/file/The_Evolution_of_Biomarker_ Use_in_Clinical_Trials_for_Cancer_Treatments.pdf 90. FDA approved antibody-drug conjugates (ADCs) up to 2023. Biopharma PEG. Published October 30, 2019. Accessed February 19, 2023. https://www.biochempeg.com/article/74.html 91. Destiny clinical trials. Destinyclinicaltrials.com. Accessed February 19, 2023. https://www.destinyclinicaltrials.com/en 92. FDA grants regular approval to fam-trastuzumab deruxtecan-nxki for breast cancer. US Food and Drug Administration. Published May 11, 2022. Accessed February 19, 2023. https://www.fda.gov/ drugs/resources-information-approved-drugs/fda-grants-regular- approval-fam-trastuzumab-deruxtecan-nxki-breast-cancer 93. FDA approves fam-trastuzumab deruxtecan-nxki for HER2- positive gastric adenocarcinomas. US Food and Drug Administration. Published January 15, 2021. Accessed February 19, 2023. https://www.fda.gov/drugs/resources-information- approved-drugs/fda-approves-fam-trastuzumab-deruxtecan- nxki-her2-positive-gastric-adenocarcinomas 94. FDA grants accelerated approval to fam-trastuzumab deruxtecan-nxki for HER2-mutant non-small cell lung cancer. US Food and Drug Administration. Published August 16, 2022. Accessed February 19, 2023. https://www.fda.gov/drugs/ resources-information-approved-drugs/fda-grants-accelerated- approval-fam-trastuzumab-deruxtecan-nxki-her2-mutant-non- small-cell-lung 95. FDA approves fam-trastuzumab deruxtecan-nxki for HER2-low breast cancer. US Food and Drug Administration. Published August 5, 2022. Accessed February 19, 2023. https://www.fda.gov/ drugs/resources-information-approved-drugs/fda-approves-fam- trastuzumab-deruxtecan-nxki-her2-low-breast-cancer 96. Eiger D, Agostinetto E, Saúde-Conde R, de Azambuja E. The exciting new field of HER2-low breast cancer treatment. Cancers (Basel). 2021;13(5):1015. doi:10.3390/cancers13051015 97. Modi S, Jacot W, Yamashita T, et al. Trastuzumab deruxtecan in previously treated HER2-low advanced breast cancer. N Engl J Med. 2022;387(1):9-20. doi:10.1056/NEJMoa2203690 98. Abuhelwa Z, Alloghbi A, Nagasaka M. A comprehensive review on antibody-drug conjugates (ADCs) in the treatment landscape of non-small cell lung cancer (NSCLC). Cancer Treat Rev. 2022;106:102393. doi:10.1016/j.ctrv.2022.102393 99. Mullin R. New day for antibody-drug conjugates. Chemical & Engineering News. May 15, 2022. 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