World Antibody Drug Conjugate Summit Europe: February 21–23, 2011; Frankfurt,Germany

The World Antibody Drug Conjugate Summit Europe, organized by Biorbis/Hanson Wade was held in Frankfurt, Germany February 21–23, 2011. Antibody drug conjugates (ADCs), also called immunoconjugates, are becoming an increasingly important class of therapeutics as demonstrated by the attendance of nearly 100 delegates at this highly focused meeting. Updates on three ADCs that are in late-stage clinical development, trastuzumab emtansine (T-DM1), brentuximab vedotin (SGN-35) and inotuzumab ozogamicin (CMC-544), were presented by speakers from ImmunoGen, Genentech, Roche, Seattle Genetics and Pfizer. These ADCs have shown encouraging therapeutic effects against solid tumors (T-DM1) and hematological malignancies (SGN-35, CMC-544). The key feature of the new generation of ADCs is the effective combination of the cytotoxicity of natural or synthetic highly potent antineoplastic agents, tumor selective monoclonal antibodies and blood-stable optimized linkers. Early clinical data for ADCs were showcased by Progenics Pharmaceuticals (PSMA ADC), Celldex (CDX-011) and Biotest (BT-062). Takeda, MedImmune and sanofi-aventis outlined their strategies for process development and analytical characterization. In addition, presentations on duocarmycin based-ADCs, α emitting immunoconjugates and antibody-conjugated nanoparticles were given by representatives from Syntarga, Algeta and the University of Stuttgart, respectively.Key words: antibody drug conjugates, immunoconjugates, trastuzumab emtansine, brentuximab vedotin, inotuzumab ozogamicin, oncology, cancer     

Fourth World Antibody-Drug Conjugate Summit

The 4th World Antibody Drug Conjugate (WADC) Summit, organized by Hanson Wade was held on February 29?March 1, 2012 in Frankfurt, Germany, which was also the location for the Antibody Drug Conjugate Summit Europe held in February 2011. During the one year between these meetings, antibody drug conjugates (ADCs) have confirmed their technological maturity and their clinical efficacy in oncology. Brentuximab vedotin (ADCETRIS^TM) gained approval by the US Food and Drug Administration in August 2011 and trastuzumab emtansine (T-DM1) confirmed impressive clinical efficacy responses in a large cohort of breast cancer patients. During the 4th WADC meeting, antibody-maytansinoid conjugates were showcased by representatives of ImmunoGen (T-DM1, SAR3419, lorvotuzumab mertansine/IMGN801, IMGN529 and IMG853) and Biotest (BT-062). Data on antibody-auristatin conjugates were presented by scientists and clinicians from Seattle Genetics and Takeda (brentuximab vedotin), Pfizer (5T4-MMAF), Agensys/Astella (AGS-16M8F), Progenics (PSMA-ADC) and Genmab (anti-TF ADCs). Alternative payloads such as calicheamicins and duocarmycin used for preparation of ADCs were discussed by Pfizer and Synthon representatives, respectively. In addition, emerging technologies, including site-directed conjugation (Ambrx), a protein toxin as payload (Viventia), hapten-binding bispecific antibodies (Roche), and use of light activated drugs (Photobiotics), were also presented. Last but not least, progresses in solving Chemistry Manufacturing and Control, and pharmacokinetic issues were addressed by scientists from Genentech, Pfizer, Novartis and Pierre Fabre.     

Fourth World Antibody-Drug Conjugate Summit: February 29–March 1, 2012, Frankfurt,Germany

The 4th World Antibody Drug Conjugate (WADC) Summit, organized by Hanson Wade was held on February 29‑March 1, 2012 in Frankfurt, Germany, which was also the location for the Antibody Drug Conjugate Summit Europe held in February 2011. During the one year between these meetings, antibody drug conjugates (ADCs) have confirmed their technological maturity and their clinical efficacy in oncology. Brentuximab vedotin (ADCETRIS^TM) gained approval by the US Food and Drug Administration in August 2011 and trastuzumab emtansine (T-DM1) confirmed impressive clinical efficacy responses in a large cohort of breast cancer patients. During the 4th WADC meeting, antibody-maytansinoid conjugates were showcased by representatives of ImmunoGen (T-DM1, SAR3419, lorvotuzumab mertansine/IMGN801, IMGN529 and IMG853) and Biotest (BT-062). Data on antibody-auristatin conjugates were presented by scientists and clinicians from Seattle Genetics and Takeda (brentuximab vedotin), Pfizer (5T4-MMAF), Agensys/Astella (AGS-16M8F), Progenics (PSMA-ADC) and Genmab (anti-TF ADCs). Alternative payloads such as calicheamicins and duocarmycin used for preparation of ADCs were discussed by Pfizer and Synthon representatives, respectively. In addition, emerging technologies, including site-directed conjugation (Ambrx), a protein toxin as payload (Viventia), hapten-binding bispecific antibodies (Roche), and use of light activated drugs (Photobiotics), were also presented. Last but not least, progresses in solving Chemistry Manufacturing and Control, and pharmacokinetic issues were addressed by scientists from Genentech, Pfizer, Novartis and Pierre Fabre.     

Novel peptide linkers for highly potent antibody-auristatin conjugate

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.     

Antibody–drug conjugates as novel anti-cancer chemotherapeutics

Over the past couple of decades, antibody–drug conjugates (ADCs) have revolutionized the field of cancer chemotherapy. Unlike conventional treatments that damage healthy tissues upon dose escalation, ADCs utilize monoclonal antibodies (mAbs) to specifically bind tumour-associated target antigens and deliver a highly potent cytotoxic agent. The synergistic combination of mAbs conjugated to small-molecule chemotherapeutics, via a stable linker, has given rise to an extremely efficacious class of anti-cancer drugs with an already large and rapidly growing clinical pipeline. The primary objective of this paper is to review current knowledge and latest developments in the field of ADCs. Upon intravenous administration, ADCs bind to their target antigens and are internalized through receptor-mediated endocytosis. This facilitates the subsequent release of the cytotoxin, which eventually leads to apoptotic cell death of the cancer cell. The three components of ADCs (mAb, linker and cytotoxin) affect the efficacy and toxicity of the conjugate. Optimizing each one, while enhancing the functionality of the ADC as a whole, has been one of the major considerations of ADC design and development. In addition to these, the choice of clinically relevant targets and the position and number of linkages have also been the key determinants of ADC efficacy. The only marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), have demonstrated their use against both haematological and solid malignancies respectively. The success of future ADCs relies on improving target selection, increasing cytotoxin potency, developing innovative linkers and overcoming drug resistance. As more research is conducted to tackle these issues, ADCs are likely to become part of the future of targeted cancer therapeutics.     

Antibody-drug conjugates: Design and development for therapy and imaging in and beyond cancer,LabEx MAbImprove industrial workshop,July 27–28, 2017, Tours,France

The annual “Antibody Industrial Symposium”, co organized by LabEx MAbImprove, MabDesign and Polepharma, was held in Tours, France on June 27–28, 2017. The focus was on antibody-drug-conjugates (ADCs), new entities which realize the hope of Paul Ehrlich's magic bullet. ADCs result from the bioconjugation of a highly cytotoxic drug to a selective monoclonal antibody, which acts as a vector. Building on knowledge gained during the development of three approved ADCs, brentuximab vedotin (Adcetris®), ado trastuzumab emtansine (Kadcyla®) and inotuzumab ozogamicin (Besponsa®), and the many ADCs in development, this meeting addressed strategies and the latest innovations in the field from fundamental research to manufacturing.     

American Association of Pharmaceutical Scientists National Biotechnology Conference Short Course: Translational Challenges in Developing Antibody-Drug Conjugates: May 24, 2012, San Diego,CA

The American Association of Pharmaceutical Scientists (AAPS) National Biotechnology Conference Short Course “Translational Challenges in Developing Antibody-Drug Conjugates (ADCs),” held May 24, 2012 in San Diego, CA, was organized by members of the Pharmacokinetics, Pharmacodynamics and Drug Metabolism section of AAPS. Representatives from the pharmaceutical industry, regulatory authorities, and academia in the US and Europe attended this short course to discuss the translational challenges in ADC development and the importance of characterizing these molecules early in development to achieve therapeutic utility in patients. Other areas of discussion included selection of target antigens; characterization of absorption, distribution, metabolism, and excretion; assay development and hot topics like regulatory perspectives and the role of pharmacometrics in ADC development. MUC16-targeted ADCs were discussed to illustrate challenges in preclinical development; experiences with trastuzumab emtansine (T-DM1; Genentech) and the recently approved brentuximab vedotin (Adcetris®; Seattle Genetics) were presented in depth to demonstrate considerations in clinical development. The views expressed in this report are those of the participants and do not necessarily represent those of their affiliations.     

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine,a lysine-linked antibody drug conjugate

Antibody–drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla®). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin®), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1.     

Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

Antibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual drug-linker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiple-level structural assessment protocols.     

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models

HER2-ECD (human epidermal growth factor receptor 2 – extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies’ propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.     

Antibody-drug conjugates in breast cancer: Marching from HER2-overexpression into HER2-low

The recent decade has witnessed a vigorous prosper of antibody-drug conjugates (ADCs) in solid tumors including breast cancer. Integrating the specificity of monoclonal antibodies and potency of cytotoxic drugs, ADCs are capable of delivering cytotoxic agents directly to tumor cells and surrounding accomplices with heterogeneous antigen expression by exerting the distinctive bystander effect. Up till now, three ADCs (T-DM1, T-DXd and SG) have attained the official approval and stepped into clinical practices in breast cancer, with numerous promising products in the pipeline. As an unprecedented breast cancer subgroup identified following solidified drug benefit, the cognitive and therapeutic paradigm of HER2-low population which was previously thought lacking definite targets and efficacious regimens has been thoroughly rewritten by ADCs, and several encouraging achievements are expected in ongoing trials. Herein, we discuss the contrived knowledge, latest advancements and future perspectives of ADCs in HER2-overexpressing and HER2-low breast cancer.     

抗体偶联药物的技术现状和展望

抗体偶联药物（antibody drug conjugate,ADC）通常由抗体通过链接体与毒素小分子偶联而成,同时具备抗体的高靶向性和小分子药物的高活性,使之作为一种新兴的靶向治疗手段,在肿瘤治疗领域展现出了优秀的疗效和潜力,成为药物研发领域的新热点。目前全球已有14款ADC药物获批上市,处于临床研究阶段的ADC候选药物分子超过140个。为了进一步提高ADC药物的安全性和有效性,近年来涌现出了各种新颖的技术。本文对ADC药物分子的关键元素,包括抗体、链接体、毒素小分子以及偶联技术等方面的最新研究进展进行总结,并讨论其优缺点。期望这些讨论能够帮助增加对ADC药物研究和开发更加系统的理解,为研发出更加高效和安全的ADC药物带来一些思考。     

Potential drugs used in the antibody–drug conjugate (ADC) architecture for cancer therapy

Cytotoxic small-molecule drugs have a major influence on the fate of antibody–drug conjugates (ADCs). An ideal cytotoxic agent should be highly potent, remain stable while linked to ADCs, kill the targeted tumor cell upon internalization and release from the ADCs, and maintain its activity in multidrug-resistant tumor cells. Lessons learned from successful and failed experiences in ADC development resulted in remarkable progress in the discovery and development of novel highly potent small molecules. A better understanding of such small-molecule drugs is important for development of effective ADCs. The present review discusses requirements making a payload appropriate for antitumor ADCs and focuses on the main characteristics of commonly-used cytotoxic payloads that showed acceptable results in clinical trials. In addition, the present study represents emerging trends and recent advances of payloads used in ADCs currently under clinical trials.     

Site-specific and hydrophilic ADCs through disulfide-bridged linker and branched PEG

Kadcyla® (T-DM1), an antibody–drug conjugates (ADCs) for HER2+ breast cancer treatment, has been approved by the Food and Drug Administration (FDA) in 2013. An ADC of random lysine conjugation, it has difficulties in DAR control and unsatisfactory PK due to uneven DAR distribution. It also gives rise to aggregation during conjugation because of the hydrophobicity nature of the cytotoxin, DM1. The linker-drug in T-DM1, SMCC-DM1 is hydrophobic and requires certain percentage of organic solvent such as DMA in the conjugation solution, limiting the manufacturing process in an organic-solvent-compatible device and adding extra costs. To address these problems, a site-specific conjugation method was developed involving full reduction of antibody and full conjugation with the bridge-like conjugator-drug, based on the work of Caddick and co-workers, to obtain a site-directed antibody-drug conjugate with DAR 4. The bridge-like conjugator was assembled with SMCC-DM1 and different lengths of hydrophilic polyethylene glycol (PEG) moiety. By applying PEG moiety in the side chain of the linker-drug, the organic solvent used in the conjugation can be reduced. When the PEG length is about 26 units, organic solvent is no longer needed in the conjugation. Reducing the amount of organic solvent in conjugation could also diminish the aggregation occurrence during the conjugation. Moreover, the conjugation configuration with the designed conjugator was also discussed in the article. The binding affinity of the resulting ADCs did not show significant decrease and the cell based assay and animal study have shown the comparable results with T-DM1.     

Antibody drug conjugates

Antibody drug conjugates (ADCs) have emerged as a viable option in targeted delivery of highly potent cytotoxic drugs in treatment of solid tumors. At the time of writing, only two ADCs have received regulatory approval with >40 others in clinical development. The first generation ADCs suffered from a lack of specificity in amino acid site-conjugations, yielding statistically heterogeneous stoichiometric ratios of drug molecules per antibody molecule. For the second generation ADCs, however, site-specific amino acid conjugation using enzymatic ligation, introduction of unnatural amino acids, and site-specific protein engineering hold promise to alleviate some of the current technical limitations. The rapid progress in technology platforms and antibody engineering has introduced novel linkers, site-specific conjugation chemistry, and new payload candidates that could possibly be exploited in the context of ADCs. A search using the Clinical Trial Database registry (www.clinicaltrials.gov), using the keyword ‘antibody drug conjugate’, yielded ~270 hits. The main focus of this article is to present a brief overview of the recent developments and current challenges related to ADC development.     

Targeting the autophagy promoted antitumor effect of T-DM1 on HER2-positive gastric cancer

Trastuzumab emtansine (T-DM1), an antibody-drug conjugate consisted of the HER2-targeted monoclonal antibody trastuzumab and the tubulin inhibitor emtansine, has shown potent therapeutic value in HER2-positive breast cancer (BC). However, a clinical trial indicated that T-DM1 exerts a limited effect on HER2-positive gastric cancer (GC), but the underlying mechanism is inconclusive. Our research attempted to reveal the probable mechanism and role of autophagy in T-DM1-treated HER2-positive GC. In this study, our results showed that T-DM1 induced apoptosis and exhibited potent therapeutic efficacy in HER2-positive GC cells. In addition, autophagosomes were observed by transmission electron microscopy. Autophagy was markedly activated and exhibited the three characterized gradations of autophagic flux, consisting of the formation of autophagosomes, the fusion of autophagosomes with lysosomes, and the deterioration of autophagosomes in autolysosomes. More importantly, autophagic inhibition by the suppressors 3-methyladenine (3-MA) and LY294002 significantly potentiated cytotoxicity and apoptosis in HER2-positive GC cells in vitro, while the combined use of LY294002 and T-DM1 elicited potent anti-GC efficacy in vivo. In mechanistic experiments, immunoblot analysis indicated the downregulated levels of Akt, mTOR, and P70S6K and confocal microscopy analysis clearly showed that autophagic inhibition promoted the fusion of T-DM1 molecules with lysosomes in GC cells. In conclusion, our research demonstrated that T-DM1 induced apoptosis as well as cytoprotective autophagy, and autophagic inhibition could potentiate the antitumor effect of T-DM1 on HER2-positive GC. Furthermore, autophagic inhibition might increase the fusion of T-DM1 with lysosomes, which might accelerate the release of the cytotoxic molecule emtansine from the T-DM1 conjugate. These findings highlight a promising therapeutic strategy that combines T-DM1 with an autophagy inhibitor to treat HER-positive GC more efficiently.Subject terms: Targeted therapies, Tumour biomarkers     

Arming antibodies: prospects and challenges for immunoconjugates

Immunoconjugates--monoclonal antibodies (mAbs) coupled to highly toxic agents, including radioisotopes and toxic drugs (ineffective when administered systemically alone)--are becoming a significant component of anticancer treatments. By combining the exquisite targeting specificity of mAbs with the enhanced tumor-killing power of toxic effector molecules, immunoconjugates permit sensitive discrimination between target and normal tissue, resulting in fewer toxic side effects than most conventional chemotherapeutic drugs. Two radioimmunoconjugates, ibritumomab tiuxetan (Zevalin) and tositumomab-131I (Bexxar), and one drug conjugate, gemtuzumab ozogamicin (Mylotarg), are now on the market. For the next generation of immunoconjugates, advances in protein engineering will permit greater control of mAb targeting, clearance and pharmacokinetics, resulting in significantly improved delivery to tumors of radioisotopes and potent anticancer drugs. Pre-targeting strategies, which separate the two functions of antibody-based localization and delivery or generation of the toxic agent into two steps, also promise to afford superior tumor targeting and therapeutic efficacy. Several challenges in optimizing immunoconjugates remain, however, including poor intratumoral mAb uptake, normal tissue conjugate exposure and issues surrounding drug potency and conditional release from mAb carriers. Nonetheless, highly promising results from preclinical models will continue to drive the clinical development of this therapeutic class.     

Antibody–drug conjugates (ADCs) for cancer therapy: Strategies,challenges, and successes

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody–drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.     

抗体偶联药物国内研发现状及企业布局分析

抗体偶联药物(antibody-drug conjugates,ADC)由单克隆抗体和小分子细胞毒药物通过连接子偶联而成,因在血液瘤、实体瘤等临床治疗中被证明是一种极有前景的技术手段,已经成为国内外抗体药物研发的新热门方向.系统阐述了ADC药物开发及工业化生产中的核心五要素,对国内ADC的研发管线进行了梳理,并对参与A...     

Site-specific antibody drug conjugates for cancer therapy

Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors—target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris^®) and ado-trastuzumab emtansine (Kadcyla^®). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0–8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.