Selected Publications for Antibody-Drug Conjugates

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: 315-337. PubMed ID: 28303026

This article provides a comprehensive and updated review of the progressions in ADC development, covering a wide topic including the basis for ADC, the chemical compounds involved in most ADC development, conjugation technologies, as well as a full list of ADCs in clinical evaluation and on the market.

Chari, R.V.J., Miller, M.L., Widdison, W.C. Antibody-Drug Conjugates: An Emerging Concept in Cancer Therapy. Angew. Chem. Int. Ed. 2014, 53: 3796-3827. PubMed ID: 24677743

This article reviews the chemical aspects of the first and second generation of ADC payloads and payload-linker complexes. The authors presented an extensive body of knowledge regarding the payload drug efficacy, linker selection, bio-conjugation method, as well as ADC potency evaluations.

Srinivasarao, M., Galliford, C.V., Low, P.S. Principles in the Design of Ligand-Targeted Cancer Therapeutics and Imaging Agents. Nat. Rev. Drug Discov. 2015, 14: 203-219. PubMed ID: 25698644

This article provides a general guideline in the designing of efficient targeting entities that employs an antibody as the guiding molecule. The authors focused on strategies in designing and preparing biochemically compatible linkers as well as in modifying antibodies to facilitate conjugation.

Storz, U. Antibody-Drug Conjugates: Intellectual Property Considerations. mAbs. 2015, 7: 989-1009. PubMed ID: 26292154

This article focuses on the intellectual property (IP) issues involved in ADC development. The author gave a detailed explanation about the complicated entanglement of the IP issues associated with different components of an ADC, including the antibody, linker, payload, as well as the payload-linker complex.

The Therapeutic Monoclonal Antibody Market

Ecker, D.M., Jones, S.D., Levine, H.L. The Therapeutic Monoclonal Antibody Market. mAbs. 2015, 7: 9-14. PubMed ID: 25529996

This article provides an overview of the expanding therapeutic antibody market and points out a clear increase in the revenue of monoclonal antibody and monoclonal antibody associated therapeutic, including ADCs.

Sassoon, I., Blanc, V. Antibody-drug Conjugate (ADC) Clinical Pipeline: A Review. Methods Mol Biol. 2013, 1045: 1-27. PubMed ID: 23913138

This review article provides a general description of the ADCs in clinical trial pipeline back in 2013. Even though updates have been made to renew the list, the strategies for clinical trial set-up, stage design, and execution described in this paper is quite important in planning such work.

Dumontet, C., Jordan, M.A. Microtubule-Binding Agents: A Dynamic Field of Cancer Therapeutics. Nat. Rev. Drug Discov. 2010, 9: 790-803. PubMed ID: 20885410

This article describes the discovery, structure, and chemical characteristic of various microtubule binding agents. The authors elaborated the mechanism of interaction between these organic agents, many of which were derived from plants, to microtubules and also focus on their clinical developability as anti-cancer chemo-therapeutic agents or as payload for ADCs.

Maderna. A., Leverett, C.A. Recent Advances in the Development of New Auristatins: Structural Modifications and Application in Antibody Drug Conjugates. Mol. Pharm. 2015, 12: 1798-1812. PubMed ID: 25697404

Auristatin derivatives are a group of toxins that is most widely used ADC developments. In this article, the authors presented the chemical structures of Auristatin and its various derivatives. Strategies for Auristatin modification to yield derivatives that are chemically more compatible with linker chemistry and bio-conjugations are also discussed in detail.

Francisco, J.A., Cerveny, C.G., Meyer, D.L., et al. cAC10-vcMMAE, An Anti-CD30-monomethyl Auristatin E Conjugate with Potent and Selective Antitumor Activity. Blood. 2003, 102: 1458-1465. PubMed ID: 12714494

This article elaborates the process in the construction of brentuximab vedotin (Adcetris), one of the FDA-approved ADCs. The authors included the steps of antibody validation, drug-linker synthesis, ADC conjugation and characterization, as well as the pre-clinic cellular and Xenograft model evaluation of the ADC efficacy. This is an excellent paper to be used as a general guideline for ADC design and evaluations.

Oroudjev, E., Lopus, M., Wilson, L., et al. Maytansinoid-Antibody Conjugates Induce Mitotic Arrest by Suppressing Microtubule Dynamic Instability. Mol. Cancer Ther. 2010, 9:2700-2713. PubMed ID: 20937595

The authors of this article tested the efficacy of maytansine derivatives, DM1 and DM4, in the concept of antibody drug conjugates and revealed the mechanism of action of the maytansine derivatives in microtubule dynamic suppression.

Lambert, J.M., Chari, R.V. Ado-trastuzumab Emtansine (T-DM1): An Antibody-Drug Conjugate (ADC) for HER2-positive Breast Cancer. J. Med. Chem. 2014, 57:6949-6964. PubMed ID: 24967516

This article contains a comprehensive description of the construction, analysis, pre-clinical and clinical evaluation of T-DM1, or Kadcyla, one of the FDA-approved ADCs on the market. The content of the article serves as a guideline for a successful ADC development project.

Tumey, L.N., Leverett, C.A., Vetelino, B., et al. Optimization of Tubulysin Antibody-Drug Conjugates: A Case Study in Addressing ADC Metabolism. ACS Med Chem Lett. 2016, 22 (11): 977-982. PubMed ID: 27882194

Authors of this article developed an ADC based on tubulysin, another microtubule inhibitor that is used as ADC payload. In this study, tubulysin was conjugated to the mAb via a non-cleavable linker and the resulted ADC was proven to be efficient in suppressing tumor growth. The authors also exploited the chemical impact of subcellular compartments on the activity of the ADC and demonstrated that the chemical modification inflected by certain intracellular pathways might affect the activity of an ADC.

de Goeij, B.E., Lambert, J.M. New Developments for Antibody-drug Conjugate-based Therapeutic Approaches. Curr Opin Immunol. 2016, 40: 14-23. PubMed ID: 26963132

In this review article the authors gave a comprehensive review of the current status of ADC development. The review focuses on aspects such as the ADCs in clinical pipeline, the expended therapeutic windows of ADCs, the potential immunogenicity raised by ADCs and more importantly, describes DNA toxins as innovative, highly efficient new payloads for ADCs.

Walker, S., Landovitz, R., Ding, W.D., et al. Cleavage Behavior of Calicheamicin Gamma 1 and Calicheamicin T. Proc Natl Acad Sci USA. 1992, 89 (10): 4608–4612. PMCID: PMC49132

Authors of this article demonstrate the mode of action by two calicheamicin derivatives: calicheamicin gamma 1 (the major derivative used in ADC development such as gemtuzumab ozogamicin and inotuzumab ozogamicin) and calicheamicin T as being the DNA cleavage agent that degrade DNA with certain sequence preferences and at a high efficiency.

Ricart, A.D. Antibody-drug Conjugates of Calicheamicin Derivative: Gemtuzumab ozogamicin and Inotuzumab ozogamicin. Clin Cancer Res. 2011, 17 (20): 6417-6427. PubMed ID: 22003069

This article offers a comprehensive review regarding the ADCs, especially Gemtuzumab ozogamicin (GO, Mylotarg), constructed by coupling of a mAb with calicheamicin derivative. Aspects such as pharmacodynamics, pharmacokinetics, safety, clinical efficacy… of the two ADCs are reviewed.

Chen, X., Ding, G., Gao, Q., et al. A Human Anti-c-Met Fab Fragment Conjugated with Doxorubicin as Targeted Chemotherapy for Hepatocellular Carcinoma. PLoS One. 2013, 8(5): e63093. PubMed ID: 23675455

Authors of this article constructed an ADC using Fab portion of the anti-c-Met antibody with doxorubicin via a non-cleavable linker to the Lys residues on the Ab. The resulted ADC showed good anti-tumor activity in HepG2 xenograft mice. The construct of this kind is considered as a “ChimtoximAb” defined by Creative Biolabs and the work in the article is a powerful demonstration of this set of useful constructs and tools in innovative ADC developments.
Link: ChimtoximAb to the ChimtoximAb platform webpage.

Boger, D.L., Garbaccio, R.M. Catalysis of the CC-1065 and Duocarmycin DNA Alkylation Reaction: DNA Binding Induced Conformational Change in the Agent Results in Activation. Bioorg Med Chem. 1997, 5(2): 263-276. PubMed ID: 9061191

This article demonstrates the mechanism of action of duocarmycin and duocarmycin derivative, namely CC-1065, from a structural point of view. Evidence in this article clearly suggests the site of interaction between duocarmycin derivatives and DNA and the impact the interaction causes on the structural changes of local dsDNA.

Dokter, W., Ubink, R., van der Lee, M., et al. Preclinical Profile of the HER2-targeting ADC SYD983/SYD985: Introduction of a New Duocarmycin-based Linker-drug Platform. Mol Cancer Ther. 2014, 13(11): 2618-2629. PubMed ID: 25189543

This article demonstrates the synthesis and conjugation of a new payload-linker complex comprised of a duocarmycin derivative, DUAB, and a peptide linker with a self-elimination module. This proprietary ADC has demonstrated excellent potential in both in vitro and in vivo evaluations and this innovative design offers a new route for integrating duocarmycin derivative into ADCs.

Tiberghien, A.C., Levy, J.N., Masterson, L.A., et al. Design and Synthesis of Tesirine, a Clinical Antibody−Drug Conjugate Pyrrolobenzodiazepine Dimer Payload. ACS Med Chem Lett. 2016, 7(11): 983-987. PubMed ID: 27882195

Authors of this article incorporated a novel pyrrolobenzodiazepine dimer payload, namely Tesirine (SG3249), with an anti-HER antibody via a linker that is comprised of a Val-Ala di-peptide module for drug release and an extended PEG8 arm to increase solubility and ADC stability. The payload-linker complex was conjugated to the Ab via Cys-based conjugation and the resulted ADC exert a DRA at 2.5, which enables the its high efficiency as evaluated in an in vitro cell viability analysis based on the high toxicity of Tesirine.

Miller, M.L., Fishkin, N.E., Li, W., et al. A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity. Mol Cancer Ther. 2016, 15(8): 1870-1878. PubMed ID: 27216304

AThis article focuses on a set of new ADCs that were developed with pyrrolobenzodiazepine dimer payload. The authors developed new non-cleavable linkers to conjugate this payload to the antibody in a one-step or two-step fashion, depending on the chemical structure of the adaptor. The resulted ADCs showed excellent in vitro efficacy while they were well tolerated in mice and demonstrated strong antitumor activity in several tumor models, indicating PDB dimer as potent payload for ADC development.

Moldenhauer, G., Salnikov, A.V., Lüttgau, S., et al. Therapeutic Potential of Amanitin-conjugated Anti-Epithelial Cell Adhesion Molecule Monoclonal Antibody Against Pancreatic Carcinoma. J Natl Cancer Inst. 2012, 104(8): 622-634. PubMed ID: 22457476

The work described in this article shows a new avenue to use amatoxin as a payload to generate ADC. The authors made a simple chemical modification to the a-amanitin molecule, enabling its conjugation to an anti- EpCAM antibody via a non-cleavable linker. The resulted ADC shows a DAR between 4~8 and it is highly efficient against pancreatic carcinoma in in vitro cellular assays as well as in in vivo Xenograft mouse model.

Puthenveetil, S., Loganzo, F., He, H., et al. Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads. Bioconjug Chem. 2016, 27(8): 1880-1888. PubMed ID: 27412791

In the article, the authors demonstrate several ways to modify and conjugate splicing inhibitors, for instance spliceostatin C and thailanstatin A, onto antibodies via non-cleavable linkers to either Lys or Cys residues to form ADCs, which are later proven in cellular assays to show high cellular toxicity against certain tumor cell lines.

Govindan, S.V., Cardillo, T.M., Moon, S., et al. CEACAM5-Targeted Therapy of Human Colonic and Pancreatic Cancer Xenografts with Potent Labetuzumab-SN-38 Immunoconjugates. Clin Cancer Res. 2009, 15(19): 6052-6061. PubMed ID: 19789330

This article documents the structure and fabrication of Labetuzumab-SN-38, a potent ADC against colon and pancreatic caners. SN38, a potent inhibitor of DNA topoisomerase I, is conjugated to the Cys residues of Labetuzumab via a unique CL2A linker, which contains a di-peptide motif and a self-elimination motif to release the drug in its natural form after lysosomal digestion.

Dawidczyk, C.M., Kim, C., Park, J.H., et al. State-of-the-Art in Design Rules for Drug Delivery Platforms: Lessons from FDA-approved Nanomedicines. J Control Release. 2014, 187: 133–144. PMCID: PMC4132889

This article provides a comprehensive review of the pros and cons of current liposome-based drug delivery system and provides a guideline towards the design and preparation of the next-generation drug delivery vehicles. Facilitated by guidance molecules such as antibodies or fragment antibodies, this review focuses on the strategies used in corporation of toxic payloads with those guidance molecules in the presence of lipid composites to maximize the efficiency of drug delivery.

Scott, R.C., Wang, B., Nallamothu, R., et al. Targeted Delivery of Antibody Conjugated Liposomal Drug Carriers to Rat Myocardial Infarction. Biotechnol Bioeng. 2007, 96(4): 795-802. PubMed ID: 17051598

In this article, the authors constructed a liposome-antibody conjugate by incorporating protein A onto the surface of the liposome. The antibody is then attached via a non-covalent interaction with protein A. The resulted liposomes show good targeting characteristics and decent circulatory time. This method is useful in guided liposome drug delivery vehicle development to accurately deliver the encapsulated drug to the lesion.

Lin, C., Wong, B.C.K., Chen, H., et al. Pulmonary Delivery of Triptolide-loaded Liposomes Decorated with Anti-Carbonic Anhydrase IX Antibody for Lung Cancer Therapy. Sci Rep. 2017, 7(1): 1097. PubMed ID: 28428618

This article describes the construction of an antibody-conjugated liposome particle to accurately deliver triptolide (TPL) into lung tumor cells. The antibody used in this study is an anti-carbonic anhydrase IX antibody and it is conjugated to the surface of the liposome via thiol-based conjugation method, which forms a covalent bond between the antibody and the liposome vesicle. The guided liposome works to the authors’ expectation and greatly enhances the accuracy of drug delivery and potency of treatment.

Liu, W., Onda, M., Lee, B., et al. Recombinant Immunotoxin Engineered for Low Immunogenicity and Antigenicity by Identifying and Silencing Human B-cell Epitopes. Proc Natl Acad Sci USA. 2012, 109(29): 11782-11787. PubMed ID: 22753489

The authors of this article constructed an immunotoxin by fusion pseudomonas exotoxin A with a scFv fragment antibody against CD22. The two proteins are connected by a small peptide linker and expressed as one fusion protein in E. coli. The immunotoxin inherits the targeting capability enabled by the scFv and the pseudomonas exotoxin A successfully increases the total toxicity of this fusion protein against CD22+ cancer cells.

Jain, N., Smith, S.W., Ghone, S., et al. Current ADC Linker Chemistry. Pharm Res. 2015, 32(11): 3526-3540. PubMed ID: 25759187

The authors of this article constructed an immunotoxin by fusion pseudomonas exotoxin A with a scFv fragment antibody against CD22. The two proteins are connected by a small peptide linker and expressed as one fusion protein in E. coli. The immunotoxin inherits the targeting capability enabled by the scFv and the pseudomonas exotoxin A successfully increases the total toxicity of this fusion protein against CD22+ cancer cells.

Hinrichs, M.J., Dixit, R. Antibody Drug Conjugates: Nonclinical Safety Considerations. AAPS J. 2015, 17(5): 1055-1064. PubMed ID: 26024656

Authors of this article give a comprehensive review regarding the safety precautions associated with ADC developments. As indicated in the article, linkers, a crucial component in an ADC, is heavily related to its off-target drug release. Toxicity accompanied by the random drug release can be decreased or avoided with more careful design of the linker.

Gorovits, B., Krinos-Fiorotti, C. Proposed Mechanism of Off-target Toxicity for Antibody-drug Conjugates Driven by Mannose Receptor Uptake. Cancer Immunol Immunother. 2013, 62(2): 217-223. PubMed ID: 23223907

This review article presents a hypothesis that one mechanism behind the off-target drug release of ADCs is associated with the cell surface mannose receptor (MR). The authors present compelling evidence showing that random ADC uptake by healthy cells is partially contributed by carbohydrates, specifically agalactosylated glycans, and their interaction with cell surface MR.

Dowell, J.A., Korth-Bradley, J., Liu, H., et al. Pharmacokinetics of Gemtuzumab Ozogamicin, an Antibody-Targeted Chemotherapy Agent for the Treatment of Patients with Acute Myeloid Leukemia in First Relapse. J Clin Pharmacol. 2001, 41(11): 1206-1214. PubMed ID: 11697753

This article documents the pharmacokinetics of gemtuzumab ozogamicin tested in clinical trial settings. Gemtuzumab ozogamicin, an ADC that is formulated by the conjugation of an anti-CD33 monoclonal antibody with calicheamicin via a pH-sensitive hydrazone linker, shows good efficacy, plasma stability, and safety in tested patients.

Choy, C.J., Geruntho, J.J., Davis, A.L., et al. Tunable pH-Sensitive Linker for Controlled Release. Bioconjug Chem. 2016, 27(3): 824-830. PubMed ID: 26886721

An article published by one of our technical consultants. In this article, scientist from Dr. Clifford E. Berkman’s group at Washington State University have developed a pH-sensitive linker that releases the payload under a series of pH environments just by adjusting the sidechain of the P-N bond, which is susceptible to hydrolysis.

Finniss, M.C., Chu, K.S., Bowerman, C.J., et al. A Versatile Acid-labile Linker for Antibody-drug Conjugates. Med. Chem. Commun. 2014, 5: 1355-1358. DOI: 10.1039/C4MD00150H.

In this article, the authors describe an acid-labile linker based on silyl ether chemistry. The resulted silyl ether linkers show good drug release under cellular acidity and the versatile side groups on the silyl ether enables the addition of an orthogonal handle for conjugation and side chains to modulate the chemical characteristics of the linkers.

Erickson, H.K., Widdison, W.C., Mayo, M.F., et al. Tumor Delivery and In Vivo Processing of Disulfide-Linked and Thioether-Linked Antibody−Maytansinoid Conjugates. Bioconjug Chem. 2010, 21(1): 84-92. PubMed ID: 19891424

The authors of this article assay the metabolites of ADCs formulated by different disulfide linker as well as a non-cleavable linker. The results showed that space hindrance of the linkers appears to have minimum effect on the final metabolites of the payload in the disulfide linker-containing ADCs undergone processes in vivo. The toxicity of lipophilic metabolites from ADCs with disulfide linkers surpasses that of an ADC with non-cleavable linker, providing some insights to the observation that disulfide linker-containing ADCs show better tumor inhibition efficacy in xenograft mouse models.

Kellogg, B.A., Garrett, L., Kovtun, Y., et al. Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage. Bioconjug Chem. 2011, 22(4): 717-727. PubMed ID: 21425776

To address the question of disulfide linker steric hindrance and ADC serum stability, the authors of this article synthesized a list of modified SPDB linker with increased steric hindrance. The formulated ADCs are tested for stability in both in vitro and in vivo settings and the results showed a clear correlation between the increased stability and increased linker steric hindrance.

Pillow, T.H., Schutten, M., Yu, S.F., et al. Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody–Drug Conjugates with Self-Immolative Disulfide Linkers. Mol Cancer Ther. 2017, 16(5): 871-878. PubMed ID: 28223423

PBD dimer is commonly conjugated to an antibody via a cleavable “Val-Ala” peptide linker. The authors in this article exploit a new avenue and designed a PBD dimer-containing ADC via a self-immolative disulfide linker. In vitro efficacy and in vivo safety evaluation results of this novel ADC showed an equivalent efficacy and improved safety, rendering this new linker a breakthrough in PBD-based ADC developments.

Pillow, T.H., Sadowsky, J.D., Zhang, D., et al. Decoupling Stability and Release in Disulfide Bonds with Antibody-small Molecule Conjugates. Chem. Sci., 2017, 8: 366-370. DOI: 10.1039/C6SC01831A

The authors of this article survey the application of disulfide linker in the preparation of various stable yet facile release ADCs. They prepared multiple ADCs bearing various small molecule payloads and demonstrated the versatile application of disulfide linkers.

Gondi, C.S., Rao, J.S. Cathepsin B as a Cancer Target. Expert Opin Ther Targets. 2013, 17(3): 281-291. PubMed ID: 23293836

As an important lysosomal enzyme, Cathepsin B degradation is the basis for many important di-peptide linkers in ADC development. Commonly used vc link and Phe-Lys linker are all based on Cathepsin B. This review article offers expert opinion in the oncological behavior of Cathepsin B in tumor and explains why Cathepsin B can serve as an excellent target as well as for ADC drug release.

Sutherland, M.S., Sanderson, R.J., Gordon, K.A., et al. Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-auristatin Conjugates. J Biol Chem. 2006, 281: 10540-10547. PubMed ID: 16484228

This article presents the intracellular trafficking pathway of an ADC constructed based on a peptide linker and demonstrated the impact of lysosomal enzymes, especially the Cysteine protease metabolic pathway, on the release of the payload and on the final cytotoxicity of the ADC.

Doronina, S.O., Bovee, T.D., Meyer, D.W., et al. Novel Peptide Linkers for Highly Potent Antibody−Auristatin Conjugate. Bioconjugate Chem., 2008, 19: 1960- 1963. PubMed ID: 18803412

This article presents several novel designs in peptide linkers to improve the efficacy of auristatin-based ADCs. As shown in tumor inhibition assay, ADCs developed based on new peptide linkers bearing core peptide sequences such as NorVal-(D)Asp or Asn-(D)Lys exert superior tumor inhibitory efficacy comparing to the ones forged with a Val-Cit linker, presumably due to the increased drug release efficiency empowered by the new linkers.

Dorywalska, M., Dushin, R., Moine, L., et al. Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design. Mol Cancer Ther. 2016, 15:958-970. PubMed ID: 26944918

Val-Cit linker is well-known and marketed for its superior serum stability comparing to both disulfide and hydrazone linkers. Authors in this article further evaluated the serum stability of this linker and pointed out that serum enzymes, such as Carboxylesterase 1C, can pose some potential risks for off-target drug release. A method for stability enhancement was proposed in this article and by a small revision, increase in serum stability was observed in the improved VC linker.

Singh, R., Setiady, Y.Y., Ponte, J., et al. A New Triglycyl Peptide Linker for Antibody-Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells. Mol Cancer Ther. 2016, 15: 1311-1320. PubMed ID: 27197308

This article presents the design and implementation of a novel peptide linker, CX, in the formation of an ADC using maytansinoid payloads. The efficacy of the ADC was compared with that constructed with the SMCC linker and the cytotoxicity results have shown that the CX linker-based ADC exerts 5-100-fold lower IC50 than the SMCC ADC. This is a significant development in maytansinoid-baesd ADCs and provided a new linker that provides new potentials for the use of maytansinoids.

Puthenveetil, S., He, H., Loganzo, F., et al. Multivalent Peptidic Linker Enables Identification of Preferred Sites of Conjugation for a Potent Thialanstatin Antibody Drug Conjugate. PLoS One. 2017, 12: e0178452. PubMed ID: 28558059

Authors of this article presents a new design of peptide linker to be used in ADC development. The multi-valent linker, similar to the concept of “Hydra linker” from Creative Biolabs, enables the addition of multiple payloads onto one ADC to increase the DAR and subsequently, the therapeutic efficacy. Payloads that are compatible with this linker system are thailanstatin and novel auristatin derivatives such as MMAD. The significance of the research lies in the successful demonstration of the linking between thailanstatin and an antibody, proving an additional choice of payload for future ADC developments.

Rock, B.M., Tometsko, M.E., Patel, S.K., et al. Intracellular Catabolism of an Antibody Drug Conjugate with a Noncleavable Linker. Drug Metab Dispos. 2015, 43: 1341-1344. PubMed ID: 26101225

Due to the stability of the payload-linker, the metabolites generated by using non-cleavable linkers are important factors to be considered in ADC development as well as the downstream ADC evaluations and PK/PD studies. This article presents a comprehensive analysis of the in vivo metabolites from an ADC bearing SMCC-DM1 as payload-linker complex by a LC-MS based assay.

Behrens, C.R., Ha, E.H., Chinn, L.L., et al. Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs. Mol Pharm. 2015, 12: 3986-3998. PubMed ID: 26101225

This article shows the development of ADCs based on a novel method using non-cleavable linkers. Using the C-lock technique, a non-cleavable linker bridges the free Cys residues between the light chains and hinge heavy chains, respectively. The resulted ADCs showed a higher degree of homogeneity comparing to that prepared using conventional Cys conjugation method.

Donaghy, H. Effects of Antibody, Drug and Linker on the Preclinical and Clinical Toxicities of Antibody-Drug Conjugates. MAbs. 2016, 8: 659–671. PubMed ID: 27045800

This article offers a comprehensive evaluation of the stability of ADCs based on their linker. Results from a collective of studies have indicated the superior stability of non-cleavable linkers comparing to that of di-sulfide, acid-liable, as well as peptide linkers. The indication is clear that to generate a stable ADC, non-cleavable linkers are first-line of choices with the validation that the attach of such entity does not compromise with the efficacy of the payload.

Li, X., Abrahams, C., Embry, M., et al. Targeting CD74 with Novel Antibody Drug Conjugates (ADCs) for the Treatment of B-Cell Non-Hodgkin's Lymphoma (NHL). Blood. 2016, 128: 464.

This article describes the development of a series of novel ADCs against CD74 overexpressed cells for NHL treatment. Theses ADCs, developed based on maytansinoid payload with a new non-cleavable linker, are forged using Cu-free click chemistry for site-specific conjugations. The final conjugates showed excellent cytotoxicity at nM range and good tumor inhibition efficacy comparing to a few ADCs developed via other methods that are currently under clinical evaluations.

McCombs, J.R., Owen, S.C. Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry. AAPS J. 2015, 17: 339-351.

This review article focuses on different aspects of ADC development, including the selection of payload, linker, as well as conventional conjugation chemistry. It also offers a brief touch of the more recent antibody engineering techniques to introducing special site-directed conjugation sites.

Kline, T., Steiner, A.R., Penta, K., et al. Methods to Make Homogenous Antibody Drug Conjugates. Pharm Res. 2015, 32: 3480–3493. PMCID: PMC4596908

Sochaj, A.M., Świderska, K.W., Otlewski, J. Current Methods for the Synthesis of Homogeneous Antibody-Drug Conjugates. Biotechnol Adv. 2015, 33: 775-784. PubMed ID: 25981886

Review articles #2 and 3 provide readers of a comprehensive understanding of the protein engineering methods, e.g. site-directed Cys engineering, unnatural amino acid incorporation, sortase A site insertion… to overcome the high heterogeneity of ADCs prepared by “conventional” Cys or Lys based conjugation methods. The pros and cons of selected methods are reviewed and it is appropriate to say that the challenges still remain while new methodologies are desired to push the homogeneity of ADCs to the next level.

Kantarjian, H.M., DeAngelo, D.J., Stelljes, M., et al. Inotuzumab Ozogamicin versus Standard Therapy for Acute Lymphoblastic Leukemia. N Engl J Med. 2016, 375: 740-753. PMCID: PMC5594743

This article documents the efficacy of Inotuzumab Ozogamicin (now Besponsa) in the treatment of ALL. Besponsa is a new ADC developed by Pfizer that gained FDA approval in 2017, making it the 4th FDA-approved ADC. It is formulated by conjugating Ozogamicin, a payload-linker containing calicheamicin, to an anti-CD22 antibody via the Lys sites. According to the clinical results of this study, Besponsa showed improved therapeutic efficacy comparing to conventional treatment plans for ALL.

Gomez-Roca, C.A., Boni, V., Moreno, V., et al. A Phase I Study of SAR566658, an Anti CA6-Antibody Drug Conjugate (ADC), in Patients (Pts) with CA6-positive Advanced Solid Tumors (STs). J Clin Oncol. 2016, 34: 2511.

This article presents the phase I clinical trial results of the ADC: SAR566658, developed by Sanofi. This ADC is formulated by the conjugation of an anti-CA6 antibody with SPDB–DM4 via Lys residues on the Ab. The Phase I results of this ADC showed “a favorable safety profile and encouraging antitumor activity and is selected as the RD for further clinical development.” This is another example of ADCs developed by Lys based conjugation chemistry.

Strop, P., Liu, S.H., Dorywalska, M., et al. Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates. Chem Biol. 2013, 20: 161-167. PubMed ID: 23438745

This article presents methods for characterizing conjugation site distribution in an ADC formulated by Lys based conjugations. LC-MS/MS in combination of enzymatic degradations provide us with a comprehensive map of the actual conjugation sites and the results from the article clearly indicate that the function or efficacy of the ADC can be compromised when the payload-linker is conjugated to the inappropriate sites of an Ab.

Nanna, A.R., Li, X., Walseng, E., et al. Harnessing a Catalytic Lysine Residue for the One-step Preparation of Homogeneous Antibody-drug Conjugates. Nat Commun. 2017, 8: 1112. PubMed ID: 29062027

This article provides an interesting method to prepare Lys based ADCs by exploiting the naturally occurring Lys residues in the dual variable domain (DVD) format. By utilizing the highly active potential of Lys residues in this region, the authors successfully prepared an anti-HER2 ADC using a one-step reaction. The resulted ADC showed high homogeneity comparing to those generated by conventional Lys conjugations and this is a breakthrough in developing Lys conjugates in a mutation-free manner.

Kelliher, M.T., Jacks, R.D., Piraino, M.S. et al. The Effect of Sugar Removal on the Structure of the Fc Region of an IgG Antibody as Observed with Single Molecule Förster Resonance Energy Transfer. Mol Immunol. 2014, 60: 103-108. PubMed ID: 24813166

This article uses a de-glycosylated antibody with glycosylation sites mutated to Cys. The authors then conjugated a dye to the Ab via the Cys residues and studied the Fc structure of the Ab using Single Molecule Förster Resonance Energy Transfer. Creative Biolabs provided the antibody materials used in this study.

Shinmi, D., Taguchi, E., Iwano, J., et al. One-Step Conjugation Method for Site-Specific Antibody–Drug Conjugates through Reactive Cysteine-Engineered Antibodies. Bioconjugate Chem. 2016, 27: 1324–1331. PubMed ID: 27074832

The authors present a novel site for Cys engineering to facilitate Cys conjugations similar to THIO-MAB. With a Lc-Q124C mutation, the modified antibody is more prone to payload conjugation and the resulted conjugates showed a 1:2 DAR with narrow distribution. This technique is apparently more superior to the existing Cys-based ADC preparation methods in terms of DAR distribution and has wide application potentials in ADC development.

Jackson, D., Atkinson, J., Guevara, C.I., et al. In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates. PLoS ONE. 2014, 9: e83865. PubMed ID: 24454709

Stability is always a major concern of an ADC preparation. The authors of this articles compared the stability of two ADCs prepared from un-natural amino acid conjugation and Cys conjugation. The results of in vitro and in vivo stability tests clearly indicated a more superior stability of ADCs generated via un-natural amino acid conjugation. This observation raises a concern regarding ADCs generated by Cys-based conjugations. Apparently, more work is needed to make these conjugates less prone to off-target drug release.

Pacholarzab, K.J., Barranb, P.E. Use of a Charge Reducing Agent to Enable Intact Mass Analysis of Cysteine-linked Antibody-drug-conjugates by Native Mass Spectrometry. EuPA Open Proteomics. 2016, 11: 23-27.

This article presents a new method to characterize Cys-based antibody drug conjugates via the usage of TEAA. TEAA, as a charge reducing agent, improves cysteine-linked ADC characterization by increase of resolution for intact mass analysis for the determination of DAR and payload distribution.

Bhakta, S., Raab, H., Junutula, J.R. Engineering THIOMABs for Site-specific Conjugation of Thiol-reactive Linkers. Methods Mol Biol. 2013, 1045: 189-203. PubMed ID: 23913148

This is the classic article describing the concept and execution of the idea of “THIOMAB”, which enables the conjugation of thiol-reactive payload-linker complexes to fixed sites on the antibody to ensure a narrow DAR and in the meantime, sustain the stability of the antibody. THIOMAB is the first approach to incorporate orthogonal conjugation handles into the Ab sequence and using this platform, ADCs bearing highly toxic payloads, such as PBD dimers, have been successfully generated and entered clinical evaluations.

Dimasi, N., Fleming, R., Zhong, H., et al. Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Cysteine Insertion. Mol Pharm. 2017, 14: 1501-1516. PubMed ID: 28245132

This article is an example of the application of Cys engineering techniques, such as THIOMAB, in the creation of new ADCs bearing novel functionalities. In this article, the authors introduced several Cys residues to substitute solvent accessible amino acids in the Ab sequence. The engineered Ab not only yielded ADCs with homogenous DAR but also maintained inactive against FcγRs in vitro, potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs.

Zammarchi, F., Corbett, S., Adams, L., et al. hLL2-Cys-PBD, a New Site-Specifically Conjugated, Pyrrolobenzodiazepine (PBD) Dimer-Based Antibody Drug Conjugate (ADC) Targeting CD22-Expressing B-Cell Malignancies. Blood. 2016, 128: 4176.

PBD dimers have emerged as a powerful payload for ADC development. The most widely used method to conjugate this payload to an Ab is via a va linker facilitated by thiol reactive maleimide as conjugation chemistry. This article gives an example of such ADC constructs bearing PBD dimer. Cys-based conjugation methods prevented the addition of excessive payload molecules onto the Ab to cause the instability of the final conjugates.

Ouyang, J. Drug-to-antibody Ratio (DAR) and Drug Load Distribution by Hydrophobic Interaction Chromatography and Reversed Phase High-performance Liquid Chromatography. Methods Mol Biol. 2013, 1045: 275-283. PubMed ID: 23913154

This is a classic article demonstrating the use of HPLC, especially HIC HPLC, to determine the DAR as well as to elucidate the portion of each payload species in an ADC prepared by Cys-based conjugation methods. Due to the limited number of Cys residues at the cross-links of light chain and heavy chain, as well as in the hinge region, HIC chromatography shows good resolution in separating each payload conjugation species to enable their characterization and quantification.

Wagner-Rousset, E., Janin-Bussat, M.C., Colas, O., et al. Antibody-drug Conjugate Model Fast Characterization by LC-MS Following IdeS Proteolytic Digestion. MAbs. 2014, 6: 273-285. PubMed ID: 2413561

This article presents a LC-MS based method to fully characterize the exact conjugation site on an ADC formulated by Cys conjugation. After digestion of the ADC by proteolytic enzymes such as trypsin, HPLC was used to separate the peptides while MS analysis accurately pin-pointed the Cys residues where payload conjugation took place. This method is useful for ADC QC as in the up-scale production and troubleshooting when instability of an ADC is observed.

Schumacher, D., Helma, J., Mann, F.A., et al. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase. Angew Chem Int Ed Engl. 2015, 54: 13787-13791. PubMed ID: 26404067

Tyr conjugation-- This article describes a novel conjugation method based on tubulin tyrosine ligase (TTL), an enzyme that adds a Tyr derivative onto a TUB-tag, which can be engineered into the sequence of an antibody or other protein molecules. The Tyr or Tyr derivative can then serve as an orthogonal conjugation handle to add chemical entities, such as ADC payload-linker, to the Ab or protein molecule in a site-directed fashion.

Ban, H., Nagano, M., Gavrilyuk, J., et al. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjug Chem. 2013, 24: 520–532. PMCID: PMC3658467

Tyr conjugation-- This article reports a new conjugation method developed based on a click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs). PTAD-based compounds are reactive to Tyr residues in a protein and due to the limited number of Tyr residues, they can serve as good conjugation sites to generate conjugates with limited DAR and payload distribution profile. This method is quite versatile. Modifications on PTAD enables the direct coupling of a payload or a step-wise coupling of the payload via another active chemical side chain group, such as azide.

Qasba, P.K. Glycans of Antibodies as a Specific Site for Drug Conjugation Using Glycosyltransferases. Bioconjug Chem. 2015, 26: 2170-2175. PubMed ID: 26065635

Glycan conjugation-- This review article focuses on the current progress of ADC and other immuno-conjugates developed using the antibody glycan module. An Ab bears a complex glycan that can be altered via chemical as well as enzymatic approaches, while the addition of payloads and other chemical entities are achieved via different glycosyltransferases.

Zhu, Z., Ramakrishnan, B., Li, J., et al. Site-specific Antibody-drug Conjugation through an Engineered Glycotransferase and a Chemically Reactive Sugar. MAbs. 2014, 6: 1190-1200. PubMed ID: 25517304

Glycan conjugation-- Authors of this article reports a fast and accurate method for the determination of DAR for ADCs prepared using Lys, Cys, as well as glycan conjugation methods. The glycan moiety on an Ab can impose difficulties in obtaining a high-resolution payload distribution profile. The method described in this paper is a good strategy to circumvent this issue.

Donaldson, J.M., Zer, C., Avery, K.N., et al. Identification and Grafting of a Unique Peptide-binding Site in the Fab Framework of Monoclonal Antibodies. Proc Natl Acad Sci USA. 2013, 110: 17456-17461. PubMed ID: 24101516

Meditope-binding site -- This article documents the identification of the meditope-binding site in the Fab region of an antibody that attracts the meditope peptide. Using this unique site, a payload can be attached to the peptide and conjugated to an antibody via non-covalent interactions. The universal presence of the meditope-binding site in majority of the antibodies opens a new avenue for ADC developments.

Hui, J.Z., Tsourkas, A. Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG. Bioconjug Chem. 2014, 25: 1709–1719. PMCID: PMC4166039

UV conjugation -- This article documents the usage of an engineered protein Z containing UV-active amino acid benzoylphenylalanine (BPA) at different locations. Protein Z interacts with the Fc domain of an Ab at high affinity and the payload is conjugated to the BPA sites on the protein Z and subsequently, to the antibody molecule to achieve drug conjugation.

Alves, N.J., Mustafaoglu, N., Bilgicer, B. Conjugation of a Reactive Thiol at the Nucleotide Binding Site for Site-specific Antibody Functionalization. Bioconjug Chem. 2014, 25: 1198-1202. PubMed ID: 24932680

UV conjugation -- This article documents the utilization of nucleotide binding site in immuno-conjugate preparation. This method is highly advantageous since the conjugation occur at a very mild condition and the bond formed by the nucleotide binding site and the indole-3-butyric acid (IBA) moiety is covalent and stable. Due to the wide presence of nucleotide binding sites in antibodies, this method is also considered as an approach towards site-directed antibody conjugations.

van Vught, R., Pieters, R.J., Breukinka, E. Site-specific Functionalization of Proteins and Their Applications to Therapeutic Antibodies. Comput Struct Biotechnol J. 2014, 9: e201402001. PMCID: PMC3995230

Enzyme mediated conjugation--This review article introduces readers to enzymes that transfer functional moieties to certain amino acid residues on their peptide substrates. The concept of site-specific protein/antibody functionalization is the basis for enzyme mediated conjugation methods for site-directed ADC preparations.

Clancy, K.W., Melvin, J.A., McCafferty, D.G. Sortase Transpeptidases: Iinsights into Mechanism, Substrate Specificity, and Inhibition. Biopolymers. 2010, 94: 385-396. PubMed ID: 20593474

Enzyme mediated conjugation-- Sortase A has been used in site-directed conjugation for ADC and other immuno-conjugate preparations. This article gives a comprehensive review of the biochemistry of the mechanism of sortase and the peptide sequences that can serve as substrates for this enzyme.

Beerli, R.R., Hell, T., Merkel, A.S., et al. Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency. PLoS One. 2015, 10: e0131177. PMCID: PMC4488448

Enzyme mediated conjugation-- This article offers an example of the application of Sortase A in ADC preparation. Authors incorporated the sortase A recognition motif LPETG into Ct of and Ab and payloads, namely MMAE and DM1, were modified accordingly to accommodate sortase A chemistry by the addition of a poly Glycine tail. The resulted ADCs show a homogenous DAR at ~4 and good potency when tested in both in vitro and in vivo settings.

Chen, L., Cohen, J., Song, X., et al. Improved Variants of SrtA for Site-specific Conjugation on Antibodies and Proteins with High Efficiency. Sci Rep. 2016, 6: 31899.

Enzyme mediated conjugation-- Since the discovery of the application of sortase A in site-directed conjugation, improvements have been constantly made to grant this method a higher labeling efficiency. In this article, the authors have identified a variant of sortase A that “require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields”. This improvement is also highlighted in its application to conjugate antibodies with larger MW entities such as PEGylated drugs.

Ahvazi, B., Steinert, P.M. A Model for the Reaction Mechanism of the Transglutaminase 3 Eenzyme. Exp Mol Med. 2003, 35: 228-242. PubMed ID: 14508061

Enzyme mediated conjugation-- As another novel enzyme that transfer functional molecules to substrate peptides, transglutaminase (TG) is an alternative in enzyme mediated conjugations for ADC preparation. This article provides a review into the biochemistry as well as substrate specificities of a TG and points out the potential of this family of enzymes in ADC and other immuno-conjugate preparations.

Dennler, P., Chiotellis, A., Fischer, E., et al. Transglutaminase-based Chemo-enzymatic Conjugation Approach Yields Homogeneous Antibody-drug Conjugates. Bioconjug Chem. 2014, 25: 569-578. PubMed ID: 24483299

Enzyme mediated conjugation-- This article describes a direct one-step or indirect two-step conjugation approach using a microbial transglutaminase (MTGase) to conjugate MMAE onto an Ab. The resulted ADC showed a DAR at 2 and when compared with ADCs prepared using conventional conjugation method, these new ADCs show high equivalence in potency against cancer cell lines, proving the viability of this new site-directed conjugation method.

Hallam, T.J., Wold, E., Wahl, A., et al. Antibody Conjugates with Unnatural Amino Acids. Mol Pharm. 2015, 12: 1848-1862. PubMed ID: 25898256

Unnatural amino acids-- Since the discovery of the bi-vector system to introduce unnatural amino acids (amino acids with functional side chains different from natural occurring amino acids) into essentially any proteins, this method has been employed into ADC development. Amber stop codon mediated UAA insertion adds UAA into desired locations on an Ab at controlled quantities to ensure site-directed conjugations of payloads. The chemical versatility of the UAA side chains opens endless options to conjugate various drugs onto Abs by most suitable chemistry.

Axup, J.Y., Bajjuri, K.M., Ritland, M., et al. Synthesis of Site-specific Antibody-drug Conjugates Using Unnatural Amino Acids. Proc Natl Acad Sci USA. 2012, 109: 16101-16106. PubMed ID: 22988081

Unnatural amino acids-- Authors of this article presents a classic work to generate ADCs using UAA incorporation method. p-Acetylphenylalanine was incorporated into selected sites within anti-HER2 Fab and full antibody and MMAE was selected as the payload to formulate the ADCs. The resulted ADCs showed good homogeneity and potency when tested against HER2+ cancer cell lines as well as in vivo PDX mice models.

Zimmerman, E.S., Heibeck, T.H., Gill, A., et al. Production of Site-specific Antibody-drug Conjugates Using Optimized Non-natural Amino Acids in a Cell-free Expression System. Bioconjug Chem. 2014, 25: 351-361. PubMed ID: 24437342

Unnatural amino acids-- Authors of this article provides a new method to express UAA containing Abs. They employed a cell-free system to simplify the UAA incorporation and the ADCs derived from Abs expressed in this system showed good homogeneity in sample profiling as well as excellent potency against selected tumor cell lines.

Hondal, R.J. Incorporation of Selenocysteine into Proteins Using Peptide Ligation. Protein Pept Lett. 2005, 12: 757–764. PMCID: PMC3683319

Unnatural amino acids-- Selenocysteine is chemically more active than cysteine and is subjected to thiol-based conjugation chemistries. Now considered as a UAA, Selenocysteine is incorporated into the Ct of the Ab via peptide ligation, a method that is reviewed in this selected article.

Li, X., Rader, C. Utilization of Selenocysteine for Site-Specific Antibody Conjugation. Methods Mol Biol. 2017, 1575: 145-164. PubMed ID: 28255878

Unnatural amino acids—Selenocysteine has been used in ADC preparation. This site-directed conjugation method adds payloads to the Ct of the antibody or antibody fragment (e.g. scFv) to form ADCs with homogenous drug loading. Since many of the current ADC payload-linkers are developed to suit Cys-based conjugations, those compounds are readily available to be applied to the Selenocysteine containing Ab for ADC formulation, reducing the R&D cost for developing novel ADCs.

Wakankar, A., Chen, Y., Gokarn, Y., et al. Analytical Methods for Physicochemical Characterization of Antibody Drug Conjugates. MAbs. 2011, 3: 161–172. PMCID: PMC3092617

This is a classic review article that provides a comprehensive review of the physicochemical parameters of an ADC that need to be characterized and introduces to the tools for such tasks. We consider this article a must-read for scientists who enter the field of ADC work.

Hinrichs, M.J.M., Dixit, R. Antibody Drug Conjugates: Nonclinical Safety Considerations. AAPS J. 2015, 17: 1055–1064. PMCID: PMC4540738

We bring this article in this section again since it focuses on the general safety consideration of an ADC prior to clinical evaluations. Both the antibody portion and the drug portion of an ADC can potentially cause safety concerns, including immunogenicity, off-target toxicity, the content of toxic API… The article also provides a review of the current regulatory expectations for ADCs, which serve as a good guideline for ADC developments.

Kamath, A.V., Iyer, S. Preclinical Pharmacokinetic Considerations for the Development of Antibody Drug Conjugates. Pharm Res. 2015, 32: 3470–3479. PMCID: PMC4596897

The authors of this article also give a comprehensive review of the general pharmacological considerations for ADC development before entering clinical trial stage. The article reviews the PK consideration of each component of the ADC and combines them as the whole molecule. At the end of the article, the authors also provide a quick recap of the preclinical PK strategies for ADC evaluations.

Ross, P.L., Wolfe, J.L. Physical and Chemical Stability of Antibody Drug Conjugates: Current Status. J Pharm Sci. 2016, 105: 391-397. PubMed ID: 26869406

In this article, the authors provide readers with aspects and methods from a good collection of literature to focus on the strategy to assess the stability of ADCs to facilitate the development of a stable ADC.

Durbin, K.R., Nottoli, M.S., Catron, N.D., et al. High-Throughput, Multispecies, Parallelized Plasma Stability Assay for the Determination and Characterization of Antibody−Drug Conjugate Aggregation and Drug Release. ACS Omega. 2017, 2: 4207−4215.

In this article, the authors report the development of a high-throughput screening platform to assay the serum stability, off-target drug release, as well as the tendency for aggregation of up to 15 ADCs using just a small quantity of the material (~100 ug). The reported system provides the community with a convenient tool for ADC stability assessment and the combined assay system is highly advantageous in providing accurate and efficient results to facilitate ADC developments.

Xu, K., Liu, L., Saad, O.M., et al. Characterization of Intact Antibody-drug Conjugates from Plasma/serum in vivo by Affinity Capture Capillary Liquid Chromatography-Mass Spectrometry. Anal Biochem. 2011, 412: 56-66. PubMed ID: 21216214

Authors of this article developed a method that combines the affinity capture capillary chromatography to capture ADCs in the serum and evaluate the DAR of the ADC during different stages in metabolism. The methods described in this paper provide a feasible route to assay and monitor the dynamics of the drug release and DAR changes of an ADC throughout the drug metabolism processes.

Kaur, S., Xu, K., Saad, O.M., et al. Bioanalytical Assay Strategies for the Development of Antibody-drug Conjugate Biotherapeutics. Bioanalysis. 2013, 5: 201-226. PubMed ID: 23330562

ADCs are the products of chemical modification of antibodies with toxic compounds that are covalently conjugated to the Ab. Due to their complicated biochemical nature, it is necessary to develop new analytical methods and invent new analytical criteria to assess the stability of those complex bio-macromolecules. This article provides a guideline towards the strategies for ADC analytical method design to allow the suitable characterization of the ADC.

Gorovits, B., Alley, S.C., Bilic, S., et al. Bioanalysis of Antibody-drug Conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group Position Paper. Bioanalysis. 2013, 5: 997-1006. PubMed ID: 23641692

This paper is a simplified version of the previous paper and it offers a general but quite clear outline of the points of consideration in new ADC analytical method development to assess the stability of the ADCs in both in vitro and in vivo settings.

Iwamoto, N., Shimomura, A., Tamura, K., et al. LC-MS Bioanalysis of Trastuzumab and Released Emtansine using Nano-surface and Molecular-orientation Limited (nSMOL) Proteolysis and Liquid-liquid Partition in Plasma of Trastuzumab Emtansine-treated Breast Cancer Patients. J Pharm Biomed Anal. 2017, 145: 33-39. PubMed ID: 28648785

This article presets a new method: nano-surface and molecular-orientation limited (nSMOL) proteolysis, for ADC metabolites analysis. The authors used a well-established ADC, Trastuzumab Emtansine (T-DM1), to demonstrate the feasibility of this analytical method and successfully established the assay to determine the metabolites of T-DM1 in cancer patients.

Shadid, M., Bowlin, S., Bolleddula, J. Catabolism of Antibody Drug Conjugates and Characterization Methods. Bioorg Med Chem. 2017, 25: 2933-2945. PubMed ID: 28438386

ADC metabolism generates a complicated library of metabolites that could potentially contribute to the safety of the ADC. The authors of this article summarized the mechanisms of cleavage/catabolism of various linkers to identify the metabolites and also provided current available in vitro and in vivo assay methods for the evaluation of ADC catabolisms.

Su, D., Kozak, K.R., Sadowsky, J., et al. Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification. Bioconjug Chem. 2018, 29: 1155-1167. PubMed ID: 29481745

This article provides a very practical approach to assay the extent of payload metabolism and linker modification by affinity capture capillary chromatography and LC-MS. In this article, the authors evaluated ADCs prepared by the THIOMAB method, which is known for good ADC homogeneity. ADCs bearing various payloads, including PNU-159682 analog, tubulysin, cryptophycin, and taxoid… were analyzed by this method, indicating the versatility of this method.

Saad, O.M., Shen, B.Q., Xu, K., et al. Bioanalytical Approaches for Characterizing Catabolism of Antibody-Ddrug Conjugates. Bioanalysis. 2015, 7: 1583-1604. PubMed ID: 26226309

The review article provides a guideline in the process design of ADC metabolite characterizations. Authors demonstrated the set-up and process of such assays using several ADCs prepared by the THIOMAB approach and T-DM1. The experimental strategies used to design bioanalytical assays are very practical in the characterization of ADC catabolism and supporting ADME studies during ADC clinical development.

Zhang, D., Yu, S., Ma, T., et al. Chemical Structure and Concentration of Intratumor Catabolites Determine Efficacy of Antibody Drug Conjugates. Drug Metab Dispos. 2016, 44: 1517–1523. PMCID: PMC4998580

Authors of this article designed experiments to assess and identify the correlation between the efficacy of an ADC and the catabolites generated after metabolism. The payload of interest was PBD dimer and the results of the analysis indicated that the use of intratumor catabolite analysis rather than systemic pharmacokinetic analysis, at least in this case, better collaborates the in vivo efficacy of the ADC.

Gebler, J. Advanced Characterization of Antibody Drug Conjugates (ADCs) by Liquid Chromatography and Mass Spectrometry (LC/MS). Waters application notes: https://pdfs.semanticscholar.org/presentation/b492/c4040113e4b61a41df69fedffd10902035b7.pdf.

This application note provides a comprehensive introduction and very detailed technical notations for ADC characterization using LC-MS based assays. Since many companies use the Waters system for their ADC characterization, this note is particularly useful and provides a direct impression of how the characterization of ADCs are performed.

Bailey, A.O., Houel, S., Scheffler, K., et al. Complete Characterization of a Lysine-linked
Antibody Drug Conjugate by Native LC/MS Intact Mass analysis and Peptide Mapping. ThermoFisher application notes: https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-72511-lc-ms-lysine-linked-adc-an72511-en.pdf.

This application note demonstrates the identification of conjugation site (payload distribution analysis) by both native MS and peptide mapping. Lys-based conjugation methods usually yield ADCs with high product heterogeneity as well as highly differed conjugation site distributions. The results from this application note proved the complicated chemistry in Lys conjugations.

Redman, E.A., Mellors, J.S., Starkey, J.A., et al. Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis–Mass Spectrometry. Anal. Chem. 2016, 88: 2220–2226.

Capillary electrophoresis is commonly used in ADC characterization. The authors of this article developed a strategy that couples capillary electrophoresis with MS to identify the content of different conjugation species in an ADC. This method has become a quite popular method in determine DAR and identify each conjugation specie to provide a high-resolution determination of the DAR of an ADC.

Rago, B., Clark, T., King, L., et al. Calculated Conjugated Payload from Immunoassay and LC-MS Intact Protein Analysis Measurements of Antibody-drug Conjugate. Bioanalysis. 2016, 8: 2205-2217. PubMed ID: 27682846.

In this article the authors described a new method to couple immuno-assays together with LC-MS to identify and characterize the DAR of an ADC. This method is similar to methods described by articles from the “Stability” section and has proven to be effective and accurate.

Xu, Y., Jiang, G., Tran, C., et al. RP-HPLC DAR Characterization of Site-Specific Antibody Drug Conjugates Produced in a Cell-Free Expression System. Org. Process Res. Dev. 2016, 20: 1034–1043.

Site-directed conjugations generate ADCs with more homogenous DAR and more defined payload-distributions. In this article the authors developed a RP-HPLC assay method to quantify those specific values of those ADCs and this method has proven to be fast and accurate for such special ADCs.

Huang, R.Y., Deyanova, E.G., Passmore, D., et al. Utility of Ion Mobility Mass Spectrometry for Drug-to-Antibody Ratio Measurements in Antibody-Drug Conjugates. J Am Soc Mass Spectrom. 2015, 26: 1791-1794. PubMed ID: 26122520.

Authors of this article describe a method for DAR assessment. Using ion mobility MS approach, the analyte is allowed to undergo a "cleanup" in the gas phase, providing significant improvement of signal-to-noise ratios of ADC intact mass spectra for accurate DAR measurements. Additionally, the protein drift time analysis is also performed at the same time that offers a new dimension in monitoring the changes of DAR on a batch-to-batch basis.

Clark, T., Han, X., King, L., et al. Insights into Antibody-Drug Conjugates: Bioanalysis and Biomeasures in Discovery. Bioanalysis. 2013, 5: 985-987. PubMed ID: 23641689

This brief review article points out the necessary evaluation methods and criteria needed for ADC development and assessment.

Goldberg, M.E., Djavadi-Ohaniance, L. Methods for Measurement of Antibody/antigen Affinity Based on ELISA and RIA. Curr Opin Immunol. 1993, 5: 278-281. PubMed ID: 8507406

This is a well-acknowledged article describing the common methods used for the affinity assay of an antibody, which is also applicable for ADC. Methods include ELISA and RIA, later is a surface characterization method with basis similar to that of SPR.

Neri, D., Montigiani, S., Kirkham, P.M. Biophysical Methods for the Determination of Antibody-Antigen Affinities. Trends Biotechnol. 1996, 14: 465-470. PubMed ID: 8987915

Antibodies can be generated from various platforms and methods. However, the evaluation of their affinity is a prerequisite for the downstream processes and to identify the most suitable candidates. This article also focuses on the conventional methods used for antibody affinity measurement. Any beginner who would like to gain more general information regarding such methods, this is a good place to start.

Bee, C., Abdiche, Y.N., Pons, J., Rajpal A. Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum. PLoS One. 2013, 8: e80501. PubMed ID: 24223227

This article presents a novel method to determine the affinity of an antibody, and potentially ADCs, in serum setting without the immobilization of the antigen. This method provides a convenient and yet valid avenue to assess the action of mAb or ADCs in vitro or in vivo.

Acchione, M., Kwon, H., Jochheim, C.M., Atkins, W.M. Impact of Linker and Conjugation Chemistry on Antigen Binding, Fc Receptor Binding and Thermal Stability of Model Antibody-Drug Conjugates. MAbs. 2012, 4: 362-372. PubMed ID: 22531451

The conjugation chemistry will inevitably impact the binding affinity of an ADC towards its target and Fc receptors, especially for ADCs prepared by Fc-engineered method. The authors of this article presented the readers with a comprehensive evaluation of the impact of the process of bio-conjugation to the characteristics of ADCs.

Li, P., Jiang, N., Nagarajan, S., et al. Affinity and Kinetic Analysis of Fcgamma Receptor IIIa (CD16a) Binding to IgG Ligands. J Biol Chem. 2007, 282: 6210-6221. PubMed ID: 17202140

Even though in the field of ADC the Fc functionality is usually considered as a redundant feature since the major toxicity of an ADC residues in the payload it carries, some ADCs still utilize the endogenous Fc effector function of their Fc domain as an additional level of toxicity. Under this circumstance, the Fc receptor binding affinity is required. Conjugation chemistry can sometimes lead to a compromised Fc function. This article provides a method to measure the Fc receptor binding affinity, which will be useful for the assessment and monitoring the changes in the Fc binding for such ADCs to achieve conjugation strategy optimization.

Smythe, E., Warren, G. The Mechanism of Receptor-Mediated Endocytosis. Eur J Biochem. 1991, 202: 689-699. PubMed ID: 1662613

This classic review article provides readers with an understanding of the molecular machinery involved in the process of Receptor-Mediated Endocytosis, a process facilitated by the clathrin protein.

Kuo, S.R., Alfano, R.W., Frankel, A.E., Liu, J.S. Antibody Internalization after Cell Surface Antigen Binding is Critical for Immunotoxin Development. Bioconjug Chem. 2009, 20: 1975-1982. PubMed ID: 19785403

In this article, the authors constructed different immunotoxin constructs and demonstrated that only the version showing internalization, facilitated by the antibody it was attached to, showed decent intercellular toxicity, proving that internalization is the prerequisite for the development of immunotoxins or ADCs.

Liao-Chan, S., Daine-Matsuoka, B., Heald, N., et al. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies Against Alexa Fluorophores. PLoS One. 2015, 10: e0124708. PubMed ID: 25894652

The authors of this article present a new tool: anti-Alexa antibodies, to the practice in quantifying the internalization of antibodies. This method shall also be applicable for that of ADCs.

Nath, N., Godat, B., Zimprich, C., et al. Homogeneous Plate Based Antibody Internalization Assay Using pH Sensor Fluorescent Dye. J Immunol Methods. 2016, 431: 11-21. PubMed ID: 26851520

Fluorescent dyes offer the option of direct visualization of the antibody/ADC internalization and they can also be used for the quantification of internalization. In this article, the authors present the use of pH sensitive dyes for the assay of Ab/ADC internalization. Such dyes only show signal in acidic pH environment. Thus, they are excellent indicators of the internalization of the labeled molecules.

Li, Y., Liu, P.C., Shen, Y., et al. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence–Quencher Probe as a Tool for Functional Antibody Screening. J Biomol Screen. 2015, 20: 869–875. PubMed ID: 26024945

The assay described in this article is a novel approach for the quantification of antibody internalization. Similar to the concept of Q-body, the antibody is first labeled with a pair of dye-quencher. The dye is set free and exert signal only after internalization.

Srivastava, S., Mishra, S., Dewangan, J., et al. Principles for In Vitro Toxicology, Chapter 2. Alok Dhawan and Seok Kwon, Ed. Academic Press, 2018, pp 21-43.

This book chapter reviews and introduces the readers with established methods to assess the in vitro toxicity of drugs in general. Since these methods are applicable for the assessment of that of ADCs, this is a good place to start to familiarize with the necessary assay formats.

McKim, J.M.J. Building a Tiered Approach to In Vitro Predictive Toxicity Screening: A Focus on Assays with In Vivo Relevance. Comb Chem High Throughput Screen. 2010, 13: 188–206. PubMed ID: 20053163

The author of this article provides a new method for the design of a high -throughput in vitro drug toxicity system and correlates that with the later in vivo assays. This assay system could be useful when there is a library of ADCs comprised of different Abs as well as payload-linker to be screened against and search for the best combinantion.

Shukla, S.J., Huang, R., Austin, C.P., Xia, M. The Future of Toxicity Testing: A Focus on In Vitro Methods Using a Quantitative High Throughput Screening Platform. Drug Discov Today. 2010, 15: 997–1007. PubMed ID: 20708096

This article also presents a high-throughput drug screening system. High-throughput systems as such are excellent tools for the identification and screening of new payloads for ADC development and as indicated before, the screening of an established ADC library to facilitate the search the optimized ADC candidates.

Vankemmelbeke, M., Durrant, L. Third-Generation Antibody Drug Conjugates for Cancer Therapy--A Balancing Act. Ther Deliv. 2016, 7: 141-144. PubMed ID: 26893243

This article provides insights into the balance between the conjugation method, conjugation site, as well as DAR on the toxicity of an ADC, which is the main focus of an ADC but can sometimes be misleading. The authors point out that a “balanced” focus of those different parameters will help determine the success of an ADC.

Zammarchi, F., Corbett, S., Adams, L., et al. ADCT-402, A PBD Dimer-Containing Antibody Drug Conjugate Targeting CD19-Expressing Malignancies. Blood. 2018, 131: 1094-1105. PubMed ID: 29298756

PBD dimer and its derivatives are famous for their extremely high potency but also notorious for their hydrophobicity, which makes it difficult to reach a high DAR in ADCs with such payloads. This article presents the construction of an ADC bearing one version of the PBD dimer and the authors performed a comprehensive determination of the toxicity of the resulted ADC to show that the addition of the payload did expand the therapeutic winddown of the Ab significantly.

Li, J.Y., Perry, S.R., Muniz-Medina, V., et al. A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy. Cancer Cell. 2016, 29: 117-129. PubMed ID: 26766593

ADCs are commonly formulated with a mAb conjugated to a payload via a linker. However, the ADC presented in this paper is comprised of a bi-specific antibody that recognizes different epitopes on HER2 protein, which enhances its internalization and the payload release thereafter. The authors demonstrated in this study the enhanced toxicity of this construct comparing to that of other anti-HER2 ADCs such as T-DM1. This article presents a new design for ADC development.

Chung, T.D.Y., Terry, D.B., Smith, L.H. In Vitro and In Vivo Assessment of ADME and PK Properties During Lead Selection and Lead Optimization – Guidelines, Benchmarks and Rules of Thumb. 2015 Sep 9. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK326710/

This article is a general guideline for the design of PK/PD as well as ADME experiments to evaluate the in vivo efficacy and pharmacokinetics of a drug. This is also applicable for that of ADC work and it is a recommended read before designing any in vivo experiments.

Haas, J., Manro, J., Shannon, H., et al. In Vivo Assay Guidelines. 2012 May 1 [Updated 2012 Oct 1]. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK92013/

This article, similar to the previous one, is also a guideline for in vivo experiments design and it is worthy of the readers’ attention before any in vivo work.

Polson, A.G., Calemine-Fenaux, J., Chan, P., et al. Antibody-Crug Cconjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection. Cancer Res. 2009, 69: 2358-2364. PubMed ID: 19258515

The authors of this article performed a series of in vivo assessment to establish optimized targets for ADC development as well as payload-linkers used in ADC constructs to achieve optimized tumor killing efficacy.

Li, F., Emmerton, K.K., Jonas, M., et al. Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models. Cancer Res. 2016, 76: 2710-2719. PubMed ID: 26921341

By-stander killing effect is a feature of ADCs. Upon the release of the payload molecules, they are in theory free to travel inside the tumor to inflect more damage. By-stander killing is also controversial in ADC development since it can result in the distribution of payloads in healthy tissues under certain circumstances. This article documents the effect of by-stander killing of the tumor cell in an in vivo setting.

Lin, K., Tibbitts, J., Shen, B.Q. Pharmacokinetics and ADME Characterizations of Antibody-Drug Conjugates. Methods Mol Biol. 2013, 1045: 117-1131. PubMed ID: 23913144

This is a review article that focuses on the theory, design, and practice of the PK/PD and ADME assays for ADCs. Due to their heterogenous nature (comprised of three different components rather than one single formula), the designing of such studies against ADCs are more challenging comparing to that of small molecule drugs or antibodies alone.

Shah, D.K., Loganzo, F., Haddish-Berhane, N., et al. Establishing in vitro-in vivo Correlation for Antibody Drug Conjugate Efficacy: a PK/PD Modeling Approach. J Pharmacokinet Pharmacodyn. 2018, 45: 339-349. PubMed ID: 29423862

Authors of this article shows methods to “transfer” the efficacy data from in vitro assessment to predict and validate that of the in vivo assays. This is worth noticing since there is usually a gap between the in vitro and in vivo works since the system set-ups are different and in vivo system is more complicated than in vitro assay systems. This article resolves this “gap” meticulously

Singh, A.P., Maass, K.F., Betts, A.M. et al. Evolution of Antibody-Drug Conjugate Tumor Disposition Model to Predict Preclinical Tumor Pharmacokinetics of Trastuzumab-Emtansine (T-DM1). AAPS J. 2016, 18: 861-875. PubMed ID: 27029797

Singh, A.P., Shah, D.K. Application of a PK-PD Modeling and Simulation-Based Strategy for Clinical Translation of Antibody-Drug Conjugates: A Case Study with Trastuzumab Emtansine (T-DM1). AAPS J. 2017, 19: 1054-1070. PubMed ID: 28374319

Articles 43 and 44 are two consecutive assays for the assessment of the in vivo efficacy and PK/PD as well as ADME studies for the well-established ADC, T-DM1. Due to the relatively high heterogeneity of the ADC (formulated by Lys-based conjugation chemistry), the results of these studies very well reflected the work of the ADC inside blood circulation.

Ait-Oudhia, S., Zhang, W., Mager, D.E. A Mechanism-Based PK/PD Model for Hematological Toxicities Induced by Antibody-Drug Conjugates. AAPS J. 2017, 19: 1436-1448. PubMed ID: 28646408

The authors collected and reviewed the pre-clinical data of the in vivo assessments of the two well-established ADCs, Brentuximab vedotin (Adcetris®, SGN-35) and adotrastuzumab emtansine (Kadcyla®, T-DM1). They proposed a model for the evaluation of the in vivo characteristics of ADCs based on the available data and this proposed model successfully captured the PK and associated myelosuppression of both ADCs and might serve as a general PK/PD platform for assessing hematological toxicities to ADCs.