The continuing advancements in bioconjugate medicines from delivery of potent cytotoxins to vaccine-, oligonucleotide-, radionuclide-, immunomodulator- and check-point inhibitor-conjugates, can start to address the large unmet medical need currently unattainable from current therapeutic approaches. As the field of bioconjugation develops, and the recent spate of antibody-drug conjugates (ADCs) approvals continues, the resulting knock-on effects for delivering a wider range of bioconjugated molecules will also continue to expand in utility.
Targeted delivery of potent cytotoxins represents a paradigm shift in precision medicine.
Precision medicine aims at maximizing therapeutic effects while minimizing undesired side effects for an individual patient. In the development of antibody-drug conjugates using cytotoxins as a precision medicine strategy, a monoclonal antibody, conjugated to a highly potent cytotoxic payload via a suitable linker, targets a specific antigen expressed in certain cancer cells.
The binding of ADCs to the antigen target on the cell surface initiates the internalization of the ADC-antigen complex. Upon entering the cell, the cytotoxic payloads are cleaved then bind to their target, disrupt the cellular function, and ultimately cause irreversible cell death.
Cytotoxic drugs have been commonly used for the pharmacotherapy of many forms of cancer in the past, however, they also often cause substantial toxicity to the patient. ADCs represent a new and promising therapeutic modality that enables the targeted delivery of these cytotoxic drugs to cancer cells.
The FDA has approved a handful of such ADC drugs, including gemtuzumab ozogamicin (Mylotarg®; Pfizer/Wyeth Pharmaceuticals), brentuximab vedotin (Adcetris®; Seattle Genetics), trastuzumab emtansine (Kadcyla®; Genentech/Roche), inotuzumab ozogamicin (Besponsa®; Pfizer/Wyeth Pharmaceuticals), polatuzumab vedotin (Polivy™; Genentech/Roche), trastuzumab deruxtecan (Enhertu®; Daiichi Sankyo/AstraZeneca), and enfortumab vedotin (Padcev™; Seattle Genetics/Astellas), and there are many other drug candidates at various stages of clinical development.
ADCs are expected to play an increasingly important role not only in oncology but also for the delivery of other classes of drugs, such as antibiotics, anti-inflammatory, and some others.
Bioconjugation of oligonucleotides can improve therapeutic benefit
Small DNA or RNA oligonucleotides have great potential as therapeutic agents because they can react to the messenger mRNA or pre-mRNA targets with high selectivity and therefore engage desirable control of gene expression through the Watson-Crick base pairing.
Antibody-oligonucleotide conjugates are a novel class of synthetic chimeric biomolecules that have been increasingly applied in different fields of modern biotechnology including imaging, detection, and therapeutics. This is based on the unique combination of the properties of their two constituents, namely the antibody’s exceptional targeting abilities and biodistribution profiles, as well as the oligonucleotides extensive functional and structural roles.
Many synthetic approaches have been developed to assemble the
antibody-oligonucleotide conjugates using chemical bioconjugation or site-specific conjugation technologies with reactive handles that are introduced into antibody sequences through protein engineering.
The chemical modification of oligonucleotides has led to increasing stability in the biological environment, improving potency, and reducing off-target effects. Because of the proven functional potential of both constituents and the rapid development of improved biological and chemical tools for bioconjugation, the antibody-oligonucleotide conjugates will continue to demonstrate their unique therapeutic benefits in modern medicine.
The benefits of bioconjugate vaccine therapy
Vaccination has become the most effective public health intervention ever developed, saving numerous lives globally every year. The first generation of vaccines contained inactivated or attenuated live microorganisms and although vaccination using these whole-microbe vaccines has been successful in preventing many infectious diseases, it is not applicable to certain vaccine settings, such as therapeutic vaccines for cancer, or not safe to other vaccine settings, such as vaccines for HIV.
In addition, most live-attenuated vaccines were developed empirically without a clear understanding of their mechanisms of action. Due to consideration of vaccine safety, the current vaccinology is more focused on the development of subunit vaccines that replace whole microbes synthetically with defined protein or polysaccharide antigens, that have no potential for infectivity or toxicity on their own, and have advantages in manufacturability, stability, and safety.
Bioconjugation has become a powerful approach to produce well-defined molecular vaccines via the incorporation of additional functionality directly into the antigen. Various vaccine bioconjugates can be synthesized, through a covalent linkage of a vaccine component to a protein, peptide, lipid, oligonucleotide, polymer, nanoparticle, or small molecule.
Depending on their chemical and molecular nature, vaccine bioconjugates can enhance vaccine efficacy via diverse mechanisms such as enabling tissue or cell-specific targeting via conjugation of an antigen or adjuvant to a ligand, generating new properties such as multivalency or controlled release via conjugation of vaccines to polymers, and changing the antigen processing pathways via conjugation of vaccines to nanoparticles. Future strategies to design bioconjugates that can produce tailored immune responses against a specific disease will expand our current understanding of how to modulate the immune system.
In addition, bioconjugates will continue to play key roles in rational design for the improvement of our current vaccines and for the development of new vaccines against challenging pathogens and diseases.
Radiolabelling via antibody chelator conjugates has applications in alternative oncology approaches
Attaching a radionuclide to an antibody for diagnostic purposes including positron emission
tomography (PET) imaging for pre-clinical and clinical studies is not new, however, the early approaches suffered from issues of sensitivity and resolution,
These issues were not resolved even when improved chimeric and humanized antibody radio-conjugates were developed, and so alternative antibody fragment radio-conjugate based approaches with improved pharmacokinetic (PK) profiles were applied. Limitations of these smaller protein carriers remained, however, and to address this, recent antibody development has given rise to their use in attaching radionucleotides as a theranostic platform for both radioimmunotherapy and diagnostic imaging. 
Two significant advances have contributed to this theranostic suitability, firstly the molecular engineering of the Fc portion of the antibody to reduce binding to both the neonatal receptor FcRn that contributes to the long circulatory half-life and to the Fcg-receptors reducing the off-target delivery; secondly in the bioconjugation approaches for site-specifically controlling the extent of chelators attached to the antibody and in the ability to stably attach these chelators.
The real benefit of this theranostic platform is the ability to profile and stratify the patient population for the suitability of treatment. New conjugation techniques using site-selective conjugation as well as enzymatic attachment have led to an array of novel radio-conjugates currently in development, and the relevance of radiolabel delivery through antibody chelator conjugates is higher today than ever before as combination therapies are added to the area of therapeutic development opportunities.
Recruiting the innate immune response through conjugation of immunomodulators
The innate immune system serves as the primary defense mechanism which is essential in recognizing pathogens and other endogenous molecules including tumor-derived antigens. It is an extremely complex mixture of signaling and protective components that includes dendritic cells, macrophages, and monocytes which act as inflammatory response activators. In the tumor microenvironment (TME) of cancer cells, understanding whether this signaling response activation is present or absent is likely to determine a successful therapeutic outcome, and there are several well-known pathways such as Toll-like receptors (TLRs) and stimulator of interferon genes (STING) that can regulate an antitumor immune response. 
Targeting the innate immune system using small molecule TLR and STING agonists is therefore relevant as an immunotherapeutic approach and has been increasingly investigated for novel therapeutic indications, however, this suffers from limitations of poor PK and tumor exposure via systemic delivery, often where only intratumoral injections show positive patient outcomes. One way to overcome these limitations is by conjugating the immunomodulator to a targeting antibody allowing specific delivery to the TME and improving the therapeutic index by improvements in PK and an increase in exposure of the immunomodulator at the tumor site.  As the magnitude of preclinical knowledge of immunomodulator antibody conjugates continues to increase, the therapeutic benefits of reduced systemic toxicity, as well as improvements in PK, will become clear.
Bioconjugation as a novel delivery approach for check-point inhibitors
Immuno-oncology (IO) covers a broad approach for therapeutic treatments by harnessing the immune system through activation of lymphocytic T-cells, and it has been well established that immune checkpoint proteins such as cytotoxic T lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1) and its ligand (PD-L1) participate in tumor proliferation by helping the evasion of the body’s immune system. The development of immune checkpoint inhibitors to modulate the immune response by removing this evasion has led to a step-change in our understanding of anti-cancer treatments, and inhibition of CTLA-4, PD-1 and PD-L1 have been shown to release the suppression signaling of T-cells leading to increases in cytokine expression and positive oncological outcomes.
The focus for much of this IO development has been antibody driven with several given recent market approval, however, there remain issues over reduced effectiveness of these drugs. One area that is undergoing significant research is in combination therapy where both IO antibody and cytotoxic ADCs are co-administered, and another is the development of small-molecule checkpoint inhibitors (Chki), both of which have the potential to lead to enhanced therapeutic benefits.  As the accumulation of knowledge from modulating the innate immune response through bioconjugate drugs grows, so does the direct implications for Chki’s being delivered as antibody conjugates, thereby further expanding the potential of bioconjugate medicines.
 What are Antibody-drug Conjugates? ADC Review | Journal of Antibody-drug Conjugates. Online.
 Wadhawan A, Chatterjee M, Singh G. Present Scenario of Bioconjugates in Cancer Therapy: A Review. Int J Mol Sci. 2019;20(21):5243. Published 2019 Oct 23. doi:10.3390/ijms20215243
 Wang C, Xu P, Zhang L, Huang J, Zhu K, Luo C. Current Strategies and Applications for Precision Drug Design. Front Pharmacol. Published 2018 Jul 18. doi.org/10.3389/fphar.2018.00787
 Parslow A, Parakh S, Lee F, Gan H, Scott A. Antibody–Drug Conjugates for Cancer Therapy. Biomedicines 2016;4(3):14. doi:10.3390/biomedicines4030014
 Nicolaou K, Rigol S. The Role of Organic Synthesis in the Emergence and Development of Antibody–Drug Conjugates as Targeted Cancer Therapies. Angew Chem Int Ed. 2019;58(33):2-38.
 Domingo O, Hellmuth I, Jäschke A, Kreutz C, Helm M. Intermolecular ‘Cross-Torque’: the N4-Cytosine Propargyl Residue Is Rotated to the ‘CH’-Edge as a Result of Watson-Crick Interaction. Nucleic Acids Res. 2015;43(11):5275–5283. doi:10.1093/nar/gkv285
 Massaad-Massade L, Boutary S, Caillaud M, et al. New Formulation for the Delivery of Oligonucleotides Using “Clickable” siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes. Bioconjug Chem. 2018;29(6):1961–1972. doi:10.1021/acs.bioconjchem.8b00205
 Dovgan I, Koniev O, Kolodych S, Wagner A. Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents. Bioconjug Chem. 2019;30(10):2483–2501. doi:10.1021/acs.bioconjchem.9b00306
 Jewell CM, Caruso F. Bioconjugate Materials in Vaccines and Immunotherapies. Bioconjug Chem. 2018;29(3):571. doi:10.1021/acs.bioconjchem.8b00134
 Kay E, Cuccui J, Wren BW. Recent Advances in the Production of Recombinant Glycoconjugate Vaccines. NPJ Vaccines. 2019;4:16. Published 2019 May 1. doi:10.1038/s41541-019-0110-z
 Feldman M., Bridwell A, Scott N, Vinogradov E, McKee S, Chavez S, Twentyman J, Stallings C, Rosen D, Harding C. A Promising Bioconjugate Vaccine against Hypervirulent Klebsiella Pneumoniae. PNAS. 2019;116(37):18655-18663.
 Hwang C, Smith L, Natori Y, Ellis B, Zhou B, Janda K. Efficacious Vaccine against Heroin Contaminated with Fentanyl. ACS Chem. Neurosci. 2018;9(6):1269−1275. DOI: 10.1021/acschemneuro.8b00079
 Goldberg DM, Sharkey RM. Novel radiolabeled antibody conjugates. Oncogene. 2007;26. 3734–3744. doi:10.1038/sj.onc.1210373
 Morais M, Ma MT. Site-specific chelator-antibody conjugation for PET and SPECT imaging with radiometals. Drug Discov Today Technol. 2018;30:91–104. doi:10.1016/j.ddtec.2018.10.002
 Fay R, Holland JP. The Impact of Emerging Bioconjugation Chemistries on Radiopharmaceuticals. J Nucl Med. 2019;60(5):587–591. doi:10.2967/jnumed.118.220806
 Peltek OO, Muslimov AR, Zyuzin MV, and Timin AS. Current outlook on radionuclide delivery systems: from design consideration to translation into clinics. J. Nanobiotechnol. 2019;17:90. doi:10.1186/s12951-019-0524-9
 Ignacio BJ, Albin TJ, Esser-Kahn AP, Verdoes M. Toll-like Receptor Agonist Conjugation: A Chemical Perspective. Bioconjug Chem. 2018;29(3):587–603. doi:10.1021/acs.bioconjchem.7b00808
 Cetinbas NM, Catcott KC, Avocetien K, Bentley KW, Bradly S, Carter T, et al. Tumor targeting of a STING agonist with an antibody-drug conjugate elicits potent anti-tumor immune responses. Online. The Society for Immunotherapy of Cancer (SITC) 34th Annual Meeting & Pre-Conference Programs (SITC 2019), Nov. 6–10, 2019, Gaylord National Hotel & Convention Center in National Harbor, Md.
 Lamichhane P, Deshmukh R, Brown JA, Jakubski S, Parajuli P, Nolan T, Raja D, Badawy M, Yoon T, Zmiyiwsky M, Lamichhane N. Novel Delivery Systems for Checkpoint Inhibitors. Medicines 2019;6;74. doi:10.3390/medicines6030074
 Coats S, Williams S, Kebble B, Dixit R, Tseng L, Yao N-S, Tice DA, Soria J-C. Antibody–Drug Conjugates: Future Directions in Clinical and Translational Strategies to Improve the Therapeutic Index. Clin Cancer Res. 2019. doi: 10.1158/1078-0432.CCR-19-0272
 Li PY, Fan Z, Cheng H. Cell Membrane Bioconjugation and Membrane-Derived Nanomaterials for Immunotherapy. Bioconjug Chem. 2018;29(3):624–634. doi:10.1021/acs.bioconjchem.7b00669
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How to cite:
Frigerio M, Kang F. Addressing the Challenges of Bioconjugate Medicines – J. ADC. April 8, 2020. DOI: 10.14229/jadc.2020.04.08.001.
Last Editorial Review: April 8, 2020