FORMULATION FORUM - Advances in Drug Delivery by Antibody Drug Conjugates (ADCs)

INTRODUCTION

With the launch of the very first Antibody Drug Candidate-based drug Mylotarg™ in 2020 for treatment of acute myeloid leukemia (AML), antibody drug conjugates (ADCs) have revolutionized the drug industry over several decades. Out of more than 267 clinical trials, 12 drugs have been approved by the FDA (Figure 1).¹

ADCs bring a new and innovative approach to target diseases, especially in the delivery of cancer drugs in a safe way. While conventional chemotherapy treatments remain widely used, ADC technology requires additional steps for the delivery of chemotherapy agents or payloads via a linker attached to a monoclonal antibody that specifically binds to a specific target expressed on cancer cells, thus sparing healthy cells from damage. Thus, ADCs are targeted medicines that deliver chemotherapy agents directly to cancer cells. After binding to the target protein or receptor at the cell surface, the ADC releases a cytotoxic drug into the cancer cells. These drugs can be encapsulated in lipid nanoparticles (LNPs) or directly tethered through linkers to antibodies.

Human monoclonal antibodies (IgG), a heterodimer protein (IgG) composed of two distinct fragments, the antigen binding fragment arms (Fab) and fragment crystallizable stem (Fc), are engineered to carry human antibody genes and act as an ideal delivery platform for ADCs. Highly cell-specific with long circulating half-life then offer minimal immunogenicity. As shown in Figure 2A tethered with chemical “linkers” as one entity, these antibodies and cytotoxic drugs are less prone to cleavage, thus making them very stable. As the drug penetrates into the tumors, these cells die by damaging their DNA or by preventing new cancer cells from forming and spreading.² In Figure 2B, the LNP carrying the payload is attached with specific site of antibody via a ligand.

The following will focus on two aspects of drug delivery through ADCs. One, where an antibody is conjugated via a ligand with functionalized LNPs carrying cytotoxic drugs; and two, where an antibody is conjugated directly with drug through a linker at the specific site. Let’s consider the latter one first as several ADCs have been approved in the recent past.

ADCS: UNDERSTANDING THE MECHANISMS

ADC delivery requires three basic necessary steps. First, an antibody binds with target antigen on the surface. Second, antigen-ADC complex is then internalized into target cell by receptor mediator endocytosis. And third, cytotoxic drug is released by lysosomal enzymes, which trigger cell death. ADC clinical activities are modulated by various factors, namely, target antigen, conjugation chemistries, linker attributes, payload potency, and tumor models.

Key Components
Target antigen – For an ADC to be completely internalized though endocytosis, the antigen density should be 10,000 copies/cell or higher. It is important for delivery of the payload to target the cell to avoid any subsequent cytotoxicity.³ In other words, overexpression of antigen at the tumor surface compared to normal cells is critical for minimizing the drug cytotoxicity and maximizing the efficacy of drug. For example, overexpression of HER2/neu antigen at the tumor tissues compared to healthy tissues leads to robust and efficient internalization of payload into HER2-target cells. Others with a lesser degree of antigen target expression on cell surface can lead to failed clinical trials when tested at biological doses, and termination of the investigation.⁴

ANTIBODY

It is a biological targeting epitope that promotes the internalization of drug through a receptor-mediated mechanism. It is highly effective as ADC biologics with respect to mere biologics’ affinity. With the antigens overexpressed on cell surface, ADC biologics could help mitigate on target/off toxicity on normal cells but can retain potency against tumor cells, suggesting further safety and higher efficacy of antibody-directed drugs. For instance, in a preclinical study, ADC RN765C demonstrated the killing of EGFR receptor mediated tumor cell lines while minimizing the toxicity against normal human cells.⁵

CONJUGATION & LINKER

Lysine (amino group) and cysteine (sulfhydryl group) are non-specific amino acids of antibodies for biologic conjugation. The amino acids moieties create more homogeneous ADC drug products with greater safety profiles and improved pharmacokinetic properties. Of the 267 clinical ADCs, 111 candidates utilized non-specific amino acids conjugation, 72 candidates utilized site specific conjugation.¹

Linkers are cleavable or non-cleavable. The cleavable linkers are designed to be unstable and deliver the payload inside the cell by hydrolysis or proteolysis, or thiol reduction among other mechanisms. Unexpected extracellular cleavage could lead to adverse effects or increased efficacy by being recognized by a tumor antigen- expressing cell leading to diffusion through plasma membrane. In non-cleavable linkers, like the approved ADC drug Kadcyla, the payload is released following proteolysis by lysosomal enzymes leading to release of modified payload-adduct that inhibits the diffusion across the plasma membranes and results in lowering of systemic toxicity and efficacy of drug. In ADC drugs like Enhertu that employs a cleavable linker, it shows much higher clinical activities in tumors with lower HER2 target expression.⁶

DRUG/PAYLOAD

Traditionally, cancer ADC drugs act by three distinct mechanisms. Those include microtubule inhibition, DNA damaging, and topoisomerase inhibition mechanisms. The potency of these payloads could dictate the ADC efficacy and toxicity. Payload ADC effectiveness is dependent upon the number of payload molecules per ADC (drug-antibody ratio, DAR), presence of multi-drug resistance efflux pump, potential untimely cleavage of payload outside cell as by-standard entity, and payload clearance. The net positive versus negative charge and hydrophobicity versus hydrophilicity of payload may influence the clearance rate, which could lead to altering the on-target efficacy and off-target toxicity of an ADC. Metabolism of ADCs can also have impact of safety and efficacy. For example, SN-38, a lactone-based molecule, is inactivated in the liver and can lead to undesired side effects by opening of lactone ring.⁷

Figure 3 shows the approved ADCs classified based on mechanism of action and tumors models.

The FDA has approved thus far 11 ADCs for hematologic and solid tumors as shown in Figure 2. Among them, Mylotarg™, approved in 2010 and withdrawn for safety concerns, was re-approved in 2017 at a much lower dose in combination with chemotherapy. Blenrep™, launched in 2020, was withdrawn in 2022 due to lack of post approval clinical data (Figure 1). Among 11 approved ADCs, 6 utilize microtubule inhibitor mechanism, 2 topoisomerase inhibition mechanism, and 3 ADCs involved with DNA damaging mechanism. These drugs or payloads with the highest potency are DNA damaging agent PBD (IC50 in picomolar range to lowest potency topoisomerase I inhibitor SN-38 with an IC50 values in nanomolar range.⁸

Table 1 shows the ADC approved drugs by indications and with other details.¹

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LNP-ADCs FOR DELIVERY OF ANTICANCER DRUGS

As noted in Table 1, all ADCs that are administered as a solution form have been approved for selective and targeted delivery of cancer drugs to achieve desired efficacy at lower loses and minimize the adverse effects. LNP-ADCs have gained ground in the recent past for selective and efficient delivery of cancer drugs.9 As shown in Figure 1, these LNP carriers require surface functionalization with their carboxyl groups to tether antibodies via known chemical reactions requiring 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) couplings with primary amines of antibodies. Other preferred reaction is via site-specific free sulfhydryl groups, created by antibody reduction or thiolation in the Fc region, which can be conjugated to maleimide-activated amino groups of the LNPs. Functionalization through covalent conjugation of antibody results in stable bond formation and allows for controls of ligand density. Considering the hydrodynamic radius of antibody (20 nm), the size of chemically functionalized/conjugated LNPs is expected to increase by 40 nm. The smaller size of antibody clearly allows deeper penetration into tumor cells.

LNPs can be activated by two different methods. First, it can be engineered together with lipid and targeting ligands, and second, a targeting ligand can be post inserted into LNPs. The latter is preferred because the ligands are better exposed at the outer surface LNPs as opposed to encapsulating the ligand within the LNPs in the previous case, allowing lesser degree of exposure to the outer surface. Typically, LNPs are designed with aims to long circulating DSPE-PEG bearing different functional groups such as amino (DSPE- PEG-NH2), carboxyl (DSPE-PEG-COOH), maleimide (DSPE-PEG-maleimide) or NHS (e.g. DSPE-PEG-NHS), followed by modification with antibody.

Table 2 shows a number of LNPs-antibody conjugates investigated in the clinical studies, but none have yet approved as drug products.

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In addition to LNPs, polymer-based PLGA-based NPs have also been investigated for delivery of drugs via ADCs. For example, Hu, et al tested the paclitaxel carrying PLGA conjugated antibody against the carcinoembryonic antigen overexpressed in 90% pancreatic tumors and observed complete internalization in BxPC-3 pancreatic cells with excellent cytotoxicity activities as opposed to non-targeted drug.20 Wei, et al also delivered effectively the PLGA-salinomycin through anti-CD44 Fab’s antibody ligand to suppress the prostrate cancer cells.²¹

FUTURE PERSPECTIVE & SUMMARY

As we continue to address the modern and yet more complicated challenges in drug development for formulation of innovative small molecules and large molecules to find cures for life-threatening diseases, the precise delivery of drugs to target cells is critical for preventing adverse effects and improving efficacy of drugs. Thus, finding the appropriate technology to deliver ADCs is challenging and remains at the forefront of the industry. To date, only 11 drugs have been approved as ADCs, primarily formulated in “aqueous solutions” containing ingredients or excipients approved in injectable drug products and are also listed in the FDA’s inactive ingredient database.

In the recent past, attempts have been made for conjugating antibodies with polymeric and lipid nanoparticles (LNPs) as carriers for oncology drugs, but no drugs have been approved yet. In spite of the challenges, the innovation continues as more drugs enter clinical trials.^22,23 The future looks brighter than ever before. It is because the functionalized LNPs can be ideal for conjugation with antibodies (Figure 1) and be used as state-of-the-art drug delivery systems for targeting specific tumor cells to improve therapeutic efficacy by selectively binding of antibodies to receptors overexpressed in angiogenic endothelial cells or cancer cells.9 Ascendia’s capabilities in LipidSol®, a lipid-based technology, can be engineered to fit with ADCs for targeted delivery of potent drugs to tumor cells.²⁴

REFERENCES

1. H. Maecker, V. Jonnalagadda, S. Bhakta, V. Jammalamadaka, and J. R. Junutula, Exploration of the antibody–drug conjugate clinical landscape, MABS 2023, 15, 2229101.
2. C. Peters and S. Brown, Antibody-drug conjugates as novel anti-cancer chemotherapeutics, Biosci Rep., 2015, 35(4):e00225. doi: 10.1042/BSR20150089.
3. M. Hammood, A. W. Craig, and J. V. Leyton, Impact of endocytosis mechanisms for the receptors targeted by the currently approved Antibody-Drug Conjugates (ADCs)-A necessity for future ADC research and development. Pharmaceuticals (Basel). 2021;14 doi:10.3390/ph14070674. PMID: 34358100.
4. C. Lemech, N. Woodward, N. Chan, J. Mortimer, L. Naumovski, S. Nuthalapati, B. Tong, F. Jiang, P. Ansell, C. K. Ratajczak, et al. A first-in-human, Phase 1, dose-escalation study of ABBV-176, an antibody-drug conjugate targeting the prolactin receptor, in patients with advanced solid tumors. Invest New Drugs. 2020, 38, 1815–25.
5. Wong, B.C.; Zhang, H.; Qin, L.; Chen, H.; Fang, C.; Lu, A.; Yang, Z. Carbonic anhydrase IX-directed immunoliposomes for targeted drug delivery to human lung cancer cells in vitro. Drug Des. Dev. Ther. 2014, 8, 993–1001.
6. S. Modi, C. Saura, T. Yamashita, Y. H. Park, S. B. Kim, K. Tamura, F. Andre, H. Iwata, Y. Ito, J. Tsurutani et al, Trastuzumab deruxtecan in previously treated HER2-positive breast cancer. N. Engl. J. Med. 2020, 382, 610–621.
7. R. H. Mathijssen, R. J. van Alphen, J. Verweij, W. J. Loos, K. Nooter, G. Stoter, and A. Sparreboom, Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clin Cancer Res. 2001, 7, 2182–2194.
8. D. M. Goldenberg and R. M. Sharkey, Antibody-drug conjugates targeting TROP-2 and incorporating SN-38: a case study of anti-TROP-2 sacituzumab govitecan. MAbs. 2019, 11, 987–95.
9. A. C. Marques, P. C. Costa, S. Velho and M. H. Amaral, Lipid nanoparticles functionalized with antibodies for anticancer drug therapy, Pharmaceutics 2023, 15, 216.
10. Kuo, Y.C.; Liang, C.T. Catanionic solid lipid nanoparticles carrying doxorubicin for inhibiting the growth of U87MG cells. Colloids Surf. B Biointerfaces, 2011, 85, 131–137.
11. Kim, J.H.; Kim, Y.; Bae, K.H.; Park, T.G.; Lee, J.H.; Park, K. Tumor-Targeted Delivery of Paclitaxel Using Low Density LipoproteinMimetic Solid Lipid Nanoparticles. Mol. Pharm. 2015, 12, 1230–1241.
12. Liu, D.; Liu, F.; Liu, Z.; Wang, L.; Zhang, N. Tumor specific delivery and therapy by double-targeted nanostructured lipid carriers with anti-VEGFR-2 antibody. Mol. Pharm. 2011, 8, 2291–2301.
13. Guo, S.; Zhang, Y.; Wu, Z.; Zhang, L.; He, D.; Li, X.; Wang, Z. Synergistic combination therapy of lung cancer: Cetuximab functionalized nanostructured lipid carriers for the co-delivery of paclitaxel and 5-Demethylnobiletin. Biomed. Pharmacother. 2019, 118, 109225.
14. Liu, Y.; Zhang, H.; Cui, H.; Zhang, F.; Zhao, L.; Liu, Y.; Meng, Q. Combined and targeted drugs delivery system for colorectal cancer treatment: Conatumumab decorated, reactive oxygen species sensitive irinotecan prodrug and quercetin co-loaded nanostructured lipid carriers. Drug Deliv. 2022, 29, 342–350.
15. Di Filippo, L.D.; Lobato Duarte, J.; Hofstätter Azambuja, J.; Isler Mancuso, R.; Tavares Luiz, M.; Hugo Sousa Araújo, V.; Delbone Figueiredo, I.; Barretto-de-Souza, L.; Miguel Sábio, R.; Sasso-Cerri, E.; et al. Glioblastoma multiforme targeted delivery of docetaxel using bevacizumab-modified nanostructured lipid carriers impair in vitro cell growth and in vivo tumor progression. Int. J. Pharm. 2022, 618, 121682.
16. Jain, S.; Deore, S.V.; Ghadi, R.; Chaudhari, D.; Kuche, K.; Katiyar, S.S. Tumor microenvironment responsive VEGF-antibody functionalized pH sensitive liposomes of docetaxel for augmented breast cancer therapy. Mater. Sci. Eng. C 2021, 121, 1118.
17. Lu, X.; Liu, S.; Han, M.; Yang, X.; Sun, K.; Wang, H.; Mu, H.; Du, Y.; Wang, A.; Ni, L.; et al. Afatinib-loaded immunoliposomes functionalized with cetuximab: A novel strategy targeting the epidermal growth factor receptor for treatment of non-small-cell lung cancer. Int. J. Pharm. 2019, 560, 126–135.
18. Merino, M.; Lozano, T.; Casares, N.; Lana, H.; Troconiz, I.F.; ten Hagen, T.L.M.; Kochan, G.; Berraondo, P.; Zalba, S.; Garrido, M.J. Dual activity of PD-L1 targeted Doxorubicin immunoliposomes promoted an enhanced efficacy of the antitumor immune response in melanoma murine model. J. Nanobiotechnol. 2021, 19, 102.
19. Yang, W.; Hu, Q.; Xu, Y.; Liu, H.; Zhong, L. Antibody fragment-conjugated gemcitabine and paclitaxel-based liposome for effective therapeutic efficacy in pancreatic cancer. Mater. Sci. Eng. C 2018, 89, 328–335.
20. Hu, C.M.; Kaushal, S.; Tran Cao, H.S.; Aryal, S.; Sartor, M.; Esener, S.; Bouvet, M.; Zhang, L. Half-antibody functionalized lipid-polymer hybrid nanoparticles for targeted drug delivery to carcinoembryonic antigen presenting pancreatic cancer cells. Mol. Pharm. 2010, 7, 914–920.
21. Wei, J.; Sun, J.; Liu, Y. Enhanced targeting of prostate cancer-initiating cells by salinomycin-encapsulated lipid-PLGA nanoparticles linked with CD44 antibodies. Oncol. Lett. 2019, 17, 4024–4033.
22. Espelin, C.W.; Leonard, S.C.; Geretti, E.; Wickham, T.J.; Hendriks, B.S. Dual HER2 targeting with trastuzumab and liposomal encapsulated doxorubicin (MM-302) demonstrates synergistic antitumor activity in breast and gastric cancer. Cancer Res. 2016, 76, 1517–1527.
23. Kasenda, B.; König, D.; Manni, M.; Ritschard, R.; Duthaler, U.; Bartoszek, E.; Bärenwaldt, A.; Deuster, S.; Hutter, G.; Cordier, D.; et al. Targeting immunoliposomes to EGFR-positive glioblastoma. ESMO Open 2022, 7, 100365.
24. J. Huang and S. Ali, LipidSol: Liposomes- Chemistry, properties, and applications of lipid nanoparticles, Drug Dev. Delivery, 2023, 23, 22-25.

Shauket Ali, PhD
Sr. Director, Scientific Affairs & Technical Marketing
Ascendia Pharmaceuticals
shaukat.ali@ascendiapharma.com
www.ascendiapharma.com

Dr. Shaukat Ali joins Ascendia Pharmaceuticals Inc. as Senior Director of Scientific Affairs and Technical Marketing after having worked in the pharma industry for many years. His areas of expertise include lipid chemistry, liposomes, lipid nanoparticles, surfactant-based drug delivery systems, SEDDS/SMEDDS, oral and parenteral, topical and transdermal drug delivery, immediate- and controlled-release formulations. He earned his PhD in Organic Chemistry from the City University of New York and carried out his post-doctoral research in Physical Biochemistry at the University of Minnesota and Cornell University. He has published extensively in scientific journals and is inventor/co-inventor of several US and European patents.

Jim Huang, PhD
Founder & CEO
Ascendia Pharmaceuticals
j.huang@ascendiapharma.com
www.ascendiapharma.com

Dr. Jim Huang is the Founder and CEO of Ascendia Pharmaceuticals, Inc. he earned his PhD in Pharmaceutics from the University of the Sciences in Philadelphia (formerly Philadelphia College of Pharmacy and Sciences) under Joseph B. Schwartz. He has more than 20 years of pharmaceutical experience in preclinical and clinical formulation development, manufacturing, and commercialization of oral and parenteral dosage forms. His research interests are centered on solubility/bioavailability improvement and controlled delivery of poorly water-soluble drugs through nano-based technologies.