As discussed in our previous article, antibody-drug conjugates (ADCs) have emerged as a highly promising class of anti-cancer drugs, and significant technical innovations are being made in all three components of the ADC, i.e. the antibody, the drug payload, and the linker joining them. The linker has been an area of particular focus, both within pharmaceutical development and the patent space. The primary considerations for effective ADC linkers are their stability during circulation followed by release at the target site and their method of attachment to the antibody. The latter can determine the ratio of payload to antibody, formulation homogeneity, and ease of manufacture, and will be the focus of the current article.

Early ADCs, including the FDA-approved Mylotarg and Kadcyla, were created by directly functionalizing solvent-accessible lysine residues on the antibody using N-hydroxysuccinimide (NHS) esters. Although this method is convenient, owing to the abundance of solvent-exposed lysine residues and mild reaction conditions required for conjugation, the resulting conjugates of this method are heterogeneous in their conjugation sites and amount of payload conjugated. Although the average drug-to-antibody ratio (DAR) of a formulation can generally be controlled to the desired range of 3:1-4:1, because there are approximately 90 accessible lysines on the antibodies typically used as scaffolds, a high degree of isomeric variability is still present in these mixtures. These mixtures can have varying pharmacokinetics, efficacy, and solubility. Especially considering the contribution of formulation heterogeneity to the temporary withdrawal of Mylotarg from the market, homogeneity is a key goal for next-generation ADC therapies.

The most common method for designing homogeneous ADCs is to conjugate the linker to cysteine residues on the antibody by reacting maleimide derivatives with cysteine sulfhydryl groups. Earlier applications, including the FDA-approved Adcetris (US7829531), were produced by reducing native interchain disulfide bonds in the antibody to provide accessible cysteines (US7837980B2). To improve antigen affinity, site-directed mutagenesis is now commonly employed to insert solvent-accessible cysteines that could be used for conjugation. These engineered antibodies, termed THIOMABs, have been used in the design of several ADCs (US8937161B2, US7723485B2, US15592072, US9000130B2).

Another promising method that has been used for the production of cysteine-linked ADCs, which does not require the engineering of mutant cysteine residues, is disulfide rebridging. In the human IgG antibodies generally used as templates for ADCs, there are many disulfide bonds. Some disulfide bonds, particularly intrachain bonds, can be important for proper binding of the target antigen, but several disulfide bonds have been shown to be reducible under mild conditions and nonessential for proper antibody function. This finding led to academic applications where ADCs were linked through cysteine residues at broken disulfide bonds. However, a more robust approach, which has seen activity in the biotech and patent arenas, is to bridge disulfide bonds with linkers attached to a cytotoxic payload. By temporarily reducing and then rebridging the disulfide bond, the structural integrity of the antibody remains intact, improving antigen binding and stability (US9884127B2, US14807234, US14437537, US13412816, WO2013190272A1, US20150283259A1, WO2015155753A3). Particularly interesting, this strategy has recently been used to generate homogeneous ADCs linked to two different payload drugs, potentially allowing for further potency (WO2018185526A1).

Yet another strategy designed to avoid the disruption of binding sites is conjugation at the N- and C-termini of the antibody. Since the termini are distant from the antigen binding domains and key structural elements for antibody stability, the termini are promising locations for the addition of the linker and payload. For example, antibodies have been engineered with a specific amino acid sequence at their C-termini, which allows for the specific conjugation of perfluoroaromatic linkers. The conjugation of a perfluoroaromatic monomethyl auristatin E (MMAE) derivative  to an engineered cysteine on trastuzumab was shown to retain the native antigen binding affinity and improve potency against HER2 breast cancer cells (WO2018140590A1).

The recent rapid progress in generating proteins with specific, genetically-encoded non-natural amino acids has led to several interesting applications in ADCs. Major advantages of non-natural amino acids are that they can contain functional groups not present on native amino acids, allowing for unique chemistry at specific sites and improved homogeneity, and potentially greater stability relative to native amino acids. Several non-natural amino acids, including p-azidomethyl-N-phenylalanine (US20170362334A1) and p-acetyl-L-phenylalanine (US14786402) have been engineered into trastuzumab to generate homogeneous ADCs with high efficacy against cancer cells. Most significantly, both of these ADCs demonstrated improved stability and safety in vivo relative to trastuzumab.

Although still in its infancy, another means of peptide side chain conjugation used in ADCs shows significant promise. Several groups have reported enzymatic methods to ligate linkers to antibodies. This method takes advantage of enzymes that generate post-translational modifications of proteins in a site-specific manner (US20180140714A1). For example, bacterial transglutaminase has been used to conjugate lysine-containing linker-payload combinations to a glutamine side chain of human IgG1 (US9717803B2). This strategy yielded a conjugate with improved targeting of tumor cells relative to chemically-conjugated molecules and is under active development.

In addition to conjugation of linkers to peptide components of the antibody, glycoengineering approaches have shown significant promise as well, and this topic will be discussed in a future article in this series. As evidenced by the diverse and robust research and IP activity focused on ADC linker chemistries, the linker provides exciting opportunities for both improvements in potency and safety as well as novel ADC IP. Especially in the ADC arena fraught with overlapping claims on naked antibodies, payloads, and established linkers, new modalities in linker chemistries provide needed avenues for innovation.

Patent or Application Number Inventor Assignee Title Issue or Publication Date
US7829531B2 Senter et al Seattle Genetics Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease 09-Nov-2010
US7837980B2 Alley et al Seattle Genetics Partially loaded antibodies and methods of their conjugation 23-Nov-2010
US8937161B2 Mao et al Genentech Inc Cysteine engineered anti-TENB2 antibodies and antibody drug conjugates 20-Jan-2015
US7723485B2 Junutula et al Genentech Inc Cysteine engineered anti-MUC16 antibodies and antibody drug conjugates 25-May-2010
US15592072 Eigenbrot et al Genentech Inc Cysteine engineered antibodies and conjugates 04-Jan-2018
US9000130B2 Bhakta et al Genentech Inc Cysteine engineered antibodies and conjugates 07-Apr-2015
US9884127B2 Miao et al Concortis Biosystems Corp Drug-conjugates, conjugation methods, and uses thereof 06-Feb-2018
US14807234 An et al Newbio Therapeutics, Inc Tridentate connexon and use thereof 21-Jan-2016
US14437537 Burt et al PolyTherics Ltd Drug-protein conjugates 15-Oct-2015
US13412816 Chang et al IBC Pharmaceuticals Inc Dimeric alpha interferon PEGylated site-specifically shows enhanced and prolonged efficacy in vivo 07-May-2013
WO2013190272A1 Godwin, Antony PolyTherics Ltd Conjugation reagents 27-Dec-2013
US20150283259A1 Burt et al PolyTherics Ltd Drug-protein conjugates 24-Oct-2015
WO2015155753A3 Zhao et al Suzhou M-Conj Biotech Co., Ltd Novel linkers and their uses in specific conjugation of drugs to biological molecule 10-Aug-2015
WO2018185526A1 Zhao et al Hangzhou Dac Biotech Co., Ltd Conjugation of a cytotoxic drug with bis-linkage 11-Oct-2018
WO2018140590A1 Pentelute et al Massachusetts Institute of Technology Self-labeling miniproteins and conjugates comprising them 02-Aug-2018
US20170362334A1 Thanos et al Sutro Biopharma Inc Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use 21-Dec-2017
US14786402 Gonzalez et al Avelas Biosciences Inc Selective drug delivery compositions and methods of use 24-Mar-2016
US20180140714A1 Dushin et al Pfizer Inc, Rinat Neuroscience Corp Stability-modulating linkers for use with antibody drug conjugates 24-May-2018
US9717803B2 Bregeon et al Scherrer Paul Institut, Innate Pharma Enzymatic conjugation of polypeptides 01-Aug-2017

-Steve Kennedy and Anthony Sabatelli, PhD, JD

Steve Kennedy is a Ph.D. Candidate in the Chemistry Department at New York University. He specializes in biophysical characterization of protein complexes and is currently focused on the role of adaptor proteins in signaling pathways. Prior to attending NYU, Steve obtained his B.S. in Chemistry with Cum Laude honors at the University of Massachusetts – Boston, during which time he conducted bioanalytical mass spectrometry method development and lipidomics research.

This article is for informational purposes, is not intended to constitute legal advice, and may be considered advertising under applicable state laws. The opinions expressed in this article are those of the author only and are not necessarily shared by Dilworth IP, its other attorneys, agents, or staff, or its clients.